[Advances in Cancer Research] Volume 94 || Apoptotic Pathways and Therapy Resistance in Human...

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Apoptotic Pathways and TherapyResistance in Human Malignancies

s in CANCERt 2005, Elsev

Kristina Viktor sson,* Rolf Lewen sohn,*and Bor is Zhivoto vsky {

*Unit of Medical Radiobiology, Department of Oncology/Pathology,

Cancer Center Karolinska, Karolinska Institute, S-171 76 Stockholm, Sweden;{Institute of Environmental Medicine, Division of Toxicology,

Karolinska Institute, S-171 77 Stockholm, Sweden

I. In
troduction II. S ignaling Pathways that Lead to Apoptosis in Mammalian Cells
A
. R eceptor-Mediated Apoptotic Pathway
B
. M itochondria-Mediated Apoptotic Pathway
C
. N uclei-Mediated Apoptotic Pathway D . E ndoplasmic Reticulum-Mediated Apoptotic Pathway
E
. L ysosomal-Mediated Apoptotic Pathway
III. M
odulators of Apoptotic Signaling A . R egulators of Receptor-Mediated Apoptosis
B
. B cl-2 Family Proteins
C
. M odulators of Caspase Activity
D
. P rotein Kinases IV. A poptosis Resistance and its Involvement in Impeded Therapy Responses
A
. D eregulated Receptor-Mediated Apoptosis
B
. F ailure of the Mitochondria-Related Death Pathway
C
. C ontribution of Deregulation of the Apoptosome Complex to Therapy Resistance D . D efective Execution of Apoptosis in Cancer Cells
E
. D eregulation of Kinase Signaling
V. R
eactivation of Apoptotic Signaling as an Approach in Anticancer Therapy A . M odulation of IAP Expression and Smac-Based Therapies in Anticancer Treatment
B
. O ngoing Clinical Trials with Heat Shock Protein Inhibitors
C
. S trategies for Targeting the Bcl-2 Family Proteins in Preclinical and Clinical Models
D
. p 53 Reactivation E . M odulation of Protein Kinase Signaling
F
. A ctivation of Death Receptor Pathways
VI. C
oncluding Remarks
R
eferences
Apoptosis and necrosis are two morphologically distinct forms of cell death that areimportant for maintaining of cellular homeostasis. Almost all agents can provoke either

response when applied to cells; however, the duration of treatment and the dose of the

used agents determine which type of death (apoptosis or necrosis) is initiated. The

response of tumors to chemo-, radio-, and hormone therapy or to treatment with

RESEARCH 0065-230X/05 $35.00ier Inc. All rights reserved. DOI: 10.1016/S0065-230X(04)94004-3

143

144 Kristina Viktorsson et al.

biologically active agents may depend at least in part on the propensity of these tumors

to undergo cell death. Some tumors, e.g., leukemias, small cell lung cancer, and semi-nomas, respond quickly to first-line therapy; this fast response is thought to result from

induction of apoptosis. Solid tumors, on the other hand, usually respond slowly and less

effectively, with cell death characterized not only by apoptosis but also by necrosis,

or mitotic catastrophe. It is likely that resistance of tumors to treatment might beassociated with defects in, or dysregulation of, different steps of the apoptotic pathways.

Several attempts were undertaken to use the knowledge of these defects to design new

drugs, which might either activate or re-activate the apoptotic machinery of tumor

cells. Here we discuss the apoptotic pathways and their role in therapy resistance ofhuman malignancies. Although such studies are still in progress, they offer great promise

for future cancer therapy. We hope that some of these agents will turn out to be

valuable additions to the future therapeutic arsenal, which will most probably include acombination of conventional cytotoxic drugs and molecular target-based pro-apoptotic

drugs. # 2005 Elsevier Inc.

I. INTRODUCTION
Current available cancer therapy regimes include radio-, chemo-, steroid,
immuno-, and gene therapies, of which the first two form the main core ofcancer treatments. Both chemotherapy and ionizing radiation can triggerthe endogenous suicide process within tumor cells. This process, also knownas apoptosis, is an active, highly ordered cell demise. In leukemia as well asin childhood tumors, both chemotherapy and ionizing radiation are ratherefficient and result in apoptosis, which in this particular case is cell cycleindependent. By contrast, solid tumors, especially from epithelial origin, areoften resistant to these treatments. However, in the case of treatmentresponse, cell death occurs after one or more cell divisions and is cell cycledependent. Although apoptotic machinery is constitutively present in bothsensitive and resistant tumors, it is likely that in nonresponsive tumors thismachinery is deregulated. Here we discuss which alterations in apoptoticpathways occur in therapy-resistant tumor cells, their significance for ther-apy response, and modern apoptosis-based anticancer therapies that are inclinical use.

II. SIGNALING PATHWAYS THAT LEAD TOAPOPTOSIS IN MAMMALIAN CELLS

Although different modes of cell death have been described in past dec-ades, the scientific community accepted that in the majority of situationscells exposed to biological, chemical, and physical agents die by one of thetwo main types of death, apoptosis or necrosis. Almost all agents can

Apoptosis and Tumor Resistance 145

provoke either response when applied to cells; however, the duration oftreatment and the dose of the used agents determine which type of death(apoptosis or necrosis) is initiated. Several protein families are involvedin the regulation of the multistep apoptotic process. Some of these proteins(a family of adaptor proteins) are required for activation of different com-plexes, such as DISC (death-inducible signaling complex) and apoptosomeand PIDDosome complexes. The second set of proteins is involved inthe activation or protection of cell death (Bcl-2 family proteins). The thirdfamily of proteins, so-called caspases (cysteine-aspartate proteases), regu-lates the activation or execution of the apoptotic process. Currently,14 different caspases have been described, and many of them have beencharacterized in detail. Caspases are proteases, which are synthesized asinactive proenzymes with different sized N-terminal prodomains. Duringapoptotic signaling, in many cases this prodomain is proteolyticallyremoved and the caspase becomes active upon organization into a tetramercomplex. Active caspases cleave their substrates after aspartic acid residuesand the dissimilar substrate specificities are determined by the four aminoacid residues N-terminal to the cleavage site (reviewed in Thornberry andLazebnik, 1998). Caspases are classified as proximal or initiator caspasesand terminal or effector caspases. Initiator caspases include caspase-1, -2,-4, -5, -8, -9, -10, and -12, which all have long N-terminal prodomains,involved in interactions with adaptor proteins. Upon such interactionsthese caspases undergo oligomerization-induced autoproteolysis leadingto their activation. Effector caspases, caspase-3, -6, -7, -11, and -13, allhave short N-terminal prodomains and are therefore unable to interact withadaptor protein (Thornberry and Lazebnik, 1998). Instead, these caspasesare activated by other proteases, generally by upstream active caspases,which by proteolytical cleavage remove the prodomain. The substrate ofthe effector caspases are either signaling or structural proteins whose cleav-age results in the morphological and functional changes associated withapoptosis.There are two major pathways through which caspases become activated:

the extrinsic, receptor-mediated pathway and the intrinsic, mitochondria-mediated pathway (Fig. 1). Accumulating evidence also shows that otherintracellular compartments and/or organelles such as the nucleus, theendoplasmatic reticulum, and the lysosomes all participate in apoptoticsignaling (Fig. 1) (Guicciardi et al., 2004; Norbury and Zhivotovsky,2004; Orrenius et al., 2003). How the signals emerging from these orga-nelles bifurcate into extrinsic and intrinsic apoptotic signaling path-ways is of major importance for cancer therapy but has been only partlyrevealed as of yet. Therefore this is an area of intensive research within theapoptotic field.


A. Receptor-Mediated Apoptotic Pathway

Extrinsic, receptor-mediated apoptotic signaling requires active participa-tion of members of the tumor necrosis factor (TNF) superfamily of receptorsand their associated ligands.Within this super family, consisting ofmore than20 members, the best-known associates in apoptotic signaling are Fas(Apo-1/CD-95), TNF, and TRAIL (Apo-2) (Igney and Krammer, 2002).Receptor-mediated apoptotic signaling is initiated by binding of theassociated ligand to the receptor. Upon such binding, the receptor–ligandcomplexes oligomerize and recruit the intracellular adaptor proteins. In thecase of the TNF receptor, the adapter protein is tumor necrosis factor receptor-associated death domain (TRADD), and in the case of Fas receptor, it isFas-associated death domain (FADD). The adaptor proteins are required forthe efficient recruitment and activation of the caspases, i.e., pro-caspase-8 and-10. Together, the intracellular proportion of the receptors, the adaptorproteins, and the pro-caspases form the death-inducible signaling complex(DISC), in which the pro-caspases are activated by autoproteolyticalprocessing (Medema et al., 1997). The subsequent steps are cell typespecific, and, accordingly, cells can be classified as type I or type II cells

Fig. 1 Cross-talk between plasma membrane, mitochondria, and nuclei in apoptotic signal-ing. Receptor-mediated apoptotic pathway starts when death ligands (exemplified here by

FasL) binds to the death receptor (exemplified here by FasR), resulting in their oligomerization.

This leads to recruitment of adaptor proteins (here exemplified with FADD) via death domain

(DD) to the receptor. A C-terminus of FADD contains a death effector domain (DED), whichinteracts with pro-caspase-8, forming a complex called FLICE. Pro-caspase-8 is activated

within this complex, and active caspase-8 can in some cells (type I) directly cleave and activate

pro-caspase-3, which cleaves many structural proteins and proteins involved in DNA mainte-

nance (exemplified here by Inhibitor of caspase-activated DNase (ICAD)). Upon this cascade ofreactions, Caspase-activated DNase (CAD) is released and induces cleavage of DNA, resulting

in nuclear apoptotic morphology. In type II cells, caspase-8 is also capable to initiate mitochon-

dria-mediated apoptotic signaling through cleavage of Bid with formation of truncated Bid(tBid). The mitochondria-mediated apoptotic signaling might be triggered by diverse stimuli

and resulted in release of several apoptogenic factors, i.e., cytochrome c, Smac/DIABLO, AIF,

Endo G, and Omi/HtrA2 from the intermembrane space of mitochondria into cytosol. This

release is regulated by the Bcl-2 family proteins. Bcl-2 and Bcl-xL block this process, whereasBak and Bax promote it. Cytosolic cytochrome c forms with Apaf-1 and pro-caspase-9 in a

presence of dATP so called the apoptosome complex. As a result, pro-caspase-9 is activated and

subsequently initiates the caspase cascade, including the activation of pro-caspase-3. The

activation/activity of pro-caspase-9 and -3 is inhibited by inhibitor of apoptosis protein (IAP),which in turn is regulated by Smac/DIABLO also released from the mitochondria. AIF and

Endo G released from mitochondria translocate to the nucleus and cause chromatin condensa-

tion and DNA fragmentation. Heat shock proteins can block apoptotic signaling at several

levels, including apoptosome formation, activation of caspases, and redistribution of Bid tomitochondria. In response to genotoxic stress, pro-caspase-2 is activated and involved in

transducing the apoptotic signal from nuclei to mitochondria.


(Scaffidi et al., 1998). In type I cells, the initiator caspases, i.e., caspase-8 and -10, directly activate the executor caspases, i.e., caspase-3 and -7(Scaffidi et al., 1998). By contrast, in type II cells, activation of pro-caspase-8 results in cleavage of the Bcl-2 family protein Bid with the formation of atruncated form, tBid, which participates in the oligomerization of Bax-likeproteins (Li et al., 1998a; Luo et al., 1998). The death ligand TRAIL canactivate several death receptors, including TRAIL-R1 (DR4) and TRAIL-R2(DR5/Killer). Within the TRAIL/DISC complex both FADD and pro-caspase-8 are detected and contribute to the signaling propensity of theirrespective complex (Igney and Krammer, 2002).

