Research Article 4-Methoxycarbonyl Curcumin: A Unique...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 329740 10 pageshttpdxdoiorg1011552013329740

Research Article4-Methoxycarbonyl Curcumin A Unique Inhibitor of BothInflammatory Mediators and Periodontal Inflammation

Ying Gu1 Hsi-Ming Lee2 Nicole Napolitano1 McKenzie Clemens1 Yazhou Zhang2

Timo Sorsa3 Yu Zhang4 Francis Johnson45 and Lorne M Golub2

1 Department of General Dentistry School of Dental Medicine Stony Brook University Stony Brook NY 11794-8706 USA2Department of Oral Biology and Pathology School of Dental Medicine Stony Brook University Stony Brook NY 11794 USA3Department of Oral and Maxillofacial Diseases Institute of Dentistry Helsinki University Central HospitalUniversity of Helsinki Helsinki Finland

4Department of Chemistry Stony Brook University Stony Brook NY 11794-3400 USA5Department of Pharmacological Sciences Stony Brook University Stony Brook NY 11794 USA

Correspondence should be addressed to Ying Gu yinggustonybrookedu

Received 22 August 2013 Revised 29 October 2013 Accepted 30 October 2013

Academic Editor Freek Zijlstra

Copyright copy 2013 Ying Gu et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Chronic inflammatory diseases such as periodontitis have been associated with increased risk for various medical conditionsincluding diabetes and cardiovascular disease Endotoxin (lipopolysaccharide LPS) derived from gram-negative periodonto-pathogens can induce the local accumulation of mononuclear cells in the inflammatory lesion increasing proinflammatorycytokines and matrix metalloproteinases (MMPs) This ultimately results in the destruction of periodontal connective tissuesincluding alveolar bone Curcumin is the principal dyestuff in the popular Indian spice turmeric and has significant regulatoryeffects on inflammatory mediators but is characterized by poor solubility and low bioactivity Recently we developed a series ofchemically modified curcumins (CMCs) with increased solubility and zinc-binding activity while retaining or further enhancingtheir therapeutic effects In the current study we demonstrate that a novel CMC (CMC 25 4-methoxycarbonyl curcumin) hassignificant inhibitory effects better than the parent compound curcumin on proinflammatory cytokines and MMPs in in vitroin cell culture and in an animal model of periodontal inflammation The therapeutic potential of CMC 25 and its congeners mayhelp to prevent tissue damage during various chronic inflammatory diseases including periodontitis and may reduce the risks ofsystemic diseases associated with this local disorder

1 Introduction

Periodontal disease is one of the most common chronicinflammatory diseases encountered in humans During thepathogenesis of this condition anaerobic gram-negativeperiodontal-associated pathogens (eg P gingivalis Tforsythia) and the lipopolysaccharide (LPS endotoxin) intheir cell walls stimulate the innate and adaptive immuneresponses in periodontal tissues [1] Inflammatory cells suchas neutrophils and monocytesmacrophages are recruited tothe lesion site and generate elevated levels of cytokines andother proinflammatorymediators such as the prostaglandinsThe resulting periodontal inflammation upregulates matrixmetalloproteinase (MMP) expression and the activity of the

latter contributes to the destruction and loss of periodontalconnective tissues including bone [2]

Curcumin [17-bis-(4-hydroxy-3-methoxyphenyl)-16-heptadiene-35-dione] is a component of the popular Indianspice turmeric and has been recommended for numerousmedical applications [3] Extensive investigations have ledto the conclusion that it is a highly pleiotropic moleculewith significant beneficial effects on inflammatory and otherdiseases including cancers such as multiple myeloma [3ndash5]This natural product has long been used as an herbal anti-inflammatory treatment to relieve pain and inflammationin the skin and muscles and for a variety of pulmonarygastrointestinal and liver diseases as well as a remedy fornonhealing wounds These therapeutic effects have been

2 Mediators of Inflammation

studied in both in vitro and in vivo model systems [6ndash8]Despite these many beneficial effects curcumin has majorlimitations including poor solubility a lack of systemicbioavailability and rapid metabolic disposition [9] Thusextremely high oral doses of the compound are neededand even then it results in only very low levels in thesystemic circulation of both animals and humans Thishas severely limited its clinical application [10] Recentlyour laboratory has developed a series of novel chemicallymodified curcumins with a carbonyl substituent at theC-4 position [11 12] Such analogues have an additionalelectron-withdrawing group which enhances their anti-inflammatory therapeutic effects One such compound(CMC 25) contains a methoxycarbonyl group at C4 showsan improved solubility better serum albumin-bindingactivity and greater acidity and enhanced zinc-bindingcharacteristics This modification has been found to enhancethe MMP-inhibitory properties of this novel compoundversus curcumin [11 12]

