INTRODUCTION

From a biological perspective carotenoids can be mosteasily classified as those that possess pro-vitamin Aactivity and those that do not From a chemical perspec-tive carotenoids are generally classified as the hydrocar-bon carotenoids of which the prototype would beβ-carotene a C40H56 hydrocarbon containing two β-ionone rings and 11 conjugated double bonds and thexanthophylls oxygenated carotenoids such as luteinC40H56O2 found in all green leaves with hydroxylgroups on each of the rings Both compounds can befound at micromolar amounts in human serum and areobtained from consumption of carotenoid-containing

fruits and vegetables1 Carotenoids may also be straight-chain molecules such as lycopene C40H56 the red pig-ment found in tomatoes The principal structural featuresof carotenoids are the possession of a methyl-substi-tuted conjugated double-bond system reflecting theirsynthesis from isoprene units in the plant2 Untilrecently carotenoids were considered to have two majorbiological functions First the possession of a β-iononering by the pro-vitamin A carotenoids allows their bio-conversion in mammals to retinoids ndash compounds withessential roles in vision and in normal growth and differ-entiation3 Second the conjugated double-bond systempossessed by all carotenoids allows them to be effectivelipid-phase antioxidants which can protect cellorganelles from free-radical mediated damage As dis-cussed later there is now evidence that carotenoids candirectly regulate the expression of genes protectiveagainst carcinogenesis and inflammation The role ofcarotenoids in the biosynthesis of retinal the visual pig-ment in the retina is a separate and discrete function thatwill not be further considered here Instead we will con-

copy W S Maney amp Son LtdRedox Report Vol 9 No 4 2004DOI 101179135100004225005967

Review

Bioactive carotenoids potent antioxidants and regulators of gene expression

Laura M Hix12 Samuel F Lockwood3 John S Bertram2

1Department of Cell and Molecular Biology and 2Cancer Research Center of Hawaii University of Hawaii at Manoa Honolulu Hawaii USA

3Hawaii Biotech Inc Aiea Hawaii USA

Carotenoi ds are plant pigments some of which act as a vital source of vitamin A to all animals thatappear to have additional benefits to primates They are potent antioxidants and photoprotectantsand can additionally modulate gene activity resulting in protection from experimentally-inducedinflammatory damage and neoplastic transformation Anti-neoplastic properties appear tightlycorrelated to their ability to induce the gap junctional protein connexin 43 (Cx43) This when up-regulated leads to decreased proliferation and decreased indices of neoplasia in animal and humancells Delivery of natural carotenoids can be compromised by poor bioavailability To overcomethis a synthetic water-dispersible derivative of astaxanthin has been synthesized and shown to behighly bioavailable a potent antioxidant protective against experimental ischemia-reperfusioninjury and capable of inducing Cx43 suggesting antineoplastic potential The ability to deliverbiologically active carotenoids at high concentration and with good reproducibility appears to havebeen achieved

Keywords Carotenoids vitamin A anti-neoplastic properties astaxanthin derivative

Received 4 April 2004Accepted 5 May 2004

Correspondence to Prof John S Bertram PhD Cancer ResearchCenter of Hawaii University of Hawaii at Manoa 1236 Laulala StHonolulu HI 96813 USATel +1 808 586 2957 Fax +1 808 586 2970Email Johncrchhawaiiedu

centrate on the role of carotenoids as antioxidants and asmodulators of gene expression

The antioxidant properties of carotenoids

The carotenoids are photoprotective in plants and manIn plants carotenoids are synthesized as essential com-ponents of the photosynthetic system They performvital roles which include (i) light-harvesting therebyimproving the efficiency of photosynthesis (ii) photo-protection (iii) singlet oxygen scavenging and (iv) dis-sipation of excess energy from excited chlorophyll4 Theprotective role of carotenoids is exemplified by thedemonstration that targeted inhibition of carotenoidbiosynthesis by herbicides leads to the rapid loss of via-bility of leaves exposed to full solar radiation5 Inhumans the xanthophylls lutein and zeaxanthin foundin all green leaves accumulate in the macula of the eyeand may also serve a similar function of photoprotectionas this region is the area subjected to greatest exposure tolight6 This is supported by evidence that individualswho consume lower amounts of these carotenoids sufferfrom an increased frequency of age-related maculardegeneration (AMD) the most frequent form of blind-ness in the elderly In further support of this associationlevels of lutein and zeaxanthin in affected retinas are sig-nificantly lower than in retinas taken from age-matchedcontrols7 It is no coincidence that the macula is alsoknown as the yellow spot by virtue of the accumulationof these yellow-absorbing carotenoids in this region ofthe retina Carotenoids also appear to have direct photo-protective properties in human skin in a manner proba-bly analogous to an internal sunscreen This property hasbeen utilized clinically in individuals suffering from ery-thropoietic protoporphyria a condition associated withacute photosensitivity Here oral administration ofeither β-carotene or canthaxanthin leads to skin pigmen-tation and decreased erythema after photo-irradiation8

Carotenoids protect against chemical oxidative damage

Cell constituents are confronted with multiple sources ofhighly-reactive oxidative species capable of causinglipid peroxidation and DNA damage In purely chemicalsystems carotenoids can be shown to be effectivequenchers of reactive oxygen species such as singletoxygen as well as radical chain breaking agents How-ever as first described by Burton and Ingold9 the situa-tion is complicated by the ability of β-carotene to act asa pro-oxidant under conditions of high pO2 Under theseconditions the initial reaction with oxidant produces anunstable intermediate which can itself react with unsatu-rated lipids for example to produce a chain reaction and

the formation of additional reactive species such asepoxides and carbonyl compounds The biological sig-nificance of this phenomenon is unclear but may be inpart responsible for the increased lung cancer ratesobserved in intervention trials conducted with high-doseβ-carotene in smokers and asbestos-exposed individu-als1011 Fortunately not all carotenoids become pro-oxi-dants under these conditions astaxanthin (33prime-dihydro-44prime-diketo-β-carotene) has been described as amore potent antioxidant than β-carotene yet does not toact as a pro-oxidant under high partial pressure of oxy-gen12 This would imply that astaxanthin and other keto-carotenoids may be inherently safer antioxidants in anycontext of high oxygen concentration such as the lungor in patients receiving supplemental oxygen

There are many examples where oxidative stress haspathological consequences in humans For examplechronic inflammation ndash either as a consequence ofautoimmune disorders or unresolved infection ndash leads tothe generation by immune cells of large amounts ofdiverse reactive oxygen species These reactive speciessuch as superoxide anion (O2

bullndash) hydrogen peroxide(H2O2) and hypochlorous acid (HOCl) have physiologi-cal functions in being cytotoxic to invading pathogenswhile the production of nitric oxide (NO) bymacrophages leads to vasodilation and increased bloodflow to infected tissues1314 In conditions of chronicinflammation however this production of reactive oxy-gen species may continue for months or years and bedamaging to local tissue and surrounding stroma Forexample chronic viral hepatitis leads to progressive lossof the liver parenchyma and replacement by fibroticlesions ndash a process known as cirrhosis Similar tissuedamage occurs in conditions of chronic gastritis and col-itis atherosclerosis and rheumatoid arthritis Unfortu-nately damage occurs not just to the architecture of thetissue but also to the genome These same reactive oxy-gen species are known to be capable of causing DNAlesions such as 8-hydroxy-deoxyguanosine and 8-oxo-guanosine both pro-mutagenic lesions They also cande-aminate DNA leading again to potential mutagenicchanges if unrepaired15 The increased cell divisioncaused by reactive hyperplasia in chronically inflamedtissues together with the increased rate of DNA damagealmost certainly results in the increased rate of malig-nancy seen in these conditions16 Indeed chronic infec-tion of gastric epithelium with Helicobacter pylori isnow known to cause most cases of chronic gastritis inthe West a condition that can lead to stomach cancer ina significant proportion of cases17

Preliminary data from the group of Nishino in Japansuggested that supplemental lycopene (10 mgday) andα-tocopherol administered orally to patients withchronic hepatitis C results in a dramatic decrease in theincidence of hepatocellular carcinoma beginning about a

182 Hix Lockwood Bertram

year after initiating treatment18 These data are consistentwith reports from the same group that liver fibrosis inrats with copper overload can be suppressed by lycopenealone19 Globally liver cancer is the most prevalent can-cer resulting most frequently from chronic viral infec-tion and aflatoxin-contaminated foods this observationtherefore is of enormous potential significance Itremains to be determined whether the chemopreventiveaction of lycopene is mediated through its antioxidantproperties or through more specific effects on gene regu-lation as will be discussed later Astaxanthin a xantho-phyll predominantly found in marine organisms andresponsible for the pigmentation of shrimp and lobsterand for the pink pigmentation of birds like the flamingowho feed on marine organisms may also possess potentanti-inflammatory activity In studies in cell culture andin mice astaxanthin was shown to inhibit the productionof inflammatory cytokines such as TNF-αprostaglandins and NO This activity resulted frominhibited activation of the nuclear transcription factorNF-κB probably a result of the scavenging of reactiveoxygen species known to activate this inflammationpathway20 While the concentrations of astaxanthin (50microM) required to produce this effect were certainly supra-physiological these concentrations are achievable bynovel highly bioavailable astaxanthin derivatives to bedescribed below

Carotenoids may protect against atherosclerosis

A second important example where oxidative damagehas severe pathological consequences is in atherosclero-sis It is now well-established that oxidation of low-den-sity lipoprotein (LDL) is a major contributing factor tothe initiation and progression of atherosclerotic lesionsthat are a major cause of the approximately 15 millionmyocardial infarctions that occur annually in the US Awidely accepted model is that damaged LDL particlesare recognized by monocytesmacrophages andengulfed an excess of oxidatively-modified LDL resultsin their conversion to a foam cell and formation of fattystreaks ndash early markers of arterial damage The presenceof foam cells in the vessel wall sets off a cycle of inflam-mation leading to additional oxidative stress prolifera-tion of the surrounding smooth muscle cells andprogressive narrowing of the artery Normalization oflipid profiles by diet and cholesterol-lowering statins canhave a major influence on risk21 There is growing evi-dence that carotenoids such as astaxanthin by acting aslipid-phase antioxidants may also be effective22 Againnot all carotenoids appear equal in this respect Forexample in three large scale trials supplemental β-carotene appeared to have no influence on incidence ofcardiovascular disease23 However epidemiological

studies have supported the role of dietary lycopene as acardiovascular risk-reducing factor24 Higher plasma lev-els of lutein were also found to be correlated inverselywith age-related increased thickening of the coronaryartery wall ndash a major risk factor for subsequent infarc-tion These authors also demonstrated the ability oflutein to decrease LDL oxidation and to decrease theattraction of monocytes to human arterial walls in cul-ture Similar results supporting the protective role oflutein against atherosclerosis were obtained in studiesutilizing a mouse model of atherosclerosis25 It may bethat lutein and other polar carotenoids are superior to β-carotene by virtue of increased gastrointestinal absorp-tion and cellular uptake as a consequence of the presenceof hydrophilic hydroxyl groups Astaxanthin is also apolar carotenoid with superior quenching ability rela-tive to both lutein and β-carotene12 and may be evenmore efficacious in cardiovascular applications22 Theability to interfere with the cycle of inflammation andtissue damage in lipoproteins and the coronary vesselwall could have a major impact on clinical outcome andlong-term consequences of other chronic diseases suchas cancer