B. Mitochondria-Mediated Apoptotic Pathway

Although mitochondria act merely as a signal amplifier in death receptor-induced apoptosis, they are of key importance for apoptosis signalinginitiated by anticancer drugs, DNA-damaging agents, kinase inhibitors,hypoxia, and growth factor withdrawal and in the cellular response to UVand ionizing radiation. A large fraction of the apoptosis-inducing agentsoperate by triggering the release of apoptogenic factors from the inter-membrane space of mitochondria. Among these factors are cytochrome c(Liu et al., 1996), AIF (apoptosis-inducing factor), SMAC/DIABLO (secondmitochondria-derived activator of caspases/direct IAP-binding proteinwith low pI), endonuclease G, and Omi/HtrA2 (Du et al., 2000; Hegdeet al., 2002; Li et al., 2001b; Martins, 2002; Srinivasula et al., 2000; Susinet al., 1999). In the cytosol, the released cytochrome c forms a so-calledapoptosome complex with the adaptor protein Apaf-1 and pro-caspase-9 inthe prescence of dATP. Within this complex, pro-caspase-9 is activated andtriggers the activation of effector caspases, most notably caspase-3, result-ing in cleavage of several substrates. This sequence of events results in theappearance of apoptosis-related morphological changes. Upon apoptoticstimuli, both AIF and endonuclease G are released from mitochondria andtranslocate to the nucleus, where they exert their effects (Li et al., 2001b;Loeffler et al., 2001; Susin et al., 1999). Nuclear localization of AIF causesperipheral nuclear condensation as well as large-scale fragmentation of theDNA, and endonuclease G generates oligonucleosome-sized DNA frag-ments resulting in the apoptosis-related nuclear morphological changes.

C. Nuclei-Mediated Apoptotic Pathway

Many of the currently available anticancer treatments, such as chemo-therapy and ionizing radiation, target the DNA. Although several mechan-isms of DNA damage-induced activation of apoptotic pathways have been


sugges ted (rev iewed in Norbur y and Zhi votovsky, 2004), too many “blackboxes” must be opened to understa nd how this pa thway is regulate d.

1. ROLE OF CASPASE-2

Casp ase-2 was the first mammali an apoptotic caspas e to be identified.This enzym e is activat ed during DN A da mage-in duced apo ptosis upstre amof mitocho ndrial event s. Subcel lular frac tionati on studies ha ve revea led thatpro-ca spase-2 is present in several intracell ular compar tmen ts, incl uding theGolgi, cytos ol, and nuc leus; howev er, it is the only pro-ca spase presen tconstitut ively in the nucle us ( Mancini et al., 2000; Zhivotovs ky et al.,1999 ). In cells, caspas e-2 is spon taneous ly recruit ed to a large proteincomplex, which is sufficient for its acti vation ( Tinel and Tschopp, 2004 ).Tinel and Tschop p also provide cle ar eviden ce that caspase-2 a ctivationmight occur without proces sing of the precur sor mol ecule. However,oligome rizatio n is an imp ortant step for caspas e-2 activat ion. Cyclin D3has been implicat ed in activation of caspas e-2. Express ion of cycl in D3 andcaspas e-2 in human cells potent iates apo ptosis compar ed with expression ofcaspas e-2 alone ( Mendelso hn et al ., 2002 ). Moreove r, expres sion of cycl inD3 increase s the amou nt of cleaved caspas e-2. It was pro posed that inter-action with cyclin D3 may stabilize caspas e-2. As it is activ e in respons e toDNA da mage, caspase-2 is invol ved in initi ation of the mi tochondri a-mediate d apop totic pathw ay. Cells stably transfec ted with pro-ca spase-2antisense , or trans iently expres sing small interferi ng (si)R NA for caspas e-2,were refracto ry to DNA damage -induc ed ap optosis and did not sho wcytoch rome c rel ease, pro-ca spase-9 an d -3 activat ion, phos phatid ylserin eexposure on the plasm a membran e, or DNA fragment ation ( Lassus et al.,2002; Rober tson et al., 2002 ). Express ion of a caspas e-2 cDN A constr uctthat is unruly to siRNA restored the ability of cells to undergo apo ptosis.How does active caspas e-2 act in the apop totic signa ling? Two groupsdemons trated that caspas e-2 can induce release of cytoc hrome c and Smacdirectly (Guo et al., 2002b; Robertson et al., 2002) or cause cleavage of thepro-apoptotic protein Bid, which moves to mitochondria and facilitatescytoch rome c rel ease (Guo et al ., 2002b) . Anot her grou p ha s shown thatactive caspase-2 engaged mitochondria by promoting translocation of Baxto the mitochondria and that Bax deficiency affects the function of caspase-2 in UV-induced apoptotic events (He et al., 2004). Thus, in several systemsand cell types, caspase-2 is suggested to be an apical caspase in the proteo-lytic cascade initiated by DNA damage, and it has also been suggested that anuclear-mitochondrial apoptotic pathway elicited by caspase-2 exists. How-ever, it is clear that many questions must be answered to understand themechanisms of caspase-2 activation in response to DNA damage. Recentlyit has been shown that caspase-2 is activated within a so-called PIDDosome


complex, which includes PIDD (the death domain-containing protein,whose expression is induced by p53) and the adaptor protein RAIDD (Tineland Tschopp, 2004). The identification of the PIDDosome as a platform forcaspase-2 activation increased the desire to understand the poorly definednucleus-to-mitochondria signaling pathway. Interestingly, in contrast toapoptosome-induced activation of caspase-9, which usually leads to apo-ptosis, PIDDosome-based activation of caspase-2 is not always toxic; cellscan survive even if a notable fraction of the caspase-2 pool is activated(Tinel and Tschopp, 2004; Verhagen et al., 2002). Most likely, a secondsignal is required for full commitment to caspase-2-mediated apoptosis. Anincrease in PIDD concentration promotes activation of caspase-2, as dostress conditions at low PIDD concentration. Inhibition of PIDD expres-sion attenuates p53-induced apoptosis, whereas overexpression of PIDDinhibits cell growth. Although PIDD appears to be a crucial target geneof a signaling pathway that is triggered by p53, it is unclear whethercaspase-2-mediated apoptosis requires p53.

2. P53-FAMILY PROTEINS

The p53 family consists of p53 and its more recently discovered rela-tives p63 and p73, which share over 60% amino acid identity within theDNA-binding region; all can induce apoptosis. In response to ionizingradiation and chemotherapeutic drugs the level of p53 is increased, mainlyas a consequence of increased p53 protein stability (Vousden and Lu,2002). The p53 protein is involved in regulation of several cellular func-tions, including gene transcription, DNA synthesis, DNA repair, cell cyclearrest, senescence, and apoptosis. How the p53 proteins decide betweeninduction of cell cycle arrest and apoptosis is still not fully understood, butrecently it was shown that the ASPP proteins are involved in directing thep53 protein to pro-apoptotic gene promoters (Bergamaschi et al., 2004).The complete function of p53 in apoptotic signaling is beyond the scope ofthis review and has been intensively discussed recently (Slee et al., 2004).Therefore here we just briefly summarize some of its effects.The role of p53 in transactivation and transrepression of pro- and anti-

apoptotic genes, respectively, is well documented. Several anti-apoptoticproteins, including Bcl-2 and survivin, an endogenous inhibitor of caspase-activity, have been shown to be transcriptionally repressed by p53(Miyashita et al., 1994). A large number of pro-apoptotic genes are regu-lated by p53. Among these proteins are Bax, PUMA, NOXA, IGF-BP3,DR5/KILLER, Fas/Apo-1, the PIGs, PERP, Apaf-1, and p53-AIP (reviewedin Vousden and Lu, 2002). During apoptosis some of these proteins (Bax,Puma, Noxa, and p53AIP1) translocate to mitochondria and promoterelease of mitochondrial intermembrane proteins to the cytosol (Oda


et al., 2000; Yu et al., 2001). The transactivation of puma and noxa by p53is of key significance for p53-induced apoptosis as documented bythe absence of radiation-induced apoptotic response in mouse embryonicfibroblasts deficient in these genes (Jeffers et al., 2003; Shibue et al., 2003;Villunger et al., 2003). p53 transactivation of PIGs may also impedemitochondrial signaling, as the product of these genes may generate reactiveoxygen species (ROS), which may damage the mitochondria (Polyaket al., 1997). p53-regulated expression of DR5/KILLER and Fas/Apo-1can interfere with receptor-mediated apoptotic signaling (Takimoto andEl-Deiry, 2000; Wu et al., 2000). p53 also induces apoptosis via an endo-plasmic reticulum (ER)-dependent mechanism by increasing the expressionof scottin, a protein located in the ER (Bourdon et al., 2002). Although ap53 knockout mice in which a transcription mutant p53 was expressedshowed impaired DNA damage-induced responses in vivo (Chao et al.,2000), it is evident that p53 transcription-independent mechanisms mightalso operate in cells. First, it has been shown that p53-induced apoptosis canproceed in the presence of transcriptional and/or translational inhibitors(Caelles et al., 1994). Second, p53 that is mutated so that it no longerlocalizes to the nucleus or is impaired in its transactivation function is stillcapable of inducing apoptosis (Marchenko et al., 2000; Moll and Zaika,2001). It was shown that a transcriptionally inactive tumor-derived mutant,p53H175, which targets to mitochondria, induced apoptosis as efficiently aswild-type p53 (Marchenko et al., 2000; Moll and Zaika, 2001). Further, ithas been reported that recombinant p53, when added to mitochondria,results in Bak oligomerization and release of cytochrome c because ofalleviation of Bcl-2 and/or Bcl-xL inhibitory effects (Mihara et al., 2003).However, recently it was shown that p53 can directly activate the pro-apoptotic Bcl-2 family members Bax and Bak and that cytosolic localizationof endogenous wild-type or trans-activation-deficient p53 was necessaryand sufficient for induction of apoptosis in the presence of either Bax orBak (Chipuk et al., 2004; Leu et al., 2004). In this case, p53 causes Bax and/or Bak oligomerization and release of cytochrome c (Chipuk et al., 2004).Importantly, the p53-mediated effects of Bax were observed to occur withkinetics and concentrations similar to those triggered by activated Bid.Therefore, when p53 accumulates in the cytosol it can function analogouslyto the BH3-only subset of proapototic Bcl-2 proteins, i.e., activate Bax andtrigger apoptosis. The authors (Chipuk et al., 2002) argued that cytosolicaccumulation of p53 was inefficient in Bax-/-mouse embryo fibroblasts(MEFs). However, a recent report suggested that in response to cellularstress p53 might interact with Bak at the level of mitochondria and causeits oligomerization (Leu et al., 2004). Notably, treatment of purifiedmitochondria from Bak-/-MEFs with recombinant p53 did not cause cyto-chrome c release, whereas this was observed in mitochondria derived from


wild-type MEFs. Immunoprecipitation experiments revealed that theobserved p53–Bak complexes resulted from a decreased association be-tween Bak and the anti-apoptotic Bcl-2 member Mcl-1, suggesting thatthe disruption of Bak–Mcl-1 association would set free Bak, which subse-quently undergoes conformational changes, forms oligomeric complexes,and promotes mitochondrial permeabilization. Although the p53-mediatedeffects on Bcl-2/Bcl-xL (Mihara et al., 2003), Bax (Chipuk et al., 2004), orBak (Leu et al., 2004) were each suggested to be the most importantp53-induced signaling event, the cell type, apoptotic stimuli, and state ofcell might determine which of these proteins p53 interacts with. As dis-cussed recently, these mechanisms are not mutually exclusive and mighteven coexi st withi n cells (Perfet tini et al., 2004).Although the p53 family members have much in common, there are also