In the current report we investigate the effect of thisnovel substance 4-methoxycarbonylcurcumin (CMC 25)on proinflammatory cytokines and MMPs in an in vivodiabetes-enhanced periodontal inflammation rat model andin a relevant cell culture model Rats with experimentaldiabetes mellitus manifest increased gingival inflammationand periodontal tissue destruction including alveolar boneloss [13ndash16] This animal model of STZ-induced diabetes asan enhancer of periodontal disease is well established inour laboratory [14 15] and has been described by othersas well [16] It was used in preclinical studies during thedevelopment of Periostat the only host modulation andMMP inhibitory therapy for periodontitis approved by theFDA This unique animal model different from traditionalrat models of experimental periodontitis using ligatures ororal pathogen infection will allow us to study the possibleassociation between this local inflammatory disease andrelevant systemic conditions We have previously demon-strated that the diabetic condition increases the levels ofcytokines and MMPs locally in the gingival tissues as wellas systemically in plasma [14 15] In addition levels ofMMP-8 in skin and both local and systemic bone loss wereincreased in this animal model [17] Therefore this willallow us to study not only the periodontal disease but alsothe systemic factors associated with this local inflammatorycondition

In addition a periodontal disease-relevant cell culturesystem involving human mononuclear cells challenged withLPS (derived from the periodontal pathogen P gingivalis)was also used to evaluate the effect of CMC25 on the inducedexcessive levels of proinflammatory MMPs and cytokinesNuclear factor-kappaB (NF-120581B) is a transcription factorinvolved in the cell signaling transduction pathway associatedwith inflammation and plays a key role in regulating the cellu-lar immune response to stimuli such as stress cytokines andbacterial or viral antigens [18] Dysregulation of NF-120581B hasbeen linked to cancer and inflammatory diseases [18] There-fore the effect of CMC 25 on the activationphosphorylationof NF-120581B was evaluated as well

2 Materials and Methods

21 Chemical Reagents All chemical reagents LPS from Pgingivalis and curcumin were purchased fromSigma-AldrichCo (St Louis MO) All cell culture reagents were purchasedfrom GibcoInvitrogen Corp (Carlsbad CA) CMC 25 wassynthesized purified (995 pure) and provided by ChemMaster Intl Inc Stony Brook NY

22 InVitroMMP InhibitionAssay (IC50) Human chromato-

graphically pure MMP-9 was purchased from CalbiochemEMD Biosciences Inc (La Jolla CA) MMP-13 was pur-chased from RampD Systems Inc (Minneapolis MN) and thesynthetic octapeptide MMP substrate (DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-dArg) was purchased from Bachem (Kingof Prussia PA) Curcumin and CMC 25 (1ndash500 120583M) wereincubated in 1mM CaCl

2 02M NaCl and 50mM TrisHCl

buffer (pH = 76) with MMP-9 (gelatinase B) or MMP-13 (collagenase-3) at 37∘C for 4 hours as described by uspreviously [11] The reaction mixture was quenched with110-phenanthroline (a zinc chelator that binds this cationin the MMP molecule) and the tripeptide degradation frag-ments of the synthetic octapeptide substrate after incubation(37∘C lmMCa2+) with each MMP were measured by high-performance liquid chromatography (HPLC) using a reverse-phase C18 column (46 times 75mm 35 120583Mmacroporous spher-ical support)The eluate was monitored at 375 nm to quantifythe DNP-labeled peptides The IC

50for each compound was

calculated from the plot of the percentage of inhibition ofenzyme activity versus the concentration of inhibitor [19 20]

23 Cell Culture Assay Human peripheral blood mononu-clear cells (PBMC) were isolated and purified from Leuko-cyte Concentrate (Long Island Blood Bank Melville NY)by density gradient centrifugation and adherence using amethod described by us previously [21] PBMC cells werethen cultured for 18 hours in serum-free macrophage media(Invitrogen Corp Carlsbad CA) at 37∘C (95 air 5 CO

2)

with either LPS derived from P gingivalis (50 ngmL) orvehicle alone Curcumin or CMC 25 was added at final con-centrations of 2 or 5 120583M Conditioned media were analyzedfor the cytokines and proinflammatory mediators TNF-120572IL-1120573 IL-6 MCP-1 and PGE

2by ELISA (see below) and for

MMP-9 by ELISA and by gelatin zymography (see below) asdescribed previously [21 22]