Novel carotenoids protect against experimentalischemia-reperfusion injury

Reperfusion injury is a relatively recently recognizedphenomenon appearing when oxygenated blood is re-introduced into an ischemic area This may occur as aresult of pathological vascular occlusion during myocar-dial infarction or stroke followed by medical interven-tion26 There is abundant evidence that a major factorcontributing to this injury is the release of reactive oxy-gen species (ROS) from mitochondria and from neu-trophils and other leukocytes trapped in the ischemictissue27 The ensuing cycle of lipid peroxidation releaseof cytokines and loss of membrane integrity leads todeath of cells in the affected area A strong rationale thusexists for the use of agents that can both quench thesereactive species and act as radical chain-breaking antiox-idants to interrupt this cycle Martin et al12 have shownthat carotenoids and in particular astaxanthin functioneffectively as chain-breaking antioxidants at low partialpressures of oxygen and low physiological concentra-tions in contrast to other lipophilic antioxidants such asvitamin E28 This suggests that carotenoids may beuniquely suited for the clinical prevention andor ame-lioration of ischemia-reperfusion injury However thehighly lipophilic nature of these compounds has effec-tively prevented their clinical use in the acute coronarysyndrome (ACS) as well in experimental settings asdelivery of effective concentrations to cells and tissues isproblematic A major achievement to overcome these

Bioactive carotenoids potent antioxidants and regulators of gene expression 183

problems was the development of the use of tetrahydro-furan (THF) as a delivery vehicle for carotenoids to cellsin culture29 but it seems unlikely that this solvent wouldbe clinically acceptable

Development of a water-dispersible derivative ofastaxanthin

In order to overcome the clinical problems associatedwith carotenoid drug delivery Hawaii Biotech Inc(HBI Aiea HI USA) has developed a novel carotenoidderivative the disodium disuccinate derivative of astax-anthin Cardaxtrade (Fig 1) This compound forms apseudo-solution in water at concentrations of up to 8mgml (~10 mM) Dispersibility is achieved in aqueoussolution secondary to self-assembly of disodium disucci-nate astaxanthin monomers into supra-molecular com-plexes Monomeric solutions of compound can also beachieved by the inclusion of ethanol at concentrations upto 50 thereby disrupting this self-assembly but pre-serving aqueous solubility30 Additionally the formationof an oil waterlecithin emulsion allows Cardax to beformulated at concentrations of greater than 50 mgmlfor oral delivery3132 These formulations of astaxanthinoffer three exciting possibilities (i) that it can be rapidlydelivered at high concentrations to tissues after eitheroral or parenteral administration (ii) aqueous-phaseROS scavenging (eg singlet oxygen and superoxideanion released by neutrophils) occurs with high effi-ciency and (iii) the ester-linkage of the succinate moi-eties should make this molecule susceptible to serum

and cell esterases leading to release of free astaxanthincapable of acting as a chain-breaking antioxidant in thelipid environment of lipoprotein or cell membrane Car-dax appears to fulfil all these expectations

Cardax is highly bioavailable after oral or intravenousadministration

Administration of Cardax as an oilwater emulsion at aconcentration of 50 mgml to mice by oral gavage atdose of 500 mgkg bodyweight requires only a volumeof about 25 microl This resulted in the rapid appearance offree astaxanthin in serum and liver with peak levelsbeing achieved approximately 6 h after administrationAt this time point levels were 1760 nM in liver 694 nMin heart while plasma levels were approximately 25 ofthat found in liver33 This demonstrates rapid absorptionand cleavage to free astaxanthin in these animalstogether with accumulation in target tissues Tissue lev-els are above the reported ED50 of 200 nM of free astax-anthin as a radical scavenger suggesting that oraladministration of this novel carotenoid derivative canachieve significant tissue protection against oxidativestress Cardax has also been administered in an aqueoussolution by intravenous tail vein injection to rats in thecardioprotective study discussed below There was adose-dependent increase in plasma astaxanthin with amean peak concentration of 612 nM being achieved 24 hafter four daily doses of 75 mgkg Levels in cardiac tis-sue were not measured31

184 Hix Lockwood Bertram

Fig 1 Structures of the three stereoisomers of the disodium salt disuccinate astaxanthin derivatives present in Cardax evaluated in the current study(shown as the all-E geometric isomers) The racemic mixture of stereoisomers contains (3S3primeS)-astaxanthin disuccinate disodium salt (3R3primeR)-astaxanthin disuccinate disodium salt and (3R3primeS meso)-astaxanthin disuccinate disodium salt in a 112 ratio

Direct scavenging of the superoxide anion

When incubated with human polymorphonuclear leuko-cytes stimulated to release superoxide by exposure tophorbol ester Cardax dose-dependently decreased thelevels of superoxide anion detected by EPR spec-troscopy utilizing the spin trap DEPMPO Completeinhibition of superoxide anion signal was achieved atmillimolar concentrations of Cardax32 This in vitro testsystem generates molar amounts of superoxide anionmuch higher than that achieved in ischemic tissue invivo In addition supramolecular assembly of Cardax inaqueous solution of the monomeric compound limits thereactivity of the monomeric compound with aqueous-phase radicals Therefore it was postulated that muchlower concentrations would be therapeutic in animalmodels in vivo results that were subsequently achievedat nanomolar concentrations in both rats (see below)31

and dogs (Lockwood unpublished results)

Protection from experimental ischemia-reperfusion injury

In a rat model of ischemiareperfusion injury (30 min ofleft anterior descending coronary artery occlusion withsubsequent reperfusion) Cardax afforded significantcardioprotection In rats pretreated with a single dose ofintravenous Cardax for each of 4 days prior to experi-mental infarction on day 5 a dose-dependent decrease inmean infarct size was observed A dose of 75 mgkgdelivered in aqueous lsquosolutionrsquo intravenously led to adecrease in infarct size of 59 over vehicle-alone con-trols a result which was statistically highly significant

(P lt 0001 Fig 2) This degree of protection frommyocardial damage was directly correlated with theserum concentrations of free astaxanthin which reachedapproximately 600 nM after 4 days of injection at thehighest concentration tested There was no apparent tox-icity to the animals during treatment31 This treatmentprotocol might allow significant protection from damagein patients at high risk for myocardial infarction or inpatients scheduled for elective surgery where inductionof reperfusion injury is a significant risk The ability todeliver astaxanthin during the lsquogolden windowrsquo ofmyocardial salvage (3ndash6 h after coronary occlusion) isalso possible a single iv dose given 5 min prior toreperfusion showed significant myocardial salvage in arodent model (Lockwood unpublished results) It islikely that additional astaxanthin derivatives withimproved pharmacokinetic profiles can be developedand be better suited to this clinical model

In addition to protection against ROS damage to mem-branes astaxanthin and for that matter other carotenoidsif they can be delivered at sufficient concentrations maybe capable of affording additional protection to the dam-aged myocardium As will be discussed below Cardaxhas been shown to increase expression of Cx43 in mouse10T12 fibroblasts34 While the studies were performedin the context of the prevention of malignant transforma-tion it is known that Cx43 plays a vital role in car-diomyocytes as the structural element of gap junctionsIn the heart gap junctions are a major component of theintercalated disc and connect myocardial cells into asyncytium allowing co-ordinated contraction35 Gapjunction toxins which include ROS decrease signaltransduction through the gap junction36 and would beexpected to cause uncoordinated contraction identifiedclinically as fibrillation and other arrhythmias Thuscarotenoids by inhibiting the propagation of ROS mayprevent fibrillation while their ability to increase syn-thesis of the structural components of the gap junctionCx43 has the potential to increase the rate of recovery ofthe myocardium after infarction These possibilitieshave yet to be evaluated

Diverse carotenoids inhibit neoplastic transformation

There is abundant epidemiological evidence that con-sumption of dietary carotenoids is associated with pro-tection against cancer in humans37 To determine if thisassociation could be confirmed experimentally and todiscover which dietary carotenoids possessed this activ-ity we began studies in transformable C3H10T12 cells(10T12) We had previously shown these cells torespond to chemical and physical carcinogens by thequantitative formation of neoplastically transformedfoci38 and had also demonstrated that cancer-preventive

Bioactive carotenoids potent antioxidants and regulators of gene expression 185

Fig 2 Mean myocardial salvage as a percentage of infarct sizearea atrisk Control mean infarct size (IS)area at risk (AAR) set at 0 salvageControl animals received vehicle injection alone Cardax-treated animalsreceived the appropriate dose once daily iv by tail vein injection for 4days prior to experimental infarction and ISAAR determinations on day 5Myocardial salvage of 56 was achieved at the highest dose administered(75 mgkg) which was statistically highly significant (P lt 0001) FromGross and Lockwood31 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

centrate on the role of carotenoids as antioxidants and asmodulators of gene expression

The antioxidant properties of carotenoids

The carotenoids are photoprotective in plants and manIn plants carotenoids are synthesized as essential com-ponents of the photosynthetic system They performvital roles which include (i) light-harvesting therebyimproving the efficiency of photosynthesis (ii) photo-protection (iii) singlet oxygen scavenging and (iv) dis-sipation of excess energy from excited chlorophyll4 Theprotective role of carotenoids is exemplified by thedemonstration that targeted inhibition of carotenoidbiosynthesis by herbicides leads to the rapid loss of via-bility of leaves exposed to full solar radiation5 Inhumans the xanthophylls lutein and zeaxanthin foundin all green leaves accumulate in the macula of the eyeand may also serve a similar function of photoprotectionas this region is the area subjected to greatest exposure tolight6 This is supported by evidence that individualswho consume lower amounts of these carotenoids sufferfrom an increased frequency of age-related maculardegeneration (AMD) the most frequent form of blind-ness in the elderly In further support of this associationlevels of lutein and zeaxanthin in affected retinas are sig-nificantly lower than in retinas taken from age-matchedcontrols7 It is no coincidence that the macula is alsoknown as the yellow spot by virtue of the accumulationof these yellow-absorbing carotenoids in this region ofthe retina Carotenoids also appear to have direct photo-protective properties in human skin in a manner proba-bly analogous to an internal sunscreen This property hasbeen utilized clinically in individuals suffering from ery-thropoietic protoporphyria a condition associated withacute photosensitivity Here oral administration ofeither β-carotene or canthaxanthin leads to skin pigmen-tation and decreased erythema after photo-irradiation8