striking differences between these proteins. In contrast to the high mutationfrequency found in p53 in human cancers, p63 and p73 are seldom mutated(Melino et al., 2003). Further, within cells both p73 and p63 are expressedas several different isoforms, some of which promote apoptosis, e.g.,TA-p73 or TA-p63, and others that are antiapoptotic, e.g., �Np73 (Melinoet al., 2003). Transcription of the different isoforms affords an intri-cate control mechanism. Like p53, the p73 stability and transcriptionalactivity in response to DNA damage are regulated by posttranslationalmodifications, mainly by phosphorylations.Several drugs, including taxol, gemcitabine, etoposide, melphalan, and

camptothecin, have been shown to increase the level of p73 and to in-duce p73-mediated gene transcription, showing the importance of p73 inchemotherapy-induced apoptosis (reviewed in Melino et al., 2003). Themechanism of p73-mediated apoptosis has not been completely elucidated;however, it has been shown that it involves an increase in transcription ofseveral known p53-induced promoters, including Bax, PUMA, and Noxa(reviewed in Levrero et al., 1999). Recently, it was reported that cells over-expressing p73� have increased levels of PUMA, which resulted in Baxactivation (Melino et al., 2004). In light of the results of the direct actionof p53 on mitochondria, it would be interesting to know if p73 can inducesimilar effects. It was reported also that p73�-induced apoptotic signaling,similarly to p53, involved induction of ER stress (Bourdon et al., 2002;Terrinoni et al., 2004). In this case, expression of p73� in Saos-2 cellscaused calnexin reorganization (a marker for ER stress) and induced theER-localized protein scotin. These events resulted in a decrease in intracel-lular calcium level as well as transcriptional activation of Gadd153, atranscription factor induced during ER stress conditions (Terrinoni et al.,2004). Hence, both p73 and p53 are capable of inducing apoptosis as aconsequence of ER stress, showing the functional redundancy of these p53family members.


In contrast to the extensive knowledge about p53-mediated apoptoticsignaling and accumulating data on p73-mediated regulation of apoptosis,very little is known about how p63 acts. The DNA-damaging agent cisplat-in has been shown to cause upregulation of the p63 protein, suggesting thatincreased stabilization of this protein in response to DNA damage may be acommon theme (Flores et al., 2002). However, it remains to be elucidatedwhether such increased stability of p63 results from posttranslational mod-ifications as described for p53 and p73. p63 has been shown to inducetranscription of some of the p53-induced genes involved in apoptotic sig-naling, including mdm2, bax, PERP, andNoxa (Flores et al., 2002). Similarto p53, both p63- and p73-mediated induction of proapoptotic genes wereenhanced after association to the ASPP proteins (Bergamaschi et al., 2004).However, it will be important to determine if p73 and p63 can induceapoptosis in a transcriptionally independent manner as p53 can. Moreover,it will also be interesting to know how these three members intervene witheach other to determine apoptotic signaling in response to DNA damageinduced by anticancer agents.

D. Endoplasmic Reticulum-MediatedApoptotic Pathway

The endoplasmic reticulum (ER) is normally an organelle wherein chaper-one-assisted polypeptide folding and modification ensure that proteins obtaintheir mature conformation. However, if a massive increase in the number ofunfolded proteins within the ER occurs, a conserved alarm system, the unfold-ed protein response (UPR), is triggered. UPR discontinues protein synthesisand increases ER-localized chaperones, which results in good opportunity forthe cell to correct its protein conformation (Travers et al., 2000). When thedamage is too severe and proper protein folding is not restored, the UPRmighttrigger apoptosis. Several years ago it was found that pro-caspase-12 is pre-dominantly localized to the ER in rodents and is specifically cleaved during ERstress (Nakagawa et al., 2000). Accordingly, it was observed that caspase-12null mice or cells were partially resistant to ER stress-induced apoptosis,although they still responded to other apoptotic stimuli. Using an antibodydirected against mouse caspase-12, it was shown that the chemotherapeuticagent cisplatin can trigger caspase-12 processing followed by caspase-3 activa-tion in a melanoma cell line (Mandic et al., 2003). The importance of caspase-12 in apoptosis signaling in human cells is still amatter of debate, as the humancaspase-12 gene is interrupted by frame shift and a premature stop codon andalso has an amino acid substitution at the site, which is critical for caspaseactivity (Fischer et al., 2002). Interestingly, sequence analysis of genomicDNAfrom people of distinct ethnic backgrounds revealed that most encoded the


truncated prodomain-only form of caspase-12 (Saleh et al., 200 4). This sug-gests that in humans it should be another enzyme that substitutes for caspase-12 in response to ER stress. Indeed, recently it was found that human caspase-4is localized to the ER membrane and is cleaved when cells are treated with ERstress-inducing agents, but not with other apoptosis inducers (Hitomi et al.,200 4). Cleavage of caspase-4 is not affected by overexpression of Bcl-2, whichprevents signal transduction through the mitochondria, suggesting that cas-pase-4 is primarily activated within the ER. Further, a reduction of caspase-4expression by siRNA decreases ER stress-induced apoptosis, but not other ERstress-independent deaths.Since mitochondr ia and the ER are interconne cted phy sically an d

physiologi cally, it is very likely that apo ptotic signa ling, when ini tiated inthe ER, migh t rel ay to mitocho ndria. It has been reported that trea tmentof cells with ER stress- inducing agents , such as tunicamy cin or brefe ldinA, causes rel ease of cytoc hrome c and mitocho ndrial depolar ization(Boy a et al., 2002; Hacki et al., 2000 ). These event s can be blocked byan ER- localized Bcl-2 ( Hacki et al., 2000 ). In add ition, cells that aredeficient in the pro-apo ptotic Bcl-2 family members Bak and Ba x show acompro mised apoptotic respon se to tunic amycin and thap sigargin (We iet al., 2001 ).Recently, it has been shown that ER- locali zed Bax and Bak are involved

in regul ation of Ca2+ rel ease from the ER (Scor rano et al ., 2003). As wasexpected, Bax- and Bak-defic ient cel ls wer e character ized by an imp airedCa2+ release and mitocho ndrial uptake of Ca 2+ in respons e to the ER stre ss-inducing agent thapsi gargin . Reconstitut ion with the Bax or Bak togetherwith the SERC A pump restored apoptoti c respons e in cells after trea tmentwith ER stress-induc ing dru gs and resul ted in mi tochondri al upt ake of Ca 2+

followed by cytoch rome c release . Once rel eased from the mi tochondri a,cytochrome c binds not only to Apaf-1 (see a bove) but also to the Ins( 1,4,5 )P3 rece ptor and blocks Ca

2+ -mediat ed inhi bition of its funct ion ( Fig. 2).This results in sustained oscillator y Ca 2+ incr ease in the cytos ol, followed bythe au gmented cytoc hrome c release an d amplific ation of the ap optoticsignal (Boeh ning et al., 2003).ER stress may trigger activa tion of the stre ss-activat ed protein kinase JNK

(see Section III.D. 1), althou gh its particip ation in ER stre ss-induce d apop to-tic signaling requ ires further examin ation. It has rece ntly been report edthat bot h p53 and p73 can cause ER stre ss (Bour don et al., 2002; Terrinon iet al., 2004). This suggests that ER might be involved in apoptotic signaling,induced by chemotherapeutic drugs, that occurs via p53- and/or p73. Thus,several evidences imply the cross-talk between the ER and mitochondriain regulation of apoptosis signaling (Orrenius et al., 2003), although addi-tional work is required to clarify their interconnections and how suchconnections inflict apoptotic signaling.

Fig. 2 Cross-talk between lysosomes, endoplasmatic reticulum, and mitochondria in apopto-

sis. During apoptosis different agents might activate lysosomes, resulting in the release oflysosomal-localized cathepsins. Subsequently cathepsins cleave and activate Bid, Bax, and

pro-caspase-3, all of which are important for progression of apoptotic signaling. Endoplasmatic

reticulum-initiated apoptotic signaling results in Ca2þ release, which in turn is essential for

release of cytochrome c and loss of mitochondrial potential. Being in cytosol, cytochrome cbinds not only to Apaf-1 (see Fig. 1), but also to the Ins (1,4,5)P3 receptor (which is involved in

the ER luminal Ca2þ regulation) and alleviates the Ca2þ inhibitory effect on the receptor, acting

as an amplificatory of the apoptotic signal. In addition, caspase-4 is activated in the ER of

human cells upon apoptotic stimuli and may be involved in release of cytochrome c or inactivation of caspase-3.


E. Lysosomal-Mediated Apoptotic Pathway

Although caspases are involved in regulation of different apoptoticpathways, several other proteolytic activities, localized in various intracel-lular compartments, may also play a role in apoptosis signaling. One of themajor intracellular storehouses for proteolytic enzymes is the lysosome,which previously has been reported to participate in necrotic and autopha-gic death, but which recently has been implicated in several modelsof apoptosis (Guicciardi et al., 2004). Partial permeabilization of the lyso-somal membrane with subsequent release of lysosomal proteases, i.e.,cathepsins, was observed in cells treated with many apoptosis-inducingagents, including TNF (Foghsgaard et al., 2001; Werneburg et al., 2002),


oxidative stress (Nilsson et al., 1997), and several lysosomotropic agents,such as ciprofloxacin, norfloxacin, and hydrochloroquinone (Boya et al.,2003a,b). Several reports suggest that once released, the lysosomal-localizedproteases mediate their effects by increasing mitochondrial permeabilityfollowed by release of apoptogenic proteins from the intermembrane space.These effects have been proposed to be a consequence of lysosome-mediatedROS generation (Zhao et al., 2003) or as an indirect effect on severalBcl-2 family proteins (Bidere et al., 2003; Boya et al., 2003a,b; Cirmanet al., 2004; Werneburg et al., 2002). Thus, cathepsin D mediates apo-ptosis by promoting Bax insertion into mitochondria as well as by causingBid cleavage (Bidere et al., 2003). Consequently, Bax/Bak double knock-out cells were resistant to apoptosis in response to several lysoso-motrophic agents (Boya et al., 2003a,b). Although these results indicatethat lysosomes might have propensity in apoptotic signaling and not solelyact as a site of protein degradation, the importance of lysosomes in thecellular response to conventional chemotherapeutic agents remains to bedetermined.

III. MODULATORS OF APOPTOTIC SIGNALING

A. Regulators of Receptor-Mediated Apoptosis

Receptor-mediated apoptosis is a multistep process that requires thepresence of receptors at the cell surface, the possibility for the ligand tobind to the receptor, oligomerization of the receptor, and, finally, recruit-ment of adaptor proteins. All these events are under the control of severalproteins, which ensure that inappropriate activation does not occur. One ofthese proteins is c-FLIP (FLICE-inhibitory protein), which has caspase-8-likeproperties but lacks the catalytical site as well as the residues that bind thecaspase-8 substrates and thus is inactive (Irmler et al., 1997). c-FLIP can alsoredirect apoptotic to pro-survival signaling via binding to the TNF receptor-associated factor 1 and 2 (TRAF1-2), which results in nuclear factor �B(NF�B) activation (Schneider et al., 1997; Kataoka et al., 1998). Receptor-mediated apoptosis might be accelerated by the aid of DAXX (Fas deathdomain-associated protein XX). This protein increases Fas-induced signalingvia interaction with the ASK1/JNK axis (Yang et al., 1997). The findingthat DAXX solely localized to the nucleus made it difficult to understandits involvement in Fas-signaling (Torii et al., 1999). Nevertheless, a recentreport showed that in response to glucose deprivation ASK-1-activated JNKis required forDAXX relocalization to the cytosol via amechanism involvinghomeodomain-interacting protein kinase (HIPK1) (Ecsedy et al., 2003; Song


and Lee, 2003 ). How ever, it remains to be determined whether this is agenera l mech anism for DAX X relocal ization and how im portant this relo -calization is for rece ptor-me diated apo ptotic signalin g.

B. Bcl-2 Fami ly Proteins

The Bcl-2 family proteins con sists of more than 30 differe nt mem bers thatfulfill anti- or pro -apoptot ic fun ctions ( Table I ). Although a maj ority ofBcl-2-f amily pr oteins operat e on the mitochondr ial leve l, some of them arelocalized to the ER and nuc lear mem branes ( Krajewski et al., 1993; Nuttet al., 2002 ; Scorran o and Kors meyer, 2003) , where they may also beinvolved in the regul ation of apop tosis (Scorrano an d Korsme yer, 2003) .All Bcl-2 family members hold at least one of four conserved Bcl-2 homo-logy domains (BH1 to BH4). The multidomain Bcl-2 family proteinsalso have a stretch of hydrophobic amino acids near their C-terminus(transmembrane domain [TM]) that anchors them to membranes. Whereasthe BH4 domain has been suggested to be important for anti-apoptotic

Table I The Bcl-2 Family Proteins

Promote apoptosis Suppress apoptosis

Bax Bcl-2

Bcl-Xs Bcl-XLBad Bcl-W

Bak Bag-1

Bar A1/Bfl-1

Bok/Mtd Mcl-1Bid BRAG-1

Bik/Nbk NR-13

Bim/Bod Boo/Diva

Hrk/DP5 BHRF-1Blk Bcl-B

Noxa

Bnip3/Nip3

Nix/Bnip3LBcl-GS

Bcl-GL

MAP-1Bcl-rambo

Bmf

Puma

BfkBRCC2 (BH3-like)


activity ( Huang et al ., 1998 ), the BH3 dom ain is esse ntial and sufficient forpro-apopot otic eff ect ( Chitten den et al., 1995 ). The pro-apop totic Bcl-2family is subdivided into the Bax subfamily (Bax , Bak , an d Bok), whichall contain BH1-BH 3 dom ains, and the “BH3-onl y” subfamily (Bid,Bad, Bim , Bik, Blk, Hrk, NO XA, and Puma). Pro-apopt otic Bcl- 2 proteinsare key regul ators of cytochrome c release . Based on experime ntal data,several not mutua lly exclusiv e mechan isms were proposed to exp lainhow cytoc hrome c release is regulate d. Acc ording to the first of thesemechanis ms, BH3-onl y pro teins interact with cardi olipin in the mitochon-drial membrane and cause cytoc hrome c release ( Lutter et al ., 2001 ). In thesecond, the BH3-onl y pr oteins trigger activation of the multid omain pro-apoptotic pro teins Bak and Bax, which sub sequent ly medi ate cytochro mec release (Cheng et al ., 2001; Wei et al ., 2001 ). The third model sugges tsthat the BH3-onl y protein s interact with and allevi ate the funct ion ofanti-apopt otic Bcl-2 fam ily mem bers ( Strasser et al ., 2000 ). Acco rding tothe fourth , the BH3-onl y proteins induce mitochon drial perm eabili zationvia interact ion with the adenine nucleoti de transloca tor (ANT) or thevoltage- dependent anion channel (V DAC), through whi ch cytoch rome cmight then be release d (Su giyama et al ., 2002; Zam zami et al., 2000 ).In many experi mental syst ems BH3-onl y proteins require Bak and/or

Bax to exert their pro-apopt otic fun ction as pro ven by the lack of cyto-chrome c rel ease an d apop totic features in Bax/Bak doubl e knockou t cel ls(Cheng et al., 2001). Thus, tBid and Bim might directly cause activation ofBak and Bax, whereas other BH3-only proteins preferentially bind anti-apoptotic Bcl-2 family members and thereby alleviate their inhibitory func-tion on Bak and Ba x (Scor rano and Korsme yer, 2003) . In ord er to beactivated, Bax translocates to mitochondria and inserts into the mitochon-drial outer membrane, whereas Bak resides in mitochondria of healthy cells(Scorrano and Kors meyer, 2003) . The entire mecha nism by which Baxtranslocation to mitochondria is controlled is not fully understood, but itmight depend on interaction with Bcl-2 (Murphy et al., 2000). Recently,it was shown that the scaffold protein 14-3-3 binds to Bax in the cytoplasmand prevents its translocation to mitochondria (Nomura et al., 2003).Stress-activated protein kinase C-Jun-NH2-terminal protein kinase (JNK)may regulate Bax translocation and thus apoptosis signaling by phos-phorylating 14-3-3 proteins, which reduces their affinity for Bax (Tsurutaet al., 2004). The nuclear protein Ku70, which is a part of the DNArepair complex DNA–PK, interacts with Bax and sequesters it in the cyto-plasm (Sawada et al., 2003b). Importantly, peptides, which mimicthe Ku70-Bax binding domain, could prevent Bax translocation and blockapoptosis in response to several anticancer drugs, including doxorubicinand cisplatin (Sawada et al., 2003a). In addition, binding of p53 to Bax orBak also results in insertion of Bax in the mitochondrial membrane and


conformational changes of both Bax and Bak (see Section II.C) (Chipuk et al.,2004; Leu et al., 2004; Mihara et al., 2003; Sawada et al., 2003b). A conse-quence of homo- and/or hetero-oligomerization of Bax and Bak in the outermitochondrialmembrane (OMM) is release of cytochrome c (Antonsson et al.,2000; Wei et al., 2000). Several mechanisms of Bax and/or Bak-mediatedcytochrome c release have been proposed. For example, oligomerized Baxand/or Bak might form a specific pore in the OMM; they might interact withANT and/or VDAC; or they might induce permeabilization of the OMM viainteractionwith“lipid” channels in the bilayer. Although it is clear that theBcl-2 family proteins play a key role in regulation of apoptosis, further studies arerequired to understand the precise mechanism by which they regulate mito-chondria-mediated apoptosis.

C. Modulators of Caspase Activity

The processing and activation of caspases are of major importancefor apoptotic signaling. However, a processed caspase is not always cata-lytically active, as their activity and processing are under the control ofseveral proteins, e.g., heat shock proteins (HSPs) and inhibitor of apoptosisproteins (IAPs).

1. HEAT SHOCK PROTEINS

Although the HSP family was first discovered as proteins whose expres-sion is controlled by heat shock, it is evident that different apoptotic stimuliinduce HSPs, which consequently might positively or negatively influenceapoptotic signaling (Parcellier et al., 2003). The mammalian HSP familyis divided into a subfamily of large HSPs (HSP90, HSP70, and HSP60) and asubfamily of small HSPs (HSP27). Overexpressed HSP27 can block apo-ptotic signaling by increasing the antioxidant defense (Mehlen et al., 1996),by sequestering cytosolic cytochrome c (Bruey et al., 2000), by inhibit-ing Bid redistribution (Paul et al., 2002), or by impeding activation ofpro-caspase-3 (Concannon et al., 2001; Pandey et al., 2000a). In addition,HSP27 can interfere with DAXX and thus inhibit FasR signaling (Charetteand Landry, 2000). Similar to HSP27, HSP70 protects cells from a broadrange of apoptotic stimuli, including chemotherapeutic drugs, by interveningwith apoptotic signaling both upstream and downstream of mitochondrialevents. Thus, HSP70 might interact with the Caspase recruitment domain(CARD) domain of Apaf-1, an event that impedes Apaf-1 oligomerizationand pro-caspase-9 activation (Beere et al., 2000; Saleh et al., 2000).However,HSP70 has also been reported to block apoptosis in Apaf-1-/-cells as a


consequence of AIF inhibition (Creagh et al., 2000; Ravagnan et al., 2001).In addition, both HSP27 and HSP70 can block activation of JNK and p38through inhibition of ASK-1 and therefore modulate apoptotic signaling(Park et al., 2001).HSP90 can prevent or promote apoptotic signaling in a stimuli-dependent

manner. Thus, in U937 cells HSP90 promotes a TNF-�-induced effect,while it inhibits staurosporine-induced apoptosis (Galea-Lauri et al., 1996;Pandey et al., 2000b). HSP90 may also influence apoptosis signaling viadephosphorylation and inactivation of Akt (Sato et al., 2000). Thus, recentevidences indicate that HSPs can interfere with apoptotic signaling at severallevels, and this might involve different functions of HSPs, such as the chaper-one activity and their propensity to target these interacting proteins forproteosomal degradation.

2. IAP FAMILY PROTEINS: THEIR REGULATORYFUNCTIONS AND THEIR REGULATORS

Inhibitor of apoptosis proteins (IAPs) is a family of proteins that isinvolved in regulation of the caspase cascade. Crystal structures revealedthat upon binding with caspases IAPs cause a steric block, resulting ininhibition of caspase-substrate binding (Shiozaki et al., 2003; Sun et al.,1999). IAPs contain one or several baculoviral IAP repeat (BIR) do-mains, which are essential for binding and inhibition of caspases (Listonet al., 2003). Thus, BIR3 mainly interacts with caspase-9, whereas BIR2more specifically inhibits caspase-3 and -7 (Maier et al., 2002; Roy et al.,1997; Takahashi et al., 1998). XIAP, cIAP-1, and cIAP-2 all bind to caspase-9, -3, and -7 and therefore act as the most potent caspase inhibitors withinthe IAP family. Another protein belonging to the IAP family is survivin,which also may bind and inhibit caspase-3 and -7 (Ambrosini et al., 1998;Grossman et al., 2001). However, it has been shown that survivin also playsa role in cell cycle regulation, with the highest expression in mitosis when itinteracts with the components of the mitotic spindle (Kobayashi et al.,1999; Li et al., 1998b).In addition to the BIR domains, some of the IAP members also contain

a RING domain, which is essential for proteasome-mediated degrada-tion. Thus, cIAP-1, cIAP-2, and XIAP all have ubiquitin ligase activity andpromote degradation of themselves at least in vitro (Verhagen et al., 2001).Cells overexpressing mutant XIAP lacking the RING domain were charac-terized by resistance in proteosome-mediated degradation and low level ofapoptosis as compared with those expressing wild-type XIAP (Yang et al.,2000). The effect of IAPs is antagonized by the mitochondria-localizedproteins Smac/DIABLO and HtrA2/Omi. Both these proteins contain an


IAP-binding motif and therefore neutralize anti-apoptotic activity of IAPs(Chai et al., 2000; Martins, 2002). Thus, within cells a balance betweenexpression of IAPs and Smac/DIABLO and/or HtrA2/Omi may determinewhether a certain apoptotic stimuli will trigger apoptosis.

D. Protein Kinases

While the apoptotic signaling depends on activation of different pathwaysdescribed above and the execution is mainly completed by caspases, theperpetuation of the apoptotic process is influenced by many other signalingsystems, of which the protein kinase network is of key importance. Proteinkinases act as transducers of both survival and death signals, and thus theycan greatly influence the apoptotic cascade.