Carotenoids protect against chemical oxidative damage

Cell constituents are confronted with multiple sources ofhighly-reactive oxidative species capable of causinglipid peroxidation and DNA damage In purely chemicalsystems carotenoids can be shown to be effectivequenchers of reactive oxygen species such as singletoxygen as well as radical chain breaking agents How-ever as first described by Burton and Ingold9 the situa-tion is complicated by the ability of β-carotene to act asa pro-oxidant under conditions of high pO2 Under theseconditions the initial reaction with oxidant produces anunstable intermediate which can itself react with unsatu-rated lipids for example to produce a chain reaction and

the formation of additional reactive species such asepoxides and carbonyl compounds The biological sig-nificance of this phenomenon is unclear but may be inpart responsible for the increased lung cancer ratesobserved in intervention trials conducted with high-doseβ-carotene in smokers and asbestos-exposed individu-als1011 Fortunately not all carotenoids become pro-oxi-dants under these conditions astaxanthin (33prime-dihydro-44prime-diketo-β-carotene) has been described as amore potent antioxidant than β-carotene yet does not toact as a pro-oxidant under high partial pressure of oxy-gen12 This would imply that astaxanthin and other keto-carotenoids may be inherently safer antioxidants in anycontext of high oxygen concentration such as the lungor in patients receiving supplemental oxygen

There are many examples where oxidative stress haspathological consequences in humans For examplechronic inflammation ndash either as a consequence ofautoimmune disorders or unresolved infection ndash leads tothe generation by immune cells of large amounts ofdiverse reactive oxygen species These reactive speciessuch as superoxide anion (O2

bullndash) hydrogen peroxide(H2O2) and hypochlorous acid (HOCl) have physiologi-cal functions in being cytotoxic to invading pathogenswhile the production of nitric oxide (NO) bymacrophages leads to vasodilation and increased bloodflow to infected tissues1314 In conditions of chronicinflammation however this production of reactive oxy-gen species may continue for months or years and bedamaging to local tissue and surrounding stroma Forexample chronic viral hepatitis leads to progressive lossof the liver parenchyma and replacement by fibroticlesions ndash a process known as cirrhosis Similar tissuedamage occurs in conditions of chronic gastritis and col-itis atherosclerosis and rheumatoid arthritis Unfortu-nately damage occurs not just to the architecture of thetissue but also to the genome These same reactive oxy-gen species are known to be capable of causing DNAlesions such as 8-hydroxy-deoxyguanosine and 8-oxo-guanosine both pro-mutagenic lesions They also cande-aminate DNA leading again to potential mutagenicchanges if unrepaired15 The increased cell divisioncaused by reactive hyperplasia in chronically inflamedtissues together with the increased rate of DNA damagealmost certainly results in the increased rate of malig-nancy seen in these conditions16 Indeed chronic infec-tion of gastric epithelium with Helicobacter pylori isnow known to cause most cases of chronic gastritis inthe West a condition that can lead to stomach cancer ina significant proportion of cases17

Preliminary data from the group of Nishino in Japansuggested that supplemental lycopene (10 mgday) andα-tocopherol administered orally to patients withchronic hepatitis C results in a dramatic decrease in theincidence of hepatocellular carcinoma beginning about a

182 Hix Lockwood Bertram

year after initiating treatment18 These data are consistentwith reports from the same group that liver fibrosis inrats with copper overload can be suppressed by lycopenealone19 Globally liver cancer is the most prevalent can-cer resulting most frequently from chronic viral infec-tion and aflatoxin-contaminated foods this observationtherefore is of enormous potential significance Itremains to be determined whether the chemopreventiveaction of lycopene is mediated through its antioxidantproperties or through more specific effects on gene regu-lation as will be discussed later Astaxanthin a xantho-phyll predominantly found in marine organisms andresponsible for the pigmentation of shrimp and lobsterand for the pink pigmentation of birds like the flamingowho feed on marine organisms may also possess potentanti-inflammatory activity In studies in cell culture andin mice astaxanthin was shown to inhibit the productionof inflammatory cytokines such as TNF-αprostaglandins and NO This activity resulted frominhibited activation of the nuclear transcription factorNF-κB probably a result of the scavenging of reactiveoxygen species known to activate this inflammationpathway20 While the concentrations of astaxanthin (50microM) required to produce this effect were certainly supra-physiological these concentrations are achievable bynovel highly bioavailable astaxanthin derivatives to bedescribed below

Carotenoids may protect against atherosclerosis

A second important example where oxidative damagehas severe pathological consequences is in atherosclero-sis It is now well-established that oxidation of low-den-sity lipoprotein (LDL) is a major contributing factor tothe initiation and progression of atherosclerotic lesionsthat are a major cause of the approximately 15 millionmyocardial infarctions that occur annually in the US Awidely accepted model is that damaged LDL particlesare recognized by monocytesmacrophages andengulfed an excess of oxidatively-modified LDL resultsin their conversion to a foam cell and formation of fattystreaks ndash early markers of arterial damage The presenceof foam cells in the vessel wall sets off a cycle of inflam-mation leading to additional oxidative stress prolifera-tion of the surrounding smooth muscle cells andprogressive narrowing of the artery Normalization oflipid profiles by diet and cholesterol-lowering statins canhave a major influence on risk21 There is growing evi-dence that carotenoids such as astaxanthin by acting aslipid-phase antioxidants may also be effective22 Againnot all carotenoids appear equal in this respect Forexample in three large scale trials supplemental β-carotene appeared to have no influence on incidence ofcardiovascular disease23 However epidemiological

studies have supported the role of dietary lycopene as acardiovascular risk-reducing factor24 Higher plasma lev-els of lutein were also found to be correlated inverselywith age-related increased thickening of the coronaryartery wall ndash a major risk factor for subsequent infarc-tion These authors also demonstrated the ability oflutein to decrease LDL oxidation and to decrease theattraction of monocytes to human arterial walls in cul-ture Similar results supporting the protective role oflutein against atherosclerosis were obtained in studiesutilizing a mouse model of atherosclerosis25 It may bethat lutein and other polar carotenoids are superior to β-carotene by virtue of increased gastrointestinal absorp-tion and cellular uptake as a consequence of the presenceof hydrophilic hydroxyl groups Astaxanthin is also apolar carotenoid with superior quenching ability rela-tive to both lutein and β-carotene12 and may be evenmore efficacious in cardiovascular applications22 Theability to interfere with the cycle of inflammation andtissue damage in lipoproteins and the coronary vesselwall could have a major impact on clinical outcome andlong-term consequences of other chronic diseases suchas cancer

Novel carotenoids protect against experimentalischemia-reperfusion injury

Reperfusion injury is a relatively recently recognizedphenomenon appearing when oxygenated blood is re-introduced into an ischemic area This may occur as aresult of pathological vascular occlusion during myocar-dial infarction or stroke followed by medical interven-tion26 There is abundant evidence that a major factorcontributing to this injury is the release of reactive oxy-gen species (ROS) from mitochondria and from neu-trophils and other leukocytes trapped in the ischemictissue27 The ensuing cycle of lipid peroxidation releaseof cytokines and loss of membrane integrity leads todeath of cells in the affected area A strong rationale thusexists for the use of agents that can both quench thesereactive species and act as radical chain-breaking antiox-idants to interrupt this cycle Martin et al12 have shownthat carotenoids and in particular astaxanthin functioneffectively as chain-breaking antioxidants at low partialpressures of oxygen and low physiological concentra-tions in contrast to other lipophilic antioxidants such asvitamin E28 This suggests that carotenoids may beuniquely suited for the clinical prevention andor ame-lioration of ischemia-reperfusion injury However thehighly lipophilic nature of these compounds has effec-tively prevented their clinical use in the acute coronarysyndrome (ACS) as well in experimental settings asdelivery of effective concentrations to cells and tissues isproblematic A major achievement to overcome these

Bioactive carotenoids potent antioxidants and regulators of gene expression 183

problems was the development of the use of tetrahydro-furan (THF) as a delivery vehicle for carotenoids to cellsin culture29 but it seems unlikely that this solvent wouldbe clinically acceptable

Development of a water-dispersible derivative ofastaxanthin

In order to overcome the clinical problems associatedwith carotenoid drug delivery Hawaii Biotech Inc(HBI Aiea HI USA) has developed a novel carotenoidderivative the disodium disuccinate derivative of astax-anthin Cardaxtrade (Fig 1) This compound forms apseudo-solution in water at concentrations of up to 8mgml (~10 mM) Dispersibility is achieved in aqueoussolution secondary to self-assembly of disodium disucci-nate astaxanthin monomers into supra-molecular com-plexes Monomeric solutions of compound can also beachieved by the inclusion of ethanol at concentrations upto 50 thereby disrupting this self-assembly but pre-serving aqueous solubility30 Additionally the formationof an oil waterlecithin emulsion allows Cardax to beformulated at concentrations of greater than 50 mgmlfor oral delivery3132 These formulations of astaxanthinoffer three exciting possibilities (i) that it can be rapidlydelivered at high concentrations to tissues after eitheroral or parenteral administration (ii) aqueous-phaseROS scavenging (eg singlet oxygen and superoxideanion released by neutrophils) occurs with high effi-ciency and (iii) the ester-linkage of the succinate moi-eties should make this molecule susceptible to serum

and cell esterases leading to release of free astaxanthincapable of acting as a chain-breaking antioxidant in thelipid environment of lipoprotein or cell membrane Car-dax appears to fulfil all these expectations

Cardax is highly bioavailable after oral or intravenousadministration

Administration of Cardax as an oilwater emulsion at aconcentration of 50 mgml to mice by oral gavage atdose of 500 mgkg bodyweight requires only a volumeof about 25 microl This resulted in the rapid appearance offree astaxanthin in serum and liver with peak levelsbeing achieved approximately 6 h after administrationAt this time point levels were 1760 nM in liver 694 nMin heart while plasma levels were approximately 25 ofthat found in liver33 This demonstrates rapid absorptionand cleavage to free astaxanthin in these animalstogether with accumulation in target tissues Tissue lev-els are above the reported ED50 of 200 nM of free astax-anthin as a radical scavenger suggesting that oraladministration of this novel carotenoid derivative canachieve significant tissue protection against oxidativestress Cardax has also been administered in an aqueoussolution by intravenous tail vein injection to rats in thecardioprotective study discussed below There was adose-dependent increase in plasma astaxanthin with amean peak concentration of 612 nM being achieved 24 hafter four daily doses of 75 mgkg Levels in cardiac tis-sue were not measured31

184 Hix Lockwood Bertram

Fig 1 Structures of the three stereoisomers of the disodium salt disuccinate astaxanthin derivatives present in Cardax evaluated in the current study(shown as the all-E geometric isomers) The racemic mixture of stereoisomers contains (3S3primeS)-astaxanthin disuccinate disodium salt (3R3primeR)-astaxanthin disuccinate disodium salt and (3R3primeS meso)-astaxanthin disuccinate disodium salt in a 112 ratio

Direct scavenging of the superoxide anion

When incubated with human polymorphonuclear leuko-cytes stimulated to release superoxide by exposure tophorbol ester Cardax dose-dependently decreased thelevels of superoxide anion detected by EPR spec-troscopy utilizing the spin trap DEPMPO Completeinhibition of superoxide anion signal was achieved atmillimolar concentrations of Cardax32 This in vitro testsystem generates molar amounts of superoxide anionmuch higher than that achieved in ischemic tissue invivo In addition supramolecular assembly of Cardax inaqueous solution of the monomeric compound limits thereactivity of the monomeric compound with aqueous-phase radicals Therefore it was postulated that muchlower concentrations would be therapeutic in animalmodels in vivo results that were subsequently achievedat nanomolar concentrations in both rats (see below)31

and dogs (Lockwood unpublished results)

Protection from experimental ischemia-reperfusion injury

In a rat model of ischemiareperfusion injury (30 min ofleft anterior descending coronary artery occlusion withsubsequent reperfusion) Cardax afforded significantcardioprotection In rats pretreated with a single dose ofintravenous Cardax for each of 4 days prior to experi-mental infarction on day 5 a dose-dependent decrease inmean infarct size was observed A dose of 75 mgkgdelivered in aqueous lsquosolutionrsquo intravenously led to adecrease in infarct size of 59 over vehicle-alone con-trols a result which was statistically highly significant

(P lt 0001 Fig 2) This degree of protection frommyocardial damage was directly correlated with theserum concentrations of free astaxanthin which reachedapproximately 600 nM after 4 days of injection at thehighest concentration tested There was no apparent tox-icity to the animals during treatment31 This treatmentprotocol might allow significant protection from damagein patients at high risk for myocardial infarction or inpatients scheduled for elective surgery where inductionof reperfusion injury is a significant risk The ability todeliver astaxanthin during the lsquogolden windowrsquo ofmyocardial salvage (3ndash6 h after coronary occlusion) isalso possible a single iv dose given 5 min prior toreperfusion showed significant myocardial salvage in arodent model (Lockwood unpublished results) It islikely that additional astaxanthin derivatives withimproved pharmacokinetic profiles can be developedand be better suited to this clinical model

In addition to protection against ROS damage to mem-branes astaxanthin and for that matter other carotenoidsif they can be delivered at sufficient concentrations maybe capable of affording additional protection to the dam-aged myocardium As will be discussed below Cardaxhas been shown to increase expression of Cx43 in mouse10T12 fibroblasts34 While the studies were performedin the context of the prevention of malignant transforma-tion it is known that Cx43 plays a vital role in car-diomyocytes as the structural element of gap junctionsIn the heart gap junctions are a major component of theintercalated disc and connect myocardial cells into asyncytium allowing co-ordinated contraction35 Gapjunction toxins which include ROS decrease signaltransduction through the gap junction36 and would beexpected to cause uncoordinated contraction identifiedclinically as fibrillation and other arrhythmias Thuscarotenoids by inhibiting the propagation of ROS mayprevent fibrillation while their ability to increase syn-thesis of the structural components of the gap junctionCx43 has the potential to increase the rate of recovery ofthe myocardium after infarction These possibilitieshave yet to be evaluated

Diverse carotenoids inhibit neoplastic transformation

There is abundant epidemiological evidence that con-sumption of dietary carotenoids is associated with pro-tection against cancer in humans37 To determine if thisassociation could be confirmed experimentally and todiscover which dietary carotenoids possessed this activ-ity we began studies in transformable C3H10T12 cells(10T12) We had previously shown these cells torespond to chemical and physical carcinogens by thequantitative formation of neoplastically transformedfoci38 and had also demonstrated that cancer-preventive

Bioactive carotenoids potent antioxidants and regulators of gene expression 185

Fig 2 Mean myocardial salvage as a percentage of infarct sizearea atrisk Control mean infarct size (IS)area at risk (AAR) set at 0 salvageControl animals received vehicle injection alone Cardax-treated animalsreceived the appropriate dose once daily iv by tail vein injection for 4days prior to experimental infarction and ISAAR determinations on day 5Myocardial salvage of 56 was achieved at the highest dose administered(75 mgkg) which was statistically highly significant (P lt 0001) FromGross and Lockwood31 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

year after initiating treatment18 These data are consistentwith reports from the same group that liver fibrosis inrats with copper overload can be suppressed by lycopenealone19 Globally liver cancer is the most prevalent can-cer resulting most frequently from chronic viral infec-tion and aflatoxin-contaminated foods this observationtherefore is of enormous potential significance Itremains to be determined whether the chemopreventiveaction of lycopene is mediated through its antioxidantproperties or through more specific effects on gene regu-lation as will be discussed later Astaxanthin a xantho-phyll predominantly found in marine organisms andresponsible for the pigmentation of shrimp and lobsterand for the pink pigmentation of birds like the flamingowho feed on marine organisms may also possess potentanti-inflammatory activity In studies in cell culture andin mice astaxanthin was shown to inhibit the productionof inflammatory cytokines such as TNF-αprostaglandins and NO This activity resulted frominhibited activation of the nuclear transcription factorNF-κB probably a result of the scavenging of reactiveoxygen species known to activate this inflammationpathway20 While the concentrations of astaxanthin (50microM) required to produce this effect were certainly supra-physiological these concentrations are achievable bynovel highly bioavailable astaxanthin derivatives to bedescribed below

Carotenoids may protect against atherosclerosis

A second important example where oxidative damagehas severe pathological consequences is in atherosclero-sis It is now well-established that oxidation of low-den-sity lipoprotein (LDL) is a major contributing factor tothe initiation and progression of atherosclerotic lesionsthat are a major cause of the approximately 15 millionmyocardial infarctions that occur annually in the US Awidely accepted model is that damaged LDL particlesare recognized by monocytesmacrophages andengulfed an excess of oxidatively-modified LDL resultsin their conversion to a foam cell and formation of fattystreaks ndash early markers of arterial damage The presenceof foam cells in the vessel wall sets off a cycle of inflam-mation leading to additional oxidative stress prolifera-tion of the surrounding smooth muscle cells andprogressive narrowing of the artery Normalization oflipid profiles by diet and cholesterol-lowering statins canhave a major influence on risk21 There is growing evi-dence that carotenoids such as astaxanthin by acting aslipid-phase antioxidants may also be effective22 Againnot all carotenoids appear equal in this respect Forexample in three large scale trials supplemental β-carotene appeared to have no influence on incidence ofcardiovascular disease23 However epidemiological

studies have supported the role of dietary lycopene as acardiovascular risk-reducing factor24 Higher plasma lev-els of lutein were also found to be correlated inverselywith age-related increased thickening of the coronaryartery wall ndash a major risk factor for subsequent infarc-tion These authors also demonstrated the ability oflutein to decrease LDL oxidation and to decrease theattraction of monocytes to human arterial walls in cul-ture Similar results supporting the protective role oflutein against atherosclerosis were obtained in studiesutilizing a mouse model of atherosclerosis25 It may bethat lutein and other polar carotenoids are superior to β-carotene by virtue of increased gastrointestinal absorp-tion and cellular uptake as a consequence of the presenceof hydrophilic hydroxyl groups Astaxanthin is also apolar carotenoid with superior quenching ability rela-tive to both lutein and β-carotene12 and may be evenmore efficacious in cardiovascular applications22 Theability to interfere with the cycle of inflammation andtissue damage in lipoproteins and the coronary vesselwall could have a major impact on clinical outcome andlong-term consequences of other chronic diseases suchas cancer

Novel carotenoids protect against experimentalischemia-reperfusion injury

Reperfusion injury is a relatively recently recognizedphenomenon appearing when oxygenated blood is re-introduced into an ischemic area This may occur as aresult of pathological vascular occlusion during myocar-dial infarction or stroke followed by medical interven-tion26 There is abundant evidence that a major factorcontributing to this injury is the release of reactive oxy-gen species (ROS) from mitochondria and from neu-trophils and other leukocytes trapped in the ischemictissue27 The ensuing cycle of lipid peroxidation releaseof cytokines and loss of membrane integrity leads todeath of cells in the affected area A strong rationale thusexists for the use of agents that can both quench thesereactive species and act as radical chain-breaking antiox-idants to interrupt this cycle Martin et al12 have shownthat carotenoids and in particular astaxanthin functioneffectively as chain-breaking antioxidants at low partialpressures of oxygen and low physiological concentra-tions in contrast to other lipophilic antioxidants such asvitamin E28 This suggests that carotenoids may beuniquely suited for the clinical prevention andor ame-lioration of ischemia-reperfusion injury However thehighly lipophilic nature of these compounds has effec-tively prevented their clinical use in the acute coronarysyndrome (ACS) as well in experimental settings asdelivery of effective concentrations to cells and tissues isproblematic A major achievement to overcome these

Bioactive carotenoids potent antioxidants and regulators of gene expression 183

problems was the development of the use of tetrahydro-furan (THF) as a delivery vehicle for carotenoids to cellsin culture29 but it seems unlikely that this solvent wouldbe clinically acceptable

Development of a water-dispersible derivative ofastaxanthin

In order to overcome the clinical problems associatedwith carotenoid drug delivery Hawaii Biotech Inc(HBI Aiea HI USA) has developed a novel carotenoidderivative the disodium disuccinate derivative of astax-anthin Cardaxtrade (Fig 1) This compound forms apseudo-solution in water at concentrations of up to 8mgml (~10 mM) Dispersibility is achieved in aqueoussolution secondary to self-assembly of disodium disucci-nate astaxanthin monomers into supra-molecular com-plexes Monomeric solutions of compound can also beachieved by the inclusion of ethanol at concentrations upto 50 thereby disrupting this self-assembly but pre-serving aqueous solubility30 Additionally the formationof an oil waterlecithin emulsion allows Cardax to beformulated at concentrations of greater than 50 mgmlfor oral delivery3132 These formulations of astaxanthinoffer three exciting possibilities (i) that it can be rapidlydelivered at high concentrations to tissues after eitheroral or parenteral administration (ii) aqueous-phaseROS scavenging (eg singlet oxygen and superoxideanion released by neutrophils) occurs with high effi-ciency and (iii) the ester-linkage of the succinate moi-eties should make this molecule susceptible to serum

and cell esterases leading to release of free astaxanthincapable of acting as a chain-breaking antioxidant in thelipid environment of lipoprotein or cell membrane Car-dax appears to fulfil all these expectations