1. MAPK/SAPK SIGNALING

Mitogen-activated protein kinases (MAPKs) are one of the best-characterized signaling cascades regulating cell survival and death (Fig. 3).Both stress- and growth-regulating signals are transduced from the cellsurface into the nucleus via these cascades. The MAPK cascades consist ofthree-kinase modules (Hagemann and Blank, 2001). The apical MAPKKKsare serine/threonine kinases, which phosphorylate and activate their sub-strates, MAPKKs. These dual-specificity kinases phosphorylate critical thre-onine and tyrosine residues in MAPKs, which are serine/threonine kinaseswhose substrates include transcription factors and cytosolic proteins. TheMAPK family consists of the extracellular signal-regulated kinase (ERK orp42/44 MAPK), c-Jun NH2-terminal protein kinase/stress-activated proteinkinase (JNK/SAPK), and p38-MAPK (Fig. 3). Mitogenic stimulation ofcells results in activation of the ERK pathway, which mediates survival,differentiation, or a proliferative response in cells. Signaling in response tochemical and environmental stress activates JNK and p38 (Davis, 2000;Tobiume et al., 2001), and their activation mostly but not exclusively resultsin apoptosis. Importantly, chemotherapeutic agents, such as cisplatin,etoposide, and ionizing radiation, have been shown to activate JNK inexperimental cell systems, and studies using anti-sense JNK, dominantnegative JNK mutants, or cells deleted in c-Jun have shown a decrea-sed cellular sensitivity to DNA-damaging treatments (Sanchez-Perez andPerona, 1999; Zanke et al., 1996). In addition to MAPKKs, the tyrosinekinase c-Abl has been implicated in JNK and in p38 activation in responseto ionizing radiation or chemotherapeutic drugs (Kharbanda et al., 1995;Sanchez-Prieto et al., 2002). Furthermore, c-Abl-deficient cells, which fail to

Fig. 3 MAPK/SAPK signaling cascades. In response to growth factors and cellular stress, the

MAPK-(mitogen-activated protein kinase) and SAPK (stress-activated protein kinase)-signalingcascades, respectively, are activated. Both these cascades are composed of three kinase modules,

which subsequently activate each other by phosphorylation. For simplicity, only the transcrip-

tional regulation of each pathway is depicted. Activation of the ERK and JNK pathways inresponse to growth factors is initiated upon binding of growth factor to the receptor. This is

followed by oligomerization and reciprocal phosphorylation of the intracellular proportion of

the receptor and recruitment of adaptor proteins. The latter bring Ras to the plasma membrane

where it is activated and is involved in the triggering of Raf and MEKKs, respectively. Inresponse to cellular stress induced by chemotherapy drugs, ASK-1 (apoptosis stimulating kinase

1) is activated, which can subsequently activate the SAPK pathway, JNK, and p38. Cellular

stress as well as DNA damage can activate c-Abl, which translocates to cytosol and triggers the

JNK pathway.


activate JNK, were more resistant to ionizing radiation than their wild-typeparental, indicating the importance of c-Abl for regulation of pro-apoptoticJNK activity (Yuan et al., 1997). In addition, both JNK and c-Abl wereimplicated in ER stress. Thus, upon stress JNK is recruited to ER and isactivated by an association with the cytoplasmatic portion of the chaperoneIre1 and adaptor protein TRAF2 (Urano et al., 2000). c-Abl wasalso detected in the ER of healthy cells, and as a result of ER stress ittranslocates to the mitochondria and mediates cytochrome c release (Ito


et al., 2001). However, it is unclear whether c-Abl causes JNK activation atthis site.The mechanisms by which JNK and/or p38 activation trigger apoptosis

may involve activation of transcription factors as well as phosphorylation ofproteins involved in regulation of apoptosis. JNK causes transcriptionalactivation of c-Jun, ATF-2, and Elk-2 of the AP-1 family and subsequentlyincreases expression of pro-apoptotic proteins (Davis, 2000). As an exam-ple, Fas-R and TNF� are transcriptional targets of JNK (Bossy-Wetzel et al.,1997). Possible candidate proteins, which act as molecular links betweenJNK and apoptotic signaling through the mitochondria, are the Bcl-2 familymembers. Several studies have demonstrated that JNK phosphorylatesBcl-2 and Bcl-xL in vitro (Kharbanda et al., 2000; Maundrell et al., 1997;Yamamoto et al., 1999). Furthermore, mutations of the JNK-targeted phos-phorylation sites within Bcl-2 and/or Bcl-xL increased the anti-apoptoticcapacity of either protein (Kharbanda et al., 2000; Yamamoto et al., 1999).The BH3-only protein Bad is also phosphorylated by JNK. This phosphory-lation of serine 128 within the Bad protein was shown to increase itspro-apoptotic function in growth factor withdrawal-induced apoptosisand to counteract the survival signaling mediated by Akt (Donovan et al.,2002). Recently, the requirement for Bax-like proteins in JNK-mediatedapoptotic signaling was implicated. Activated JNK was sufficient to pro-mote cytochrome c release and apoptosis, but failed to do so in cells lack-ing expression of Bax-like proteins (Lei and Davis, 2003). Although nophosphorylation of Bax has been reported, it was shown that JNK-deficientcells had an impaired activation of Bax, cytochrome c release, and inductionof apoptosis (Lei et al., 2002), suggesting that JNK via an indirect mecha-nism is regulating Bax. As described previously, Bax activation requires itstranslocation to the mitochondria, which is controlled by the 14-3-3 pro-teins (Henshall et al., 2002; Nomura et al., 2003). Recently it was reportedthat JNK promotes translocation of Bax to mitochondria by phosphorylat-ing the 14-3-3 proteins, thus alleviating their inhibitory effect on Bax(Tsuruta et al., 2004). The Bcl-2 family member Bim is another target ofJNK. Normally Bim is associated with dynein filaments, and upon phos-phorylation by JNK Bim is translocated to mitochondria and induces acti-vation of Bak/Bax followed by mitochondrial permeabilization (Lei andDavis, 2003).Less is known about by which mechanism p38 might use to regulate

apoptotic signaling. However, it has been shown that p38 can stimulatethe translocation of Bax to mitochondria (Ghatan et al., 2000). p38 mayalso stimulate transcription of the CHOP/GADD153 gene, which results inincreased levels of pro-apoptotic Gadd-family proteins (Wang and Ron,1996).


2. PI3K-AKT

The phosphatidylinositol 3-kinase (PI3K) is deregulated in a variety ofhuman cancers (Mills et al., 2001; Shayesteh et al., 1999) and causesincreased survival signaling, which may lead to suppression of apoptosis.Once activated, PI3K mediates synthesis of phosphoinositols at the innerleaflet of the plasma membrane, which is involved in the activation of thedownstream substrates of PI3K. The most pronounced anti-apoptotic effectof PI3K is activation of the serine/threonine kinase Akt, which by severaldifferent mechanisms exhibits an anti-apoptotic propensity. Via phosphory-lation Akt inactivates several key pro-apoptotic proteins, including the Bcl-2family member Bad, pro-caspase-9, and forkhead transcription factor 1(FKHRL1) (Pommier et al., 2004). In the presence of survival factors, Aktphosphorylates Bad at serine 136, an event that causes Bad interaction with14-3-3 protein and sequestration in the cytoplasm (Zha et al., 1996). Onceassociated with 14-3-3, Bad is phosphorylated by protein kinase A (PKA),an event that disrupts the association between Bad and other Bcl-2 proteins.Consequently, Bcl-2/Bcl-xL is free to exert its anti-apoptotic effect (Lizcanoet al., 2000). Akt can phosphorylate FKHRL1 and sequester it in the cytosolalso by binding to 14-3-3 proteins, which impedes the transactivation ofpro-apoptotic genes, including Bim and FasL (Brunet et al., 1999; Dijkerset al., 2000). In addition, Akt can block JNK and p38 activation(Berra et al., 1998; Cerezo et al., 1998). Thus, it is evident that differentprotein kinase signaling pathways intervene with each other as well as withthe core apoptotic machinery and might greatly influence the apoptoticresponse to a given anticancer therapeutic agent.

IV. APOPTOSIS RESISTANCE AND ITS INVOLVEMENTIN IMPEDED THERAPY RESPONSES

Anticancer drugs are designed to eradicate tumor cells via activationof cell death machinery. Although leukemia and some solid tumors, suchas small cell lung carcinomas and seminomas, respond rather efficientlyto treatment, other solid tumors mainly of epithelial origin are therapyresistant. One possible explanation is that these tumors have a defect intheir apoptotic machinery. If so, which pro-apoptotic mechanisms aredownregulated and which anti-apoptotic mechanisms are upregulated insolid tumors? Can reintroduction of pro-apoptotic signaling componentsor alleviation of anti-apoptotic mechanisms improve anticancer treat-ment responses? Current research in apoptosis deals with these questions,


and in the following sections we discuss some of the latest achievementswithin the field.

A. Deregulated Receptor-Mediated Apoptosis

The death receptor-mediated pathway is activated in response to avariety of anticancer drugs (Debatin and Krammer, 2004). Impaired signal-ing through this pathway might result in a decreased apoptotic responseand mediate resistance to anticancer treatments. Indeed, in doxorubicin-resistant CEM cells no upregulation of the FasR after treatment wasobserved (Friesen et al., 1996). Interestingly, cells that were resistant tochemotherapeutic drugs showed a cross-resistance to anti-Fas antibody.As was mentioned above, for receptor-mediated apoptosis the presence,processing, and activation of pro-caspase-8 and -10 are essential. Loss ofpro-caspase-8 expression has been observed in small-cell lung carcinomas(SCLCs) as well as in neuroblastomas as a result of methylation of thecaspase-8 promoter (Fulda et al., 2001; Joseph et al., 1999). Treatment ofthese cell lines with 5-aza-20-deoxycytidine (5-dAzaC), which blocks methy-lation, restored expression of the caspase-8 gene and protein and sensitizedtumor cells to drug-induced apoptosis (Fulda et al., 2001).In clinical specimens of head and neck cancer, a mutated form of the

caspase-8 gene was found (Mandruzzato et al., 1997). These mutationsreduced the efficacy of caspase-8 to induce apoptosis, although it was notcompletely blocked. In human vulvae squamous carcinoma cells, a deletionwithin the caspase-8 gene resulted in expression of pro-caspase-8, whichshowed an impaired interaction with FADD (Liu et al., 2002a). Further,cells expressing this mutated caspase-8 were characterized by decreasedapoptotic potential. Thus, several human tumor cell types, includinglung carcinoma, neuroblastoma, and melanomas, may partially escapedrug-induced apoptosis through caspase-8 inactivation, and restoration ofthis pathway may increase the efficacy of anticancer treatment.FLIP is a protein that can inhibit caspase-8 activity, and an increased

expression of FLIP has been observed in HeLa cells selected for cisplatinresistance (Kamarajan et al., 2003). Furthermore, this high expressionof FLIP made cells cross-resistant to Fas signaling. Constitutively highFLIP expression abrogated the death receptor signaling in non-Hodgkin’slymphomas (Irisarri et al., 2000). Importantly, a reduction of c-FLIP expres-sion by protein synthesis inhibitors in these cells restored their sensitivityto Fas-mediated apoptosis. Overexpression of FLIP causes apoptosisresistance in different tumor cells, including multiple myeloma, coloncarcinoma, and B-chronic lymphocyte leukemia (Mitsiades et al., 2002;


Olsson et al., 2001). Although a deregulated receptor-mediated apoptoticpathway may influence the efficiency of some anticancer agents, severaldrugs as well as ionizing radiation do not depend on the death receptorsfor initiation of apoptosis. The fact that some tumors in which the deathreceptor pathway is intact are still resistant to treatment suggests thatdefects in other apoptotic pathways may also account for the lack of theirresponse to chemotherapeutic interventions.