Cardax is highly bioavailable after oral or intravenousadministration

Administration of Cardax as an oilwater emulsion at aconcentration of 50 mgml to mice by oral gavage atdose of 500 mgkg bodyweight requires only a volumeof about 25 microl This resulted in the rapid appearance offree astaxanthin in serum and liver with peak levelsbeing achieved approximately 6 h after administrationAt this time point levels were 1760 nM in liver 694 nMin heart while plasma levels were approximately 25 ofthat found in liver33 This demonstrates rapid absorptionand cleavage to free astaxanthin in these animalstogether with accumulation in target tissues Tissue lev-els are above the reported ED50 of 200 nM of free astax-anthin as a radical scavenger suggesting that oraladministration of this novel carotenoid derivative canachieve significant tissue protection against oxidativestress Cardax has also been administered in an aqueoussolution by intravenous tail vein injection to rats in thecardioprotective study discussed below There was adose-dependent increase in plasma astaxanthin with amean peak concentration of 612 nM being achieved 24 hafter four daily doses of 75 mgkg Levels in cardiac tis-sue were not measured31

184 Hix Lockwood Bertram

Fig 1 Structures of the three stereoisomers of the disodium salt disuccinate astaxanthin derivatives present in Cardax evaluated in the current study(shown as the all-E geometric isomers) The racemic mixture of stereoisomers contains (3S3primeS)-astaxanthin disuccinate disodium salt (3R3primeR)-astaxanthin disuccinate disodium salt and (3R3primeS meso)-astaxanthin disuccinate disodium salt in a 112 ratio

Direct scavenging of the superoxide anion

When incubated with human polymorphonuclear leuko-cytes stimulated to release superoxide by exposure tophorbol ester Cardax dose-dependently decreased thelevels of superoxide anion detected by EPR spec-troscopy utilizing the spin trap DEPMPO Completeinhibition of superoxide anion signal was achieved atmillimolar concentrations of Cardax32 This in vitro testsystem generates molar amounts of superoxide anionmuch higher than that achieved in ischemic tissue invivo In addition supramolecular assembly of Cardax inaqueous solution of the monomeric compound limits thereactivity of the monomeric compound with aqueous-phase radicals Therefore it was postulated that muchlower concentrations would be therapeutic in animalmodels in vivo results that were subsequently achievedat nanomolar concentrations in both rats (see below)31

and dogs (Lockwood unpublished results)

Protection from experimental ischemia-reperfusion injury

In a rat model of ischemiareperfusion injury (30 min ofleft anterior descending coronary artery occlusion withsubsequent reperfusion) Cardax afforded significantcardioprotection In rats pretreated with a single dose ofintravenous Cardax for each of 4 days prior to experi-mental infarction on day 5 a dose-dependent decrease inmean infarct size was observed A dose of 75 mgkgdelivered in aqueous lsquosolutionrsquo intravenously led to adecrease in infarct size of 59 over vehicle-alone con-trols a result which was statistically highly significant

(P lt 0001 Fig 2) This degree of protection frommyocardial damage was directly correlated with theserum concentrations of free astaxanthin which reachedapproximately 600 nM after 4 days of injection at thehighest concentration tested There was no apparent tox-icity to the animals during treatment31 This treatmentprotocol might allow significant protection from damagein patients at high risk for myocardial infarction or inpatients scheduled for elective surgery where inductionof reperfusion injury is a significant risk The ability todeliver astaxanthin during the lsquogolden windowrsquo ofmyocardial salvage (3ndash6 h after coronary occlusion) isalso possible a single iv dose given 5 min prior toreperfusion showed significant myocardial salvage in arodent model (Lockwood unpublished results) It islikely that additional astaxanthin derivatives withimproved pharmacokinetic profiles can be developedand be better suited to this clinical model

In addition to protection against ROS damage to mem-branes astaxanthin and for that matter other carotenoidsif they can be delivered at sufficient concentrations maybe capable of affording additional protection to the dam-aged myocardium As will be discussed below Cardaxhas been shown to increase expression of Cx43 in mouse10T12 fibroblasts34 While the studies were performedin the context of the prevention of malignant transforma-tion it is known that Cx43 plays a vital role in car-diomyocytes as the structural element of gap junctionsIn the heart gap junctions are a major component of theintercalated disc and connect myocardial cells into asyncytium allowing co-ordinated contraction35 Gapjunction toxins which include ROS decrease signaltransduction through the gap junction36 and would beexpected to cause uncoordinated contraction identifiedclinically as fibrillation and other arrhythmias Thuscarotenoids by inhibiting the propagation of ROS mayprevent fibrillation while their ability to increase syn-thesis of the structural components of the gap junctionCx43 has the potential to increase the rate of recovery ofthe myocardium after infarction These possibilitieshave yet to be evaluated

Diverse carotenoids inhibit neoplastic transformation

There is abundant epidemiological evidence that con-sumption of dietary carotenoids is associated with pro-tection against cancer in humans37 To determine if thisassociation could be confirmed experimentally and todiscover which dietary carotenoids possessed this activ-ity we began studies in transformable C3H10T12 cells(10T12) We had previously shown these cells torespond to chemical and physical carcinogens by thequantitative formation of neoplastically transformedfoci38 and had also demonstrated that cancer-preventive

Bioactive carotenoids potent antioxidants and regulators of gene expression 185

Fig 2 Mean myocardial salvage as a percentage of infarct sizearea atrisk Control mean infarct size (IS)area at risk (AAR) set at 0 salvageControl animals received vehicle injection alone Cardax-treated animalsreceived the appropriate dose once daily iv by tail vein injection for 4days prior to experimental infarction and ISAAR determinations on day 5Myocardial salvage of 56 was achieved at the highest dose administered(75 mgkg) which was statistically highly significant (P lt 0001) FromGross and Lockwood31 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

problems was the development of the use of tetrahydro-furan (THF) as a delivery vehicle for carotenoids to cellsin culture29 but it seems unlikely that this solvent wouldbe clinically acceptable

Development of a water-dispersible derivative ofastaxanthin

In order to overcome the clinical problems associatedwith carotenoid drug delivery Hawaii Biotech Inc(HBI Aiea HI USA) has developed a novel carotenoidderivative the disodium disuccinate derivative of astax-anthin Cardaxtrade (Fig 1) This compound forms apseudo-solution in water at concentrations of up to 8mgml (~10 mM) Dispersibility is achieved in aqueoussolution secondary to self-assembly of disodium disucci-nate astaxanthin monomers into supra-molecular com-plexes Monomeric solutions of compound can also beachieved by the inclusion of ethanol at concentrations upto 50 thereby disrupting this self-assembly but pre-serving aqueous solubility30 Additionally the formationof an oil waterlecithin emulsion allows Cardax to beformulated at concentrations of greater than 50 mgmlfor oral delivery3132 These formulations of astaxanthinoffer three exciting possibilities (i) that it can be rapidlydelivered at high concentrations to tissues after eitheroral or parenteral administration (ii) aqueous-phaseROS scavenging (eg singlet oxygen and superoxideanion released by neutrophils) occurs with high effi-ciency and (iii) the ester-linkage of the succinate moi-eties should make this molecule susceptible to serum

and cell esterases leading to release of free astaxanthincapable of acting as a chain-breaking antioxidant in thelipid environment of lipoprotein or cell membrane Car-dax appears to fulfil all these expectations

Cardax is highly bioavailable after oral or intravenousadministration

Administration of Cardax as an oilwater emulsion at aconcentration of 50 mgml to mice by oral gavage atdose of 500 mgkg bodyweight requires only a volumeof about 25 microl This resulted in the rapid appearance offree astaxanthin in serum and liver with peak levelsbeing achieved approximately 6 h after administrationAt this time point levels were 1760 nM in liver 694 nMin heart while plasma levels were approximately 25 ofthat found in liver33 This demonstrates rapid absorptionand cleavage to free astaxanthin in these animalstogether with accumulation in target tissues Tissue lev-els are above the reported ED50 of 200 nM of free astax-anthin as a radical scavenger suggesting that oraladministration of this novel carotenoid derivative canachieve significant tissue protection against oxidativestress Cardax has also been administered in an aqueoussolution by intravenous tail vein injection to rats in thecardioprotective study discussed below There was adose-dependent increase in plasma astaxanthin with amean peak concentration of 612 nM being achieved 24 hafter four daily doses of 75 mgkg Levels in cardiac tis-sue were not measured31

184 Hix Lockwood Bertram

Fig 1 Structures of the three stereoisomers of the disodium salt disuccinate astaxanthin derivatives present in Cardax evaluated in the current study(shown as the all-E geometric isomers) The racemic mixture of stereoisomers contains (3S3primeS)-astaxanthin disuccinate disodium salt (3R3primeR)-astaxanthin disuccinate disodium salt and (3R3primeS meso)-astaxanthin disuccinate disodium salt in a 112 ratio

Direct scavenging of the superoxide anion

When incubated with human polymorphonuclear leuko-cytes stimulated to release superoxide by exposure tophorbol ester Cardax dose-dependently decreased thelevels of superoxide anion detected by EPR spec-troscopy utilizing the spin trap DEPMPO Completeinhibition of superoxide anion signal was achieved atmillimolar concentrations of Cardax32 This in vitro testsystem generates molar amounts of superoxide anionmuch higher than that achieved in ischemic tissue invivo In addition supramolecular assembly of Cardax inaqueous solution of the monomeric compound limits thereactivity of the monomeric compound with aqueous-phase radicals Therefore it was postulated that muchlower concentrations would be therapeutic in animalmodels in vivo results that were subsequently achievedat nanomolar concentrations in both rats (see below)31

and dogs (Lockwood unpublished results)

Protection from experimental ischemia-reperfusion injury

In a rat model of ischemiareperfusion injury (30 min ofleft anterior descending coronary artery occlusion withsubsequent reperfusion) Cardax afforded significantcardioprotection In rats pretreated with a single dose ofintravenous Cardax for each of 4 days prior to experi-mental infarction on day 5 a dose-dependent decrease inmean infarct size was observed A dose of 75 mgkgdelivered in aqueous lsquosolutionrsquo intravenously led to adecrease in infarct size of 59 over vehicle-alone con-trols a result which was statistically highly significant

(P lt 0001 Fig 2) This degree of protection frommyocardial damage was directly correlated with theserum concentrations of free astaxanthin which reachedapproximately 600 nM after 4 days of injection at thehighest concentration tested There was no apparent tox-icity to the animals during treatment31 This treatmentprotocol might allow significant protection from damagein patients at high risk for myocardial infarction or inpatients scheduled for elective surgery where inductionof reperfusion injury is a significant risk The ability todeliver astaxanthin during the lsquogolden windowrsquo ofmyocardial salvage (3ndash6 h after coronary occlusion) isalso possible a single iv dose given 5 min prior toreperfusion showed significant myocardial salvage in arodent model (Lockwood unpublished results) It islikely that additional astaxanthin derivatives withimproved pharmacokinetic profiles can be developedand be better suited to this clinical model

In addition to protection against ROS damage to mem-branes astaxanthin and for that matter other carotenoidsif they can be delivered at sufficient concentrations maybe capable of affording additional protection to the dam-aged myocardium As will be discussed below Cardaxhas been shown to increase expression of Cx43 in mouse10T12 fibroblasts34 While the studies were performedin the context of the prevention of malignant transforma-tion it is known that Cx43 plays a vital role in car-diomyocytes as the structural element of gap junctionsIn the heart gap junctions are a major component of theintercalated disc and connect myocardial cells into asyncytium allowing co-ordinated contraction35 Gapjunction toxins which include ROS decrease signaltransduction through the gap junction36 and would beexpected to cause uncoordinated contraction identifiedclinically as fibrillation and other arrhythmias Thuscarotenoids by inhibiting the propagation of ROS mayprevent fibrillation while their ability to increase syn-thesis of the structural components of the gap junctionCx43 has the potential to increase the rate of recovery ofthe myocardium after infarction These possibilitieshave yet to be evaluated

Diverse carotenoids inhibit neoplastic transformation

There is abundant epidemiological evidence that con-sumption of dietary carotenoids is associated with pro-tection against cancer in humans37 To determine if thisassociation could be confirmed experimentally and todiscover which dietary carotenoids possessed this activ-ity we began studies in transformable C3H10T12 cells(10T12) We had previously shown these cells torespond to chemical and physical carcinogens by thequantitative formation of neoplastically transformedfoci38 and had also demonstrated that cancer-preventive

Bioactive carotenoids potent antioxidants and regulators of gene expression 185

Fig 2 Mean myocardial salvage as a percentage of infarct sizearea atrisk Control mean infarct size (IS)area at risk (AAR) set at 0 salvageControl animals received vehicle injection alone Cardax-treated animalsreceived the appropriate dose once daily iv by tail vein injection for 4days prior to experimental infarction and ISAAR determinations on day 5Myocardial salvage of 56 was achieved at the highest dose administered(75 mgkg) which was statistically highly significant (P lt 0001) FromGross and Lockwood31 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

Direct scavenging of the superoxide anion

When incubated with human polymorphonuclear leuko-cytes stimulated to release superoxide by exposure tophorbol ester Cardax dose-dependently decreased thelevels of superoxide anion detected by EPR spec-troscopy utilizing the spin trap DEPMPO Completeinhibition of superoxide anion signal was achieved atmillimolar concentrations of Cardax32 This in vitro testsystem generates molar amounts of superoxide anionmuch higher than that achieved in ischemic tissue invivo In addition supramolecular assembly of Cardax inaqueous solution of the monomeric compound limits thereactivity of the monomeric compound with aqueous-phase radicals Therefore it was postulated that muchlower concentrations would be therapeutic in animalmodels in vivo results that were subsequently achievedat nanomolar concentrations in both rats (see below)31

and dogs (Lockwood unpublished results)

Protection from experimental ischemia-reperfusion injury

In a rat model of ischemiareperfusion injury (30 min ofleft anterior descending coronary artery occlusion withsubsequent reperfusion) Cardax afforded significantcardioprotection In rats pretreated with a single dose ofintravenous Cardax for each of 4 days prior to experi-mental infarction on day 5 a dose-dependent decrease inmean infarct size was observed A dose of 75 mgkgdelivered in aqueous lsquosolutionrsquo intravenously led to adecrease in infarct size of 59 over vehicle-alone con-trols a result which was statistically highly significant

(P lt 0001 Fig 2) This degree of protection frommyocardial damage was directly correlated with theserum concentrations of free astaxanthin which reachedapproximately 600 nM after 4 days of injection at thehighest concentration tested There was no apparent tox-icity to the animals during treatment31 This treatmentprotocol might allow significant protection from damagein patients at high risk for myocardial infarction or inpatients scheduled for elective surgery where inductionof reperfusion injury is a significant risk The ability todeliver astaxanthin during the lsquogolden windowrsquo ofmyocardial salvage (3ndash6 h after coronary occlusion) isalso possible a single iv dose given 5 min prior toreperfusion showed significant myocardial salvage in arodent model (Lockwood unpublished results) It islikely that additional astaxanthin derivatives withimproved pharmacokinetic profiles can be developedand be better suited to this clinical model

In addition to protection against ROS damage to mem-branes astaxanthin and for that matter other carotenoidsif they can be delivered at sufficient concentrations maybe capable of affording additional protection to the dam-aged myocardium As will be discussed below Cardaxhas been shown to increase expression of Cx43 in mouse10T12 fibroblasts34 While the studies were performedin the context of the prevention of malignant transforma-tion it is known that Cx43 plays a vital role in car-diomyocytes as the structural element of gap junctionsIn the heart gap junctions are a major component of theintercalated disc and connect myocardial cells into asyncytium allowing co-ordinated contraction35 Gapjunction toxins which include ROS decrease signaltransduction through the gap junction36 and would beexpected to cause uncoordinated contraction identifiedclinically as fibrillation and other arrhythmias Thuscarotenoids by inhibiting the propagation of ROS mayprevent fibrillation while their ability to increase syn-thesis of the structural components of the gap junctionCx43 has the potential to increase the rate of recovery ofthe myocardium after infarction These possibilitieshave yet to be evaluated

Diverse carotenoids inhibit neoplastic transformation

There is abundant epidemiological evidence that con-sumption of dietary carotenoids is associated with pro-tection against cancer in humans37 To determine if thisassociation could be confirmed experimentally and todiscover which dietary carotenoids possessed this activ-ity we began studies in transformable C3H10T12 cells(10T12) We had previously shown these cells torespond to chemical and physical carcinogens by thequantitative formation of neoplastically transformedfoci38 and had also demonstrated that cancer-preventive

Bioactive carotenoids potent antioxidants and regulators of gene expression 185

Fig 2 Mean myocardial salvage as a percentage of infarct sizearea atrisk Control mean infarct size (IS)area at risk (AAR) set at 0 salvageControl animals received vehicle injection alone Cardax-treated animalsreceived the appropriate dose once daily iv by tail vein injection for 4days prior to experimental infarction and ISAAR determinations on day 5Myocardial salvage of 56 was achieved at the highest dose administered(75 mgkg) which was statistically highly significant (P lt 0001) FromGross and Lockwood31 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

retinoids could inhibit neoplastic transformation in thesecells39 Because of the problems of drug delivery of theselipophilic molecules the first carotenoids to be testedwere those available in a lsquobeadletrsquo formulation how-ever this limited studies to β-carotene and canthaxan-thin40 In order to extend studies to a more diverse seriesof dietary carotenoids we developed THF as a deliverysolvent Use of THF results in the formation of a pseudo-solution of carotenoids in cell culture medium ndash a formthat is highly bio-available When carotenoids wereadded 1 week after removal of the chemical carcinogenall carotenoids regardless of their pro-vitamin A activitywere capable of inhibiting the development of neoplastictransformation Moreover just as in our earlier studieswith retinoids removal of the carotenoid led to emer-gence of neoplastic transformed foci some 3ndash4 weekslater41 This indicated that we were not dealing withselective cytotoxicity but with a reversible inhibition ofthe process of neoplastic transformation

These studies demonstrated that at least in the model cellculture system employed dietary carotenoids had the fol-lowing properties (i) they inhibited neoplastic transforma-tion in the post-initiation phase (ii) their action wasreversible and thus not a consequence of selective cytotoxi-city (iii) their action was not the result of selective growthinhibition of transformed cells (iv) their action was inde-pendent of conversion to chemopreventive retinoids sinceactivity was observed even with straight-chain hydrocar-bons such as lycopene and (v) activity did not correlatewith their antioxidant properties

Carotenoids increase gap junctional communication

A consistent finding in studies of human or animaltumor cell lines and in studies of neoplastic transforma-tion in vitro is that tumor cells communicate poorly if atall with their normal counterparts42 These results led tothe original hypothesis of growth control through junc-tional communication so eloquently proposed byLowenstein43 Gap junctions are water-filled pores calledconnexons that connect adjacent cells in most organs ofthe body These pores allow direct cytoplasmic-to-cyto-plasmic communication of water-soluble molecules andions Because of the limiting size of the pore only mole-cules lt ~1000 Da can pass excluding molecules such asmRNA and protein and thus maintaining genetic identityof the cells The existence of this network of communi-cation creates a syncytium through which cells canexchange nutrients waste products and signaling mole-cules such as cAMP Ca2+ etc44 There is evidence thatgap junctions also serve to transmit growth-inhibitorysignals that can inhibit the aberrant proliferation of car-cinogen-initiated and fully transformed cells45 This wasderived in part from studies in the 10T12 cell line and

was extended by the observation that the inhibitoryaction of retinoids on neoplastic transformation in10T12 cells was closely linked to their ability toincrease gap junctional communication (GJC) viaincreased expression of Cx43 at the mRNA and proteinlevel46 It is of interest that many classes of tumor pro-moters ndash agents that accelerate the process of carcino-genesis but are not themselves carcinogenic ndash inhibitcommunication through gap junctions47 These and otherobservations lead to our hypothesis of growth control viaGJC48 shown diagrammatically in Figure 3

The structural element of a gap junction is a trans-membrane protein called a connexin six of these con-nexin molecules are known to assemble radially toenclose the central pore This structure can then dockwith a similar structure on a contacting adjacent cell toform a complete connexon Thus the structural unit ofthe gap junction is composed of 12 connexin moleculescontributed equally by each of the communicating part-ners49 This arrangement is shown diagrammatically inFigure 4 Passage of molecules or ions through the cen-tral pore appears to be via passive diffusion down con-centration gradients At present over 20 connexinfamily members have been recognized which are differ-entially expressed according to cell type and at differentperiods of development50 Cx43 is the most widelyexpressed connexin and is the family member inducedby retinoids and as we later discovered carotenoids