B. Failure of the Mitochondria-Related Death Pathway

Since signaling through mitochondria is of major importance for in-duction of the intrinsic apoptotic pathway, it is not surprising that altera-tions in mitochondria of tumor cells will influence their response totreatment. A comparison of mitochondria isolated from normal and2-chlorodeoxyadenosine-resistant leukemia cells revealed in the latter a lackof Ca2+-dependent cytochrome c release and mitochondrial depolarization(Chandra et al., 2000). Some tumor cells are also characterized by a highermitochondrial potential than normal cells, which can impede their responseto chemotherapeutic agent-induced mitochondrial depolarization (Chen,1988). Moreover, overexpression of hexokinase II and peripheral benzodi-azepine receptors, proteins involved in the regulation of mitochondrialdepolarization, was observed in several tumor cells (Casellas et al., 2002;Smith, 2000). Deregulation of the Bcl-2 family proteins may also contributeto “mitochondrial resistance” of tumor cells. Thus, increased expression ofthe anti-apoptotic Bcl-2 proteins or improper function of the pro-apoptoticBcl-2 members is associated with the decreased susceptibility of manytumor cells to undergo apoptosis in response to anticancer treatments(Buchholz et al., 2003; Mandic et al., 2001b; Panaretakis et al., 2002;Viktorsson et al., 2003). The delicate balance between pro- and anti-apoptotic Bcl-2 proteins often determines whether tumor cells will respondto treatment, and an increased Bcl-2/Bax ratio is often observed in tumorcells. Nevertheless, although overexpression of Bcl-2 and/or Bcl-xL has beenreported in a number of different tumor types and correlated with inferiorclinical outcome in hematological as well as in nonhematological malig-nancies, the prognostic significance of Bcl-2 overexpression appears todepend on the tumor type (Gobe et al., 2002; Krajewska et al., 1996;Porwit-MacDonald et al., 1995). In some cases, overexpression of anti-apoptotic Bcl-2 proteins does not correlate with progression of disease(Casado et al., 2002; Gradilone et al., 2003; Stavropoulos et al., 2002).At first sight, these results seem paradoxical. However, overexpression ofBcl-2 can slow cell growth and promote cell death, and hyperphosphoryla-tion of Bcl-2 (irrespective of the level of this protein in cells) abolishes its


anti-apoptotic functions. All these data might explain why this “Bcl-2paradox” may be irrelevant to cell death (Blagosklonny, 2001).Tumor cells may escape apoptotic signaling by modulating the expression

and/or activation of the pro-apoptotic Bcl-2 family members. Indeed, a lowlevel of Bax expression has been reported in breast cancer and in hepatocel-lular carcinomas compared to the nonmalignant tissues (Bargou et al.,1996; Beerheide et al., 2000). In some colon cancer cells a somatic frameshift mutation leads to no detectable level of Bax protein (Rampino et al.,1997). Although tumor cells have decreased Bax levels, they may still beable to transmit apoptotic signals, as other pro-apoptotic Bcl-2 proteins,such as Bak, might overlap with the function of Bax (Wei et al., 2001). Asdiscussed above, an important step in the activation of Bax and Bak is achange in conformation of these proteins. Recently, a failure of Bak and Baxto undergo conformational changes was observed in a radio-resistant lungcarcinoma cell line (Viktorsson et al., 2003). However, it remains unclearhow general this observation is for treatment resistance of lung tumors.Although the role of other Bcl-2 family proteins in tumorigenesis has beenintensively investigated, the contribution of their deregulation in tumor cellsto treatment resistance requires additional studies.

C. Contribution of Deregulation of the ApoptosomeComplex to Therapy Resistance

In mitochondria-mediated apoptosis the apoptosome complex plays acentral role, and cells from Apaf-1 or pro-caspase-9 knockout mice areresistant to a variety of apoptotic stimuli, including chemotherapeuticagents such as etoposide and dexamethasone (Hakem et al., 1998; Yoshidaet al., 1998). Overexpression of Apaf-1 in leukemia cells increased theirsensitivity to paclitaxel and etoposide (Perkins et al., 1998). A complete lossof Apaf-1 expression has been reported in many melanoma cells as well as inmelanoma clinical specimens as a result of deletions of one allele encodingApaf-1 and methylation-caused silencing of the other (Soengas et al., 1999).All these samples were characterized by a decreased apoptotic response tothe chemotherapeutic agent adriamycin. The expression and activity ofApaf-1 were restored in these cells after treatment with the methylationinhibitor 5-dAzaC, and such treatment as well as Apaf-1 gene transfersensitized melanomas to chemotherapy-induced apoptosis (Soengas et al.,1999). Thus, a deregulated Apaf-1 activity may contribute to therapyresistance to different chemotherapeutic drugs.Several recent observations indicate that deregulation of pro-caspase-9

can also contribute to treatment resistance. Thus, low amounts of pro-caspase-9 were associated with Apaf-1 in ovarian cancer cell lines,


which influenced proper apoptosome function and resulted in impairedcisplatin-induced apoptotic signaling (Liu et al., 2002b).In some cells pro-caspase-9 is co-expressedwith its truncated form, caspase-

9S. The latter binds to Apaf-1 and acts as a dominant negative protein,inhibiting pro-caspase-9 activation (Seol and Billiar, 1999; Srinivasulaet al., 1999). Expression of caspase-9S effectively inhibits apoptosis inducedby various stimuli (Seol and Billiar, 1999). Increased expression of caspase-9Swas detected in several lung and gastric tumor cell lines (Izawa et al., 1999;Srinivasula et al., 1999). In gastric cancer cell lines, increased expression ofcaspase-9S correlated with resistance to apoptosis induction (Izawa et al.,1999). Thus, it is evident that deregulation of the apoptosome function intumors can render them resistant to different chemotherapy treatments.

D. Defective Execution of Apoptosis in Cancer Cells

The activity of execution caspases is controlled by IAP family proteins, andhigh IAP expression has been linked with worse clinical prognosis. However,in a comprehensive analysis of clinical material from patients with cervicalcarcinoma, no correlation was observed between the levels of cIAP-1 orXIAP and proliferation, apoptotic index, stage of disease, or survival of thepatients (Liu et al., 2001). Another study, in which the expression levels ofcIAP-1, cIAP-2, or XIAP and the sensitivity to chemotherapy was assessed ina clinical samples of advancedNSCLC, also turned out empty (Ferreira et al.,2001). Similar results were observed in a panel of SCLC andNSCLC cell lines(Ekedahl et al., 2002). Although low XIAP levels in acute myeloid leukemia(AML) cell lines or in a clinical material from AML patients correlated withincreased chemosensitivity (Carter et al., 2003b; Tamm et al., 2000), oppo-site results were observed in completely resected NSCLC tumors, in whichhigh XIAP levels correlated with increased survival (Ferreira et al., 2001). Inmultidrug-resistant human HL60 leukemia cells, an increased expression ofcIAP1, cIAP2, XIAP, and survivin on both the mRNA and the protein levelwas observed, suggesting that upregulation of IAPs may be associated withacquired resistance (Notarbartolo et al., 2004). Examination of a panel offive pancreatic carcinoma cell lines showed that elevated levels of cIAP-2,XIAP, and survivin correlated with their resistance to TRAIL or FasL treat-ment (Trauzold et al., 2003). Importantly, transfection of XIAP into the mostTRAIL/FasL-responsive pancreatic cell lines decreased their sensitivity tothese agents, suggesting that increased IAP expression, at least in pancreaticcells, may confer resistance to apoptosis. Immunohistochemical analysis ofhuman prostate cancer tissue revealed an increased expression of cIAP-1,cIAP-2, XIAP, and survivin compared to the normal prostate epithelium. Asan increased IAP level was observed in prostate carcinoma in situ it was


proposed that deregulation of IAP might be an early event in prostate cancerdevelopment (Krajewska et al., 2003). However, no correlation betweenIAP level and anticancer therapy responses was seen in this study. In leuke-mia cell lines as well as in AML primary cells XIAP antisense treatmentresulted in an increased apoptotic response to Ara-C (Carter et al., 2003b).Nevertheless, in samples obtained from AML patients expression of survi-vin or XIAP was not related to remission or overall survival of these patients(Carter et al., 2003a). From these studies, one can conclude that although acorrelation between IAP expression and chemosensitivity and/or survivalcan be observed in some tumor types, this is not always the case. Further, thefact that in XIAP knockout mice that developed normally, tissues werecharacterized by an increased expression of cIAP-1 and cIAP-2 arguesin favor of possible functional IAP substitutions (Harlin et al., 2001). Itwould indeed be interesting to know whether these mice show an increasedresponse to chemotherapy or radiation.Heat shock proteins that can interfere with apoptotic signaling at several

levels (see Section III.C.1) have also been reported to be overexpressedin several tumor types of different origin, such as breast, uterine, renal,endometrial cancer, osteosarcoma, and leukemia (Jaattela, 1999). In someof these tumors, high expression of HSPs has been correlated to inferiorprognosis. Antisense expression of HSP70 resulted in massive cell death,suggesting that this protein is important for tumor cell survival (Nylandstedet al., 2000). Several attempts were undertaken to understand whethera sublethal level of HSP70 antisense could sensitize tumors to chemo-therapy. Using a siRNA approach, downregulation of HSP72 in NSCLCresulted in increased basal apoptosis but did not potentiate the effect ofchemo- or radiation-induced death in these cells (Ekedahl et al., 2003).By contrast, antisense HSP70 was able to sensitize human colon cancer cellsto curcumin-induced apoptosis (Rashmi et al., 2004). It is likely that tumorsof various origin may have different requirements for HSPs for theirsurvival and therefore respond in different ways to concomitant HSPdownregulation and anticancer treatment.In addition to the inhibitory effect of IAPs andHSPs on activity of executor

caspases, a decreased expression of the executor caspases or a complete lossof their expression might interfere with the apoptotic process, resulting inresistance to treatment. Indeed, immunohistochemical analysis of prostatetumors of various grades revealed that moderately and poorly differentiatedprostate tumors had less expression of caspase-3 protein compared to well-differentiated prostate adenocarcinomas or normal prostate (Winter et al.,2001). However, it is unclear if this decreased expression has prognosticvalue in either disease progression or tumor responsiveness to treatment.It was shown that in the absence of caspase-3, apoptosis in the

breast cancer cell line MCF-7 proceeds through sequential activation of


caspase-6 and -7 (Liang et al., 2001), suggesting the functional redundancyof executor caspases.In some tumors, the presence of processed or active caspase-3 does not

result in any substrate cleavage as a consequence of cytosolic aggregation ofthe activated caspase-3 (Kottke et al., 2002). However, in other tumortypes, the apoptotic signaling induced by anticancer treatments was blockeddownstream of caspase-3 substrate cleavage (DeChant et al., 2002; Jaattela,1999). In this case increased expression of HSP70 was responsible forsuppression of tumor cell death in which caspase-3-mediated cleavage ofPARP was observed (Jaattela, 1999). Raf-mediated survival signaling couldalso abrogate apoptosis signaling downstream of pro-caspase-3 activation(DeChant et al., 2002). In addition, a defect in caspase-3 relocalization tothe nucleus of NSCLC cells in response to radiotherapy, which contributedto their radio-resistance, was reported (Joseph et al., 2001). It seems thattumor cells might be characterized by the presence of a variety of factorsinfluencing activation and/or activity of execution caspases, which mightlead to resistance of these tumors to treatment.