Carotenoids induce Cx43 irrespective of their pro-vitamin A or antioxidant properties

At the time we were characterizing the cancer chemo-preventive properties of carotenoids the only knowncommon property shared by these carotenoids was theability to act as lipid-phase anti-oxidants ndash as discussedabove a plausible mechanism for prevention Howeverwhen we examined the antioxidant activities of diversecarotenoids in 10T12 cell cultures we discovered thatwhereas all carotenoids indeed did prevent oxidativedamage as indicated by a decrease in the formation ofthiobarbituric acid reactive-substances (TBARS) thisability did not correlate with their activities as inhibitorsof transformation Furthermore the non-carotenoidlipid-phase antioxidant α-tocopherol was found to be avery poor inhibitor of neoplastic transformation Even atconcentrations of 10ndash4 M 10-fold higher than that usedfor carotenoids it failed to induce Cx43 expression yetwas the most effective inhibitor of TBARS formation51

Thus within the limits of this particular assay system forlipid-phase oxidative damage it appeared that while allcarotenoids were as expected antioxidants this did notappear to be the major factor responsible for their activityas inhibitors of transformation

186 Hix Lockwood Bertram

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

Nor did activity correlate with the pro-vitamin Aactivity of active carotenoids as may be expected fromthe similar actions of retinoids and carotenoids on Cx43expression Indeed recent studies have shown that thesetwo classes of chemopreventive agents induce Cx43

expression by different mechanisms Gene induction at thelevel of protein and mRNA by retinoids but not carotenoidsis inhibited by pharmacological antagonists of the nuclearretinoic acid receptors (RARs) In contrast induction bynon-pro-vitamin A carotenoids is inhibited by antagonists

Bioactive carotenoids potent antioxidants and regulators of gene expression 187

Fig 3 Hypothetical model of growth control by junctional communication A central carcinogen-initiated cell is surrounded by growth-inhibited normalcells (red nuclei) In the left panel the initiated cell is not in communication with normal cells and undergoes inappropriate proliferation (green nucleus)This will allow clonal expansion and the progressive accumulation of additional mutations resulting in malignancy In the right panel junctionalcommunication of growth inhibitory signals from surrounding cells is up-regulated by retinoids or carotenoids the initiated cell becomes itself growtharrested and progression to malignancy is delayed

Fig 4 Organization of connexins into the plasma membrane (A) Diagrammatic cross section through an area of cellcell contact containing gap junctionsConnexin proteins are shown in yellow traversing the phospholipid bilayer in the plasma membrane (blue) Each cell contributes 6 connexins to form acylinder enclosing a central water-filled pore seen in cross-section (foreground) (B) A single connexin molecule traverses the plasma membrane 4 timeswith both N- and C-terminal ends in the cytoplasm Connexins assemble to form a connexon by forming 3 sulfhydryl bonds between the highly conservedcysteine resides present in each opposing loop (C) Thus each connexon is bound by 18 sulfhydryl bonds to produce a tight seal blocking the entry ofextracellular ions such as Ca2+ Reprinted from Science and Medicine with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

of the peroxisome proliferator activated nuclear receptors(PPAR Vine and Bertram in preparation)

The water-dispersible astaxanthin derivative Cardaxalso up-regulates Cx43 expression in 10T12 cells

Problems associated with delivery of dietary carotenoidsto biological systems were discussed above To deter-mine if the disuccinate astaxanthin derivative Cardaxhad activity in addition to its antioxidant properties wetreated 10T12 cells with various formulations of Car-dax To enhance both solubility and bioavailability sev-eral EtOHwater formulations were tested foraggregation with UVvisible spectroscopy The lsquosolubil-ityrsquo of the derivatives was significantly enhanced by theuse of 11 (50 EtOH) and 12 (33 EtOH)EtOHwater formulations These formulations have beenpreviously demonstrated to maintain the carotenoidderivatives in monomeric form32 Induction levels ofCx43 as determined by immunoblotting were higherwith the EtOH formulation at 10ndash5 M than for formula-tions in sterile water alone demonstrating enhanced bio-logical availability using EtOH as a co-solvent assuggested by the previous physicochemical studies Themixture of stereoisomers of Cardax in pure aqueous for-mulation was able to up-regulate Cx43 expression withequivalent or greater potency than that previouslyobserved for other carotenoids in organic vehicle5152

Importantly treated cells were found to assemble Cx43into immunoreactive plaques in regions of cellcell con-tact consistent with formation of gap junctions Thiswas confirmed by functional studies utilizing a dyemicroinjection technique which demonstrate thattreated cells were more extensively coupled than sol-vent-alone control-treated cells34

Forced expression of Cx43 in human carcinoma cellsreduces markers of malignancy

The studies discussed above relating connexin-medi-ated functional GJC with growth control relied on corre-lations to prove the association However thesecorrelations do not prove a cause and effect relationshipFor example the actions of carotenoids on Cx43 geneexpression may go hand-in-hand with actions ofcarotenoids on growth control but be functionally unre-lated to these actions To more firmly establish the roleof up-regulated Cx43 expression and enhanced junc-tional communication as central to the role of retinoidsand carotenoids as anti-proliferative and cancer preven-tive agents we embarked upon the development of cellsin which Cx43 was inducible not by carotenoids orretinoids but by using a bacterial promoter system in

which activity of the artificially introduced gene is con-trollable by picogram amounts of doxycycline which atthese concentrations is not known to produce othereffects in mammalian cells The major advantage of thisapproach is that cells in the non-induced situation canserve as their own controls and the effects of Cx43expression can be determined in the absence of con-comitant exposure to carotenoids Unfortunatelybecause of technical difficulties these studies have beenlimited to the genetic engineering of established humantumor cell lines Three such lines have now been createdndash one from a cervical carcinoma53 one from a fibrosar-coma54 and one from a breast adenocarcinoma (Chenand Bertram in preparation) In all cell lines Cx43 hasbeen shown to be rapidly inducible from the bacterialpromoter to be integrated into the plasma membraneand to form functional gap junctions with adjacentinduced tumor cells The consequences of this inductionhas also been consistent in all three engineered celllines expression of Cx43 resulted in a dramatic decreasein anchorage-independent growth ndash that is growth asspheroids suspended in a semi-solid medium The sig-nificance of this observation is that growth in suspensionhas been shown to tightly correlate with malignantpotential of human tumor cells55 Thus Cx43 expressionstrongly inhibits a major in vitro marker of malignancyA more direct test for malignancy is the ability to growas a tumor when injected as a xenograft into immuno-compromised mice The results for one such study withthe human cervical carcinoma cells is shown in Figure 5It can be clearly seen that in mice administered doxycy-cline in the drinking water in order to induce Cx43expression in the injected cells subcutaneous tumorsgrew much more slowly than in control animals53 Thusboth in vivo and in vitro Cx43 expression reducesindices of neoplasia in human carcinoma cells

In monolayer culture where cells grow as a two-dimen-sional layer on plastic Cx43 induction did not causechanges in growth rate or saturation density This was incontrast to the results obtained with non-transformed10T12 cells where increased junctional communicationafter retinoid or carotenoid treatment led to decreased pro-liferation The lack of response of cells growing in mono-layer culture to Cx43 induction may be attributed to the factthat these cells derived from human tumors and which hadbeen extensively passaged in vitro had lost the ability toeither transmit or respond to junctionally mediated cell sig-naling Although none of these cell lines could be inducedto junctionally communicate with growth-inhibited10T12 cells induced human breast carcinoma cellsformed functional junctions with a growth-inhibited ratkidney cell line which constitutively expressed Cx43Under these conditions cancer cell proliferation wasinhibited The implication of these studies is that thebreast carcinoma cells can no longer generate growth

188 Hix Lockwood Bertram

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

inhibitory signals but can still respond to signals sup-plied by the growth inhibited rat cells (Chen andBertram in preparation)

We have shown that down-regulated expression of Cx43in both the human cervix and oral epithelium is an earlyevent being observed even in dysplasia a pathologyknown to predispose to malignancy but is not yet malig-nant53 Our data demonstrating that up-regulated expres-sion of Cx43 achieved by pharmacological or molecularmeans results in decreased proliferation of normal andmalignant cells suggests that if the observed down-regu-lated expression of Cx43 in dysplasia can be correctedprogression to malignancy may be delayed Indeed in clin-ical intervention studies retinoids in the case of cervicaldysplasia56 and oral leukoplakia57 and carotenoids in thecase of oral leukoplakia58 have been shown to retard car-cinogenic progression significantly The role of gap junc-tional communication in these responses has not beenthoroughly evaluated but the data are strongly suggestivethat agents capable of normalizing junctional communica-tion would have cancer preventive properties

CONCLUSIONS

The low intrinsic toxicity of carotenoids and their potentantioxidant properties makes them potentially usefuldrugs with which to counter chronic or acute pathology

cause by ROS Carotenoids also up-regulate expressionof Cx43 a tumor suppressor gene which allows directcellcell communication and suppresses indices ofmalignancy Problems associated with therapeutic drugdelivery of these highly lipophilic agents have been cur-rently solved for cell culture systems and mammalian invivo models by the development of a water-dispersibleastaxanthin derivative This novel compound retainsantioxidant activity in vitro is protective in a preclinicalmodel of myocardial infarction and has the ability to up-regulate Cx43 expression suggestive of cancer preven-tive activity

REFERENCES

1 Beecher GR Khachik F Qualitative relationship of dietary andplasma carotenoids in human beings Ann NY Acad Sci 1992669 320ndash321

2 Britton G Structure and properties of carotenoids in relation tofunction FASEB J 1995 9 1551ndash1558

3 Means AL Gudas LJ The roles of retinoids in vertebratedevelopment Annu Rev Biochem 1995 64 201ndash233

4 Frank HA Cogdell RJ Carotenoids in photosynthesisPhotochem Photobiol 1996 63 257ndash264

5 Bramley PM Carotenoid biosynthesis a target site for bleachingherbicides Biochem Soc Trans 1994 22 625ndash629

6 Bernstein PS Khachik F Carvalho LS et al Identification andquantitation of carotenoids and their metabolites in the tissues ofthe human eye Exp Eye Res 2001 72 215ndash223

Bioactive carotenoids potent antioxidants and regulators of gene expression 189

Fig 5 Expression of Cx43 reduces the growth rate of human cervical carcinoma cells in the lsquonudersquo mouse HeLa cells were engineered to express Cx43under the influence of a bacterial promoter driven by doxycycline immunocompromised nude mice were injected subcutaneously with HeLa cells thenrandomized to receive doxycycline (02 mgml in 5 sucrose) in the drinking water or sucrose alone as controls Tumor volumes were measured bycalipers at the indicated times Open circles doxycycline-treated filled circles sucrose controls Numbers by each data point represent total number oftumorsnumber of tumor injections From King et al53 with permission

7 Bernstein PS Zhao DY Wintch SW et al Resonance Ramanmeasurement of macular carotenoids in normal subjects and inage-related macular degeneration patients Ophthalmology 2002109 1780ndash1787

8 Matthews-Roth MM Recent progress in the medical applicationsof carotenoids Pure Appl Chem 1991 63 147ndash156

9 Burton GW Ingold KU β-Carotene an unusual type of lipidantioxidant Science 1984 224 569ndash573

10 Omenn GS Goodman GE Thornquist MD et al Effects of acombination of beta carotene and vitamin A on lung cancer andcardiovascular disease N Engl J Med 1996 334 1150ndash1155

11 Albanes D Heinonen OP Taylor PR et al α-Tocopherol and β-carotene supplements and lung cancer incidence in the Alpha-Tocopherol Beta-Carotene Cancer Prevention Study effects ofbase-line characteristics and study compliance J Natl CancerInst 1996 88 1560ndash1570

12 Martin HD Jager C Ruck C et al Anti- and prooxidantproperties of carotenoids J Prakt Chem 2004 341 302ndash308

13 Victor VM Rocha M De La FM Immune cells free radicals andantioxidants in sepsis Int Immunopharmacol 2004 4 327ndash347

14 Baran CP Zeigler MM Tridandapani S Marsh CB The role ofROS and RNS in regulating life and death of blood monocytesCurr Pharm Des 2004 10 855ndash866

15 Marnett LJ Oxyradicals and DNA damage Carcinogenesis2000 21 361ndash370

16 Ames BN Shigenaga MK Gold LS DNA lesions inducibleDNA repair and cell division three key factors in mutagenesisand carcinogenesis Environ Health Perspect 1993 101 (Suppl5) 35ndash44

17 Kuipers EJ Meuwissen SG Helicobacter pylori and gastriccarcinogenesis Scand J Gastroenterol Suppl 1996 218103ndash105

18 Nishino H Jinno K Prevention of hepatitis C virus-induced livercancer by natural carotenoids and vitamin E Proceedings ofFrontiers in Cancer Prevention Research AACR Conference2002 107 202

19 Watanabe S Kitade Y Masaki T et al Effects of lycopene andSho-saiko-to on hepatocarcinogenesis in a rat model ofspontaneous liver cancer Nutr Cancer 2001 39 96ndash101

20 Lee SJ Bai SK Lee KS et al Astaxanthin inhibits nitric oxideproduction and inflammatory gene expression by suppressingI(kappa)B kinase-dependent NF-kappaB activation Mol Cells2003 16 97ndash105

21 von Baeyer H Hopfenmuller W Riedel E Affeld KAtherosclerosis current concepts of pathophysiology andpharmacological intervention based on trial outcomes ClinNephrol 2003 60 (Suppl 1) S31ndashS48

22 Iwamoto T Hosada K Hirano R et al Inhibition of low-densitylipoprotein oxidation by astaxanthin J Atheroscler Thromb 20007 216ndash222

23 Clarke R Armitage J Antioxidant vitamins and risk ofcardiovascular disease Review of large-scale randomised trialsCardiovasc Drugs Ther 2002 16 411ndash415

24 Rissanen TH Voulilanen S Nyyssonen K et al Low serumlycopene concentration is associated with an excess incidence ofacute coronary events and stroke the Kuopio Ischaemic HeartDisease Risk Factor Study Br J Nutr 2001 85 749ndash754

25 Dwyer JH Navab M Dwyer KM et al Oxygenated carotenoidlutein and progression of early atherosclerosis ndash The Los AngelesAtherosclerosis Study Circulation 2001 103 2922ndash2927

26 Maxwell SR Lip GY Reperfusion injury a review of thepathophysiology clinical manifestations and therapeutic optionsInt J Cardiol 1997 58 95ndash117

27 Cobbold CA Sherratt JA Maxwell SR Lipoprotein oxidationand its significance for atherosclerosis a mathematical approachBull Math Biol 2002 64 65ndash95

28 Burton GW Ingold KU Vitamin E as an in vitro and in vivoantioxidant Ann NY Acad Sci 1989 570 7ndash22

29 Cooney RV Kappock TJ Pung A Bertram JS Solubilizationcellular uptake and activity of β-carotene and other carotenoidsas inhibitors of neoplastic transformation in cultured cellsMethods Enzymol 1993 214 55ndash68

30 Zsila F Simonyi M Lockwood SF Interaction of the disodiumdisuccinate derivative of meso-astaxanthin with human serumalbumin from chiral complexation to self-assembly Bioorg MedChem Lett 2003 13 4093ndash4100

31 Gross GJ Lockwood SF Cardioprotection and myocardialsalvage by a disodium disuccinate astaxanthin derivative Life Sci2004 75 215ndash224

32 Cardounel AJ Dumitrescu C Zweier JL Lockwood SF Directsuperoxide anion scavenging by a disodium disuccinateastaxanthin derivative relative efficacy of individualstereoisomers versus the statistical mixture of stereoisomers byelectron paramagnetic resonance imaging Biochem Biophys ResCommun 2003 307 704ndash712

33 Showalter LA Weinman SA Osterlie M Lockwood SF Plasmaappearance and tissue accumulation of non-esterified freeastaxanthin in C57Bl6 mice after oral dosing of a disodiumdisuccinate diester of astaxanthin (Heptax) Comp BiochemPhysiol [C] Toxicol Pharmacol 2004 137 227ndash236

34 Hix LM Lockwood SF Bertram JS Upregulation of connexin 43protein expression and increased gap junctional communicationby water soluble disodium disuccinate astaxanthin derivativesCancer Lett 2004 211 25ndash37

35 Yeager M Structure of cardiac gap junction intercellularchannels J Struct Biol 1998 121 231ndash245

36 Kang KS Yun JW Yoon BS et al Preventive effect ofepicatechin and ginsenoside Rb2 on the inhibition of gapjunctional intercellular communication by TPA and H2O2 CancerLett 2000 152 97ndash106

37 Mayne ST Beta-carotene carotenoids and disease prevention inhumans FASEB J 1996 10 690ndash701

38 Reznikoff CA Bertram JS Brankow DW Heidelberger CQuantitative and qualitative studies of chemical transformation ofcloned C3H mouse embryo cells sensitive to postconfluenceinhibition of cell division Cancer Res 1973 33 2339ndash2349

39 Merriman R Bertram JS Reversible inhibition by retinoids of 3-methylcholanthrene-induced neoplastic transformation inC3H10T12 cells Cancer Res 1979 39 1661ndash1666

40 Pung A Rundhaug JE Yoshizawa CN Bertram JS β-Caroteneand canthaxanthin inhibit chemically- and physically-inducedneoplastic transformation in 10T12 cells Carcinogenesis 19889 1533ndash1539

41 Bertram JS Pung A Churley M et al Diverse carotenoidsprotect against chemically induced neoplastic transformationCarcinogenesis 1991 12 671ndash678

42 Neveu M Bertram JS Gap junctions and neoplasia Adv MolCell Biol 2000 30 221ndash262

43 Loewenstein WR Junctional intercellular communication and thecontrol of growth Biochim Biophys Acta 1979 560 1ndash65

44 Nicholson BJ Weber PA Cao F et al The molecular basis ofselective permeability of connexins is complex and includes bothsize and charge Braz J Med Biol Res 2000 33 369ndash378

45 Mehta PP Bertram JS Loewenstein WR Growth inhibition oftransformed cells correlates with their junctional communicationwith normal cells Cell 1986 44 187ndash196

46 Hossain MZ Wilkens LR Mehta PP Loewenstein WR BertramJS Enhancement of gap junctional communication by retinoidscorrelates with their ability to inhibit neoplastic transformationCarcinogenesis 1989 10 1743ndash1748

47 Matesic DF Rupp HL Bonney WJ Ruch RJ Trosko JEChanges in gap-junction permeability phosphorylation and

190 Hix Lockwood Bertram

number mediated by phorbol ester and non-phorbol-ester tumorpromoters in rat liver epithelial cells Mol Carcinogenesis 199410 226ndash236

48 King TJ Fukushima L Bertram JS Connexins andcarcinogenesis upregulated expression of connexin 43 by cancerpreventive agents or by gene transfer decreases proliferation andexpression of neoplasia In Werner R (ed) Gap JunctionsAmsterdam IOS Press 1998 357ndash361

49 Beyer EC Paul DL Goodenough DA Connexin family of gapjunction proteins J Membr Biol 1990 116 187ndash194

50 Sohl G Willecke K An update on connexin genes and theirnomenclature in mouse and man Cell Commun Adhes 2003 10173ndash180

51 Zhang L-X Cooney RV Bertram JS Carotenoids up-regulateconnexin 43 gene expression independent of their pro-vitamin Aor antioxidant properties Cancer Res 1992 52 5707ndash5712

52 Zhang L-X Cooney RV Bertram JS Carotenoids enhance gapjunctional communication and inhibit lipid peroxidation inC3H10T12 cells relationship to their cancer chemopreventiveaction Carcinogenesis 1991 12 2109ndash2114

53 King TJ Fukushima LH Hieber AD et al Reduced levels of

connexin 43 in cervical dysplasia inducible expression in acervical carcinoma cell line decreases neoplastic potential withimplications for tumor progression Carcinogenesis 2000 211097ndash1109

54 King TJ Fukushima LH Yasui Y Lampe PD Bertram JSInducible expression of the gap junction protein connexin 43decreases the neoplastic potential of HT-1080 humanfibrosarcoma cells in vitro and in vivo Mol Carcinog 2002 3529ndash41

55 Sutherland BM Bennett PV Transformation of human cells byDNA transfection Cancer Res 1984 44 2769ndash2772

56 Meyskens FL Surwit E Moon Jr TE et al Enhancement ofregression of cervical intraepithelial neoplasia II (moderatedysplasia) with topically applied all-trans- retinoic acid Arandomized trial J Natl Cancer Inst 1994 86 539ndash543

57 Hong WK Lippman SM Itri LM et al Prevention of secondprimary tumors with isotretinoin in squamous-cell carcinoma ofthe head and neck N Engl J Med 1990 323 795ndash801

58 Garewal H Meyskens Jr F Katz RV β-carotene produces sustainedremissions in oral leukoplakia results of a 1 year randomizedcontrolled trial Proc Am Soc Clin Oncol 1995 14 496

Bioactive carotenoids potent antioxidants and regulators of gene expression 191