E. Deregulation of Kinase Signaling

Different tumor types are characterized by an increased Akt activity, as aresult of deregulation of the receptor tyrosine kinase itself and/or increasedRas signaling (Jimenez et al., 1998; Shayesteh et al., 1999; Vivanco andSawyers, 2002). Fibroblasts that overexpress Akt are resistant to stauros-porine and to etoposide-induced apoptosis (Tang et al., 2001). In lungcancer cells, topotecan treatment caused dephosphorylation of Akt, fol-lowed by appearance of an increased number of apoptotic cells. Conse-quently, transfection of a constitutively active Akt mutant reduced theirapoptotic response to topotecan (Nakashio et al., 2000).Increased Akt activitymay also result as a consequence of inactivation of the

tumor suppressor phosphatase and a tensin homolog deleted from chromo-some 10 (PTEN), which encodes a phosphatase that normally inactivates Akt(Di Cristofano and Pandolfi, 2000). Such inactivation has been reported in anumber of tumors, including melanomas, glioblastomas, and prostate, endo-metrial, and breast cancers (Wu et al., 2003). In line with these observations,tumors from PTEN knockout mice were able to escape apoptosis upon treat-ment with different chemotherapy drugs (Wu et al., 2003).Activation of JNK and p38 was described in a number of cell systems after

treatmentwith chemotherapy drugs, suggesting their importance for apoptoticsignaling (Fan and Chambers, 2001). However, increased survival signalingthrough Akt can inhibit cisplatin-induced JNK/p38 and Bax activation bysuppressing the upstream kinase ASK-1 (Yuan et al., 2003). An impairedactivation of JNK and p38 was observed in a radiation-resistant NSCLC,


although the precise mechanism and significance of this finding remain to beunderstood (Viktorsson et al., 2003). The levels of p38 and its upstreamregulator MKK6 were significantly reduced in specimens obtained from 20patients with liver cancer as compared with samples from adjacent non-neoplastic live tissues. Although reintroduction of active MKK6 in HepG2and HuH7 human hepatoma cell lines resulted in apoptosis, it is still un-clear whether downregulation of MKK6 and p38 is related to the resistanceof hepatocellular carcinoma specimens to treatment and if this impedeschemotherapy-induced apoptotic signaling (Iyoda et al., 2003).An increased ERK activity may also contribute to decreased apo-

ptosis in response to chemotherapy, and pharmacological inhibition of theRaf/MEK/ERK pathway enhances tumor response to a variety of chemo-therapeutic drugs and ionizing radiation (Belka et al., 2000; Hayakawaet al., 1999; Mandic et al., 2001a). Thus, there are several indica-tions supporting the hypothesis that an increased activity of pro-survivalregulating protein kinases and/or a concomitant decrease of pro-apoptoticsignaling kinases within tumors might impair their response to therapy.

V. REACTIVATION OF APOPTOTIC SIGNALING AS ANAPPROACH IN ANTICANCER THERAPY

It is evident that a defect in apoptotic signaling is a hallmark for most ifnot all tumor cells of solid origin (Hanahan and Weinberg, 2000). Themajority of currently available anticancer drugs act at least in part throughinduction of apoptosis; therefore, a defect in the apoptotic propensity of thetumors should affect their response to treatment. Various tumor cells areresistant to apoptosis although they are not completely devoid of death.Since many components of cell death machinery are still present in tumorcells, these tumors might be sensitized to chemo- and/or radiation therapyby modulation of death signaling pathways. At present, a number of anti-cancer therapies are being designed with the aim of adjusting the expressionand/or activity of factors that regulate apoptosis. Some of these drugs haveeven reached the stage of clinical trials (Table II).

A. Modulation of IAP Expression and Smac-BasedTherapies in Anticancer Treatment

As mentioned previously, tumor tissues are often characterized by anincreased expression of IAPs. Therefore, reducing of the level of IAPs byusing either the antisense technique or the more recently developed siRNAapproach might lead to successful tumor treatment responses. Indeed, recent

Table II Apoptosis-Based Anticancer Drugs in Development or in Clinical Trials

Drug Company Target Technology Indications Status

Apoptosis machinery targets

GX01 Gemin X Biotechnologies Bcl-2 Small molecule Solid tumors PreclinicalHA14-1 Bcl-2 Small molecule Preclinical

Antimycin Bcl-2 Small molecule Preclinical

INGN201 Introgen Therapeutics TP53 Adenovirus Head and neck, ovarian,non-small cell lung

cancer and other

advanced solid tumors

Phase III

SCH58500 Schering-Plough TP53 Adenovirus Advanced solid tumors Phase IIIONYX-015 Onyx Pharmaceuticals TP53 Mutant adenovirus Head and neck, colorectal,

lung, and pancreatic

cancers

Phase III

PRIMA TP53 Small molecule PreclinicalTRAIL Genetech/Immunex DR4, DR5 Recombinant protein Solid tumors Preclinical

Smac Smac Peptide Preclinical

PS-341 Millenium Proteasomeinhibitor

Peptide Hematological tumors Phase II

Pharmaceuticals

E1A-Lipid complex Targeted Genetics E1A Liposomal-encapsulated

plasmid DNA

Ovarian, head and neck

cancers, and peritonealcarcinomatosis

Phase I

Exusulind Cell pathways PDE5A/PKG Small molecule (NSAID) Solid tumors Phase II

172

SAHA Aton Pharma HDAC Small molecule Solid tumors Phase I

EMD121974 E-Merck �5�3-integrin Cyclic pentapeptide Angiogenesis (cancer) Phase IIIEndostatin EntreMed EC Recombinant protein Angiogenesis (cancer) Phase III

17-AAG NCI Hsp90 Small molecule Advanced solid tumors Phase I

Inhibitors of growth factors that kill cells via apoptosis

CDDO National Cancer Institute PPAR�, IKK

and others

Small molecule Solid tumors Phase I

UCN-01 National Cancer Institute PKC STS analog Solid tumors Phase I

ISIS 3521 ISIS Pharmaceutical Inc. PKC Antisense

oligonucleotide

Solid tumors Phase II

ISIS 5132 ISIS Pharmaceutical Inc. Raf-1 Antisense

oligonucleotide

Solid tumors Phase II

ISIS 2503 ISIS Pharmaceutical Inc. Ras Antisense

oligonucleotide

Variety of tumors Phase II

Trastuzumab Genetech Her2/neu Monoclonal antibody Breast cancer Launched as

Herceptin

Cetuximab Merk EGFR Monoclonal antibody Breast cancer, othersolid tumors

Phase III

ZD 1830 Astra Zeneca EGFR Small molecule Breast cancer Launched as

Iressa

Non-small cell lungcancer

Imatinib Novartis Oncology Abl Small molecule Gastrointestinal

stromal tumors,

chronic myeloidleukemia

Launched as

Gleevec

173


experimental studies with ovarian cancer cell lines showed that antisense-mediated downregulation of XIAP resulted in increased sensitivity to cisplatin(Holcik et al., 2000). Using the same approach, an increased cell death inNSCLC cells after radiation was observed (Li et al., 2001a). Although thesepreclinical data are promising, it is still uncertain if this approach can beapplied in the clinic as a method of increasing anticancer therapy responses.Another strategy for influencing the anti-apoptotic effects of IAPs is to

increase the cytosolic level of Smac/DIABLO, which might interact with IAPs.Several attempts to develop cell-permeable N-terminal peptides of differentsizes derived from the Smac sequence were undertaken (Arnt et al., 2002;Fulda et al., 2002; Guo et al., 2002a; Yang et al., 2003). Coadministration ofthese Smac peptides with etoposide, doxorubicin, and TRAIL resulted inalleviation of IAP effects as well as an increased apoptotic response. Thiswas observed in several tumor cell lines, including breast, neuroblastoma,melanoma, and NSCLC as well as in a malignant glioma xenograft model invivo (Arnt et al., 2002; Fulda et al., 2002;Guo et al., 2002a; Yang et al., 2003).Like other peptides, Smac peptides might be difficult to use in clinical settings.Therefore, additional work is required to develop them into efficient drugs.

B. Ongoing Clinical Trials with Heat ShockProtein Inhibitors

Since HSPs can interfere with apoptotic signaling in a multitude of ways(for details see Section III.C.1), they have also emerged as favorable targetsfor anticancer therapies (Sreedhar and Csermely, 2004). So far, only mole-cules that target HSP90 have reached clinical trials. The first developeddrug, Geldanamycine, had a clear antitumor effect. However, when testedin animal models it caused high hepatotoxicity (Supko et al., 1995).The geldanamycin analogue, 17-AAG, is less toxic. In preclinical models17-AAG was able to increase the therapeutic response to doxorubicin andto paclitaxel (Munster et al., 2001; Sausville, 2001). Although 17-AAG hasentered phase I clinical trials, it remains to be shown if targeting HSPs is aclinically valuable strategy to circumvent therapy resistance.

C. Strategies for Targeting the Bcl-2 Family Proteins inPreclinical and Clinical Models

Overexpression of Bcl-2 and/or Bcl-xL or loss of Bak and/or Bax functionhas been linked to acquired resistance of tumors to radiation and/or chemo-therapy. Several strategies either to reactivate pro-apoptotic Bcl-2 members orto suppress the anti-apoptotic ones have been developed with the aim ofincreasing anticancer treatment responses. However, only the Bcl-2 antisense


approach has so far developed into a clinical regime. The G3139, an 18-merphosphorothioate oligonucleotide, also known as Oblimersen or Genasense,was able to decrease Bcl-2 levels and increase the effect of doxorubicin inpreclinical studies of breast cancer cell lines (Chi et al., 2000). Further, coad-ministration of G3139 with chemotherapy drugs increased therapy responsesof cells derived from patients with lymphoma and multiple myeoloma (van deDonk et al., 2003). As a first clinical trial, G3139 was given to nine patientswith relapsing Bcl-2-positive non-Hodgkin’s lymphoma (Webb et al., 1997).The antitumor effect of G3139 was limited since a downregulation of Bcl-2was observed in only 50% of the patients (Webb et al., 1997). Because G3139had low cytotoxicity, clinical trials with G3139 are ongoing either as a singletreatment regime or in combination with chemotherapy (Klasa et al., 2002).Attempts to target Bcl-xL by a similar approach revealed a capacity for

growth inhibition of melanoma and colon cancer cells and high sensitiza-tion to cisplatin and ionizing radiation, respectively (Heere-Ress et al.,2002; Olie et al., 2002; Strasberg Rieber et al., 2001).An additional way to inactivate Bcl-2/Bcl-xL in tumor cells is to use

peptides resembling the BH3-domain of pro-apoptotic members, e.g., Bax.Such peptides should bind to Bcl-2/Bcl-xL and neutralize their anti-apoptoticeffect (Wang et al., 2000b). Indeed, treatment of human prostate carci-noma cells with these peptides resulted in a decreased formation of Bak-Bcl-2 oligomers and massive apoptosis. However, as was mentionedabove, there are difficulties in delivering peptides into patients. Therefore,screening of a large number of small molecular weight compounds thatcould bind to Bcl-2 was performed. As a result, two drugs, HA14-1 andantimycin A that triggered apoptosis in Bcl-2 overexpressing cells weregenerated (Kirkin et al., 2004; Wang et al., 2000a).The third approach is to increase the amount of pro-apoptotic Bcl-2 mem-

bers within cells, thereby increasing the apoptotic potential. Adenoviraladministration of either Bak and/or Bax to several tumor cell lines includingprostate, ovarian, and mesothelioma caused a reduced viability of these cells(Arafat et al., 2000; Lowe et al., 2001; Pataer et al., 2001). In ovariancancer cells, treatment with Bax and radiation showed a significantly bettereffect than either treatment alone (Arafat et al., 2000). Thus, althoughintroduction of pro-apoptotic Bcl-2 members is a promising approach forsensitizing tumors to conventional therapy, there is a long way to go beforeit can be used in clinical trials.

D. p53 Reactivation

Mutations and thereby inactivation of p53, which are observed in about50% of all human tumors, significantly impair therapy responses. There-fore, several approaches to restoring the p53 function have been developed.


One approach is the p53 replacement therapy, in which wild-type p53(wt-p53) is reintroduced into tumors either having mutant p53 or lack-ing this protein. In preclinical studies the reintroduction of wt-p53 intothese tumors resulted in apoptosis (Yonish-Rouach et al., 1991). Further,injection of wt-p53 into animal xenograft models of different tumororigin, including NSCLC, leukemia, glioblastoma, head and neck, breast,liver, ovarian, colon, and kidney cancers, resulted in increased cell death(Lebedeva et al., 2003). This effect was potentiated after co-treatment withconventional chemotherapeutic agents, including cisplatin, 5-FU, metho-trexate, and etoposide (Nguyen et al., 1996; Nielsen and Maneval, 1998).These promising preclinical results paved the way for p53 replacementregimes using adenoviral constructs in the clinic (reviewed in Lebedevaet al., 2003). Multiple clinical trials in which wt-p53 gene transfer is usedin combination with chemo- and/or radiation therapy in advanced cancerpatients are in progress. In several of these trials, reintroduction of wt-p53resulted in stabilization of disease, partial remission, or increased patientsurvival.Another therapeutic strategy is based on the ability of adenovirus to

preferentially target cells with mutant p53. ONYX-015 contains a geneti-cally modified adenoviral E1B gene in which a part, responsible for sup-pression of the p53-mediated apoptotic response, is deleted. In wt-p53 cellswith an intact p14ARF-signaling pathway, introduction of ONYX-015causes apoptosis and thus suppresses viral replication. By contrast, in cellsexpressing mutant p53 or having a block in p14ARF signaling, ONYX-015replicates and induces cell death. Preclinical human tumor xenograft studiesdemonstrated significant response to injection of ONYX-015 into tumorswith mutated p53 (Heise et al., 1997). More pronounced effects wereobtained when ONYX-015 was used in combination with 5-FU, cisplatin,or ionizing radiation (Heise et al., 1997; Rogulski et al., 2000). Ongoingclinical trials using ONYX-015 include several tumor types, such as headand neck, liver, ovarian, colorectal, and pancreatic cancers (Lebedeva et al.,2003). Preliminary results indicate that ONYX-015 per se is ineffective;however, the combination of ONYX-015 with cisplatin or 5-FU induces asignificant antitumor effect (Khuri et al., 2000).Several pharmacological activators were recently used for reactivating

p53. Peptide 46, which corresponds to residues 361–382 in human p53,was found to induce apoptosis in tumor cells with mutant p53. This effectwas mediated by restoration of proper core domain conformation and/orp53 DNA-binding properties (Selivanova et al., 1999). A smaller nine-residue peptide, CDB3, which binds the core domain of mutated p53, alsostabilizes p53 through a chaperone-like mechanism, resulting in an activeconformation of p53 (Bykov et al., 2003). Although results obtained with


these peptides using tumor cells are promising, delivery problems mightimpede their usefulness in clinical settings. Therefore, screening of chemicallibraries for small chemical compounds, which could reactivate mutant p53,has been performed. In such screenings, several compounds with the abilityto restore p53 function were found (reviewed in Bykov et al., 2003). One ofthese compounds is PRIMA-1, which was able to suppress growth of Saos-2cells expressing mutant p53 but did not have any effect on Saos-2 cellsexpressing wt-p53. PRIMA-1 stimulates DNA binding of several mutantp53 forms, activates several p53 target genes, including PUMA, and inducesapoptosis in cells expressing mutant p53 (Bykov et al., 2002). None ofthese events were observed in cells with wt-p53. In vivo studies revealedthat PRIMA-1 induces an antitumor effect in a human xenograft of Saos-2p53 mutant cells in mice without any toxic consequences (Bykov et al.,2002).Using a model system employing a p53 protein fused to a mutant steroid

binding domain of the murine estrogen receptor, it was found that PRIMA-1induces p53-mediated transcription-independent apoptosis (Chipuk et al.,2003). Pharmacologically active p53 activates Bax, allowing for mitochon-drial membrane permeabilization and cytochrome c release, followed bycaspase activation and apoptosis. In parallel, p53 can release pro-apoptoticBcl-2 proteins sequestered by Bcl-XL. Ongoing studies with PRIMA-1 and/or its analogues aim to characterize the specificity of PRIMA in in vivomodels as well as its usefulness in combinations with conventional chemoand/or radiotherapy.As emphasized above, in many cells a block in apoptotic signaling appears

downstream of p53. Despite the promising results obtained with thep53-replacement strategy, it will not be applicable to all tumors. Given therecent report of the p53 transcription-independent effect, it is also unclear ifreactivation of p53 will restore tumor response to treatment.

E. Modulation of Protein Kinase Signaling

Receptor tyrosine kinases (RTK) are of major importance for the trig-gering of several protein kinase-signaling cascades, including the Raf/MEK/ERK and PI3K/AKT pathways, both implicated in apoptosis. Therefore,blocking of RTK signaling might be a good strategy in increasing theefficiency of anticancer therapy. Different approaches have been usedfor targeting the EGFR-signaling pathway. In one of them, monoclonalantibodies (Cetuximab) against the extracellular ligand-binding domainof the receptor, which compete with EGF for receptor binding, are used.In the other, small molecules (ZD 1839 or Iressa), that act as competitive


inhibitors for ATP binding at the intracellular receptor-associated tyrosinekinase are applied. Preclinical studies revealed that Iressa not only inhibitsproliferation, but also induces apoptosis in tumor cells (Gilmore et al.,2002). Treatment of breast cancer cells with Iressa resulted in decreasedphosphorylation of the pro-apoptotic Bcl-2 family member Bad via aMAPK-signaling mechanism (Gilmore et al., 2002). Iressa significantly in-creases the therapeutic efficacy of platinum-based drugs and taxanes. Incombination with cisplatin or 5-FU in head and neck cancer cell lines,Iressa-mediated apoptosis involves Bax expression, caspase-3 activation,and a decreased Akt activity compared to either treatment alone (Magneet al., 2003).Another compound, Imatinib (STI571 or Gleevec), which caused apopto-

sis via inhibition of kinase signaling, was also suggested for clinical use. Thiscompound was designed as a BCR-Abl kinase inhibitor. Long ago it wasshown that BCR-Abl-expressing cells are characterized by high activity ofthe PI3K/Akt pathway, display increased Bcl-xL expression, and as a failureof mitochondria-mediated apoptosis are refractory to DNA-damagingdrugs (Amarante-Mendes et al., 1998; Dubrez et al., 1998; Shuai et al.,1996; Skorski et al., 1997). Treatment of these cells with Imatinib resultedin accelerated apoptosis (Dan et al., 1998; Fang et al., 2000), suggestingthat inhibition of BCR-Abl could revert resistance to apoptosis. Imatinibcan potentiate apoptotic effects induced by ara-C and doxorubicin (Fanget al., 2000), an effect that is characterized by cytosolic accumulation ofcytochrome c, increase in caspase-3 activity, and cleavage of PARP, allhallmarks of the mitochondria-mediated pathway. Nowadays Imatinib issuccessfully used in the treatment of patients with CML (chronic myeloicleukemia) and causes complete remission (Druker, 2001). Thus, anticancertherapy strategy based on blocking of protein kinase signaling has emergedas an effective method either alone or in combination with conventionalchemotherapy. However, a search for better and less toxic compounds isrequired to improve therapeutic potential.

F. Activation of Death Receptor Pathways

Several attempts to use activation of receptor-mediated pathways incancer therapy have been made. Long ago it was observed that TNF-� killsa number of tumor cells; however, a side effect, characterized by septicshock-like syndrome, significantly impedes the use of this ligand in the clinic.In a number of different cell lines, addition of recombinant TNF-�-relatedapoptosis inducing ligand TRAIL, which binds to TRAIL-R1 (DR4) andTRAIL-R2 (DR5/Killer), has been shown to cause apoptosis (Ashkenazi,


2002). In addition, in patient-derived multiple myeloma (MM) cells, recom-binant TRAIL induced apoptosis irrespective of their sensitivity to othertypes of chemotherapy (Mitsiades et al., 2001). Further, in a xenograft modelof these MM cells, TRAIL caused a significant antitumor effect and was welltolerated, implicating that it might be used systemically (Mitsiades et al.,2001). In the same way, patient-derived colon tumors were inoculated inSCID mice and TRAIL was applied either alone or in combination withchemotherapy (Naka et al., 2002). Histological analysis of the tumors trea-ted with a combination of TRAIL and 5-FU or irinotecan showed a markeddecrease in number of tumor cells and an increase in fibrotic areas (Nakaet al., 2002). In leukemia cells, a synergistic effect between TRAIL and low-dose radiation has also been reported (Gong and Almasan, 2000). Althoughrecombinant TRAIL for clinical use has been developed and tested in animalmodels, its therapeutic potential in humans awaits further research.

VI. CONCLUDING REMARKS

In 1972, Kerr, Wyllie, and Currie introduced the term apoptosis todescribe a distinct form of cell death with wide-ranging implications intissue kinetics (Kerr et al., 1972). The authors suggested that hyperplasiamight result from decreased apoptosis rather than increased mitosis. More-over, they implied apoptosis not only in tumor progression but also inspontaneous elimination of potentially malignant cells and therapeuticallyinduced tumor regression. Increasing evidence indicates that resistance ofmany types of cancer to treatment may be associated with defects in, ordysregulation of, different steps of the apoptotic pathway. Usingthis knowledge, many attempts have been made to design new drugs thatmay either activate or re-activate the apoptotic machinery in tumor cells.Several genes and proteins involved in the inhibition of apoptosis have alsobeen shown to play a role in tumorigenesis and vice versa. Thus, the down-regulation of anti-apoptotic genes and upregulation of pro-apoptoticgenes (proteins) were successfully used in the treatment of several tumors.However, cell death pathways in tumor cells appear to be much morecomplicated than was originally anticipated. Components of the apoptoticmachinery do not operate in isolation, and the activation of just one ofthe apoptotic pathways may not be sufficient to kill all types of tumorcells. Moreover, several recent reports suggest the existence of, in addi-tion to classical apoptosis and necrosis, a mixture of these two forms of celldeath. Among these are caspase-dependent and -independent death, cas-pase-independent programmed cell death, apoptosis-like and necrosis-like


programmed cell death, paraptosis, anoikis, onkosis, autophagy, and manyother examples of cell death. Depending on cell type and death trigger, thecell death process may proceed via activation of different pathways. Sup-porting this theory is accumulating evidence showing that the induction of,for example, caspase-dependent and -independent pathways might be moreeffective at killing cancer cells than either pathway alone. Interestingly, anaccurate regulation of caspase activity might result in either apoptosis orautophagic death, both of which are potentially important for the removalof tumor cells (Yu et al., 2004). Another example is TNF-�, which mightkill some cancer cells directly via apoptosis or indirectly via ligation of TNF-R on tumor endothelial cells to cause constriction of cancer cells bloodsupply, resulting in necrosis. Together with individual tumor typing andmatching with type-specific treatments, effective anticancer treatment strat-egy might require combinatorial approaches. Indeed, using genetically de-fined cells it was recently shown that DNA-alkylating agents cause necroticcell death, which is equally effective in cells with and without apoptoticdefects and does not require p53 or Bax and Bak (Thompson et al., 2004).Since many solid tumors and their metastases are heterogeneous at thecellular level, it is likely that combinatorial approaches using some agentsthat activate apoptosis and some agents that activate necrosis or other typesof death might be very useful. Thus, the understanding of the exact route bywhich all known, or yet unknown, death pathways in tumor cells operateand how these pathways might be destroyed to cure cancer is very impor-tant. Although such studies are still in progress, they offer great promise forfuture cancer therapy.

ACKNOWLEDGMENTS

Work in the authors’ laboratory is supported by grants from the Swedish and Stockholm

Cancer Societies, The Swedish Research Council, Swedish Heart and Lung Foundation, and theEuropean Commission (QLK3-CT-2002-01956). We wish to express our gratitude to Professor

Sten Orrenius for permanent support. We apologize to authors whose primary references could

not be cited due to space limitations.

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