24 Gelatin Zymography The gelatin zymography systemand SDS-PAGE gels containing polyacrylamide copolymer-ized with gelatin at a final concentration of 1mgmL werepurchased from Invitrogen Corp (Carlsbad CA) After elec-trophoresis (120V) the gels were washed with 25 TritonX-100 incubated at 37∘C overnight in calcium assay buffer(40mM TrisHCl 200mMNaCl 10mM CaCl

2 and pH 75)

and then stained with Coomassie Brilliant Blue R-250 Asdescribed by us earlier [19] clear zones of lysis against a bluebackground indicate gelatinolytic activity and were scanneddensitometrically to assess gelatinase activity MMP-2 andMMP-9 standards were purchased from RampD Systems Inc(Minneapolis MN)

Mediators of Inflammation 3

25 ELISA Assay ELISA kits for TNF-120572 IL-1120573 IL-6 MCP-1 PGE

2 and MMP-9 were purchased from RampD Systems

Inc (Minneapolis MN) Fifty or 100 120583L of the reconstitutedstandards or samples of conditioned medium was platedinto wells coated with anti-human primary antibody andthen incubated with 50120583L of a biotinylated detection anti-body reagent at room temperature for two hours Afterincubation the plates were washed three times and 100 120583Lof streptavidin-HRP solution was added to each well andincubated for 30 minutes at room temperature Followingthree further washes 331015840 5510158401015840-tetramethylbenzidine (TMB)substrate solution (100 120583L) was added to each well and theplate was allowed to develop at room temperature in the darkAfter 30 minutes 100 120583L of stop solution was added and theabsorbance of the samples was measured at 450 nm [20]

26 NF120581B Activation Assay The phosphorylation of NF120581Bwas measured by means of a Cellular Activation of SignalingELISA (CASE) kit (SABiosciences Frederick MD) PBMCcells were cultured in serum-free macrophage media (37∘C95 air 5 CO

2) for 18 hours with LPS (P gingivalis

50 ngmL) or vehicle alone and CMC 25 was added at afinal concentration of 5120583M Following the incubation thecells were treated with 4 cell fixing buffer The wells werewashed quenched and blocked for 1 hour at 22∘C and thenincubated with anti-human primary antibodies specific toeither phosphorylated or total NF120581B protein for 1 hour atroom temperature After incubation the plate was washedthree times with a buffered surfactant (phosphate bufferedsaline containing Tween 20) HRP-conjugated secondaryantibody solution was added to each well and the plate wasincubated for 60 minutes at room temperature Followingthree further washes a color developing solution was addedto each well and the plate was allowed to develop at roomtemperature in the dark After 10 minutes stop solution wasadded and the absorbance of each sample was measured at450 nm [23]

27 Animal Studies All of the experimental proceduresinvolving animals were approved by Stony Brook UniversityrsquosInstitutional Animal Care and Use Committee (IACUC)Twelve male Sprague-Dawley rats (275ndash300 g body weightviral antibody free Charles River Labs) were injected ivwith streptozotocin (STZ) 70mgkg to induce diabetes anddiabetes-enhanced periodontal disease in diabetic rats [1415] Diabetic status was confirmed weekly using a glucosetest strip which showed gt2 glucose in urine within 24ndash48 h after STZ injection Nondiabetic control rats (NDC119899 = 6) were injected iv with the vehicle (citric buffer)alone One to two days after STZ injection when glucosuriahad been established six of the STZ-diabetic rats were dailyadministered for 3 weeks by oral gavage a 1mL suspensionof CMC 25 (100mgkg body weight suspended in 2carboxymethylcellulose) or 1mL of vehicle alone (119899 = 6 rats)At the end of the treatment period the rats were sacrificedby exsanguination blood samples were collected and gingivawere dissected and pooled by group because insufficientgingival tissue is available for individual analysis Bloodglucose levels were analyzed by a blood glucose monitoring

system (Johnson and Johnson Milpitas CA) Blood samplesand gingival tissues were stored at minus80∘C until analyzed forMMPs and cytokines by gelatin zymography and ELISArespectively

28 Gingival Extracts The pooled gingival tissues fromeach group of rats were weighed minced (all procedures at4∘C) and extracted with Tris-NaCl-CaCl

2buffer (pH 76

100mg wet weight gingival tissue5mL buffer) containing5M urea [14 15] After centrifugation the supernatant wasdialyzed exhaustively against the TrisNaClCaCl

2buffer

and the extract was partially purified by precipitation withammonium sulfate added to 60 saturation Aliquots ofeach gingival extract were measured for MMP-2 and MMP-9 by gelatin zymography and were scanned densitometricallyto quantify gelatinase activity IL-1120573 was measured using acommercial ELISA kit (RampD systems Minneapolis MN)

29 Western Blot Analysis Samples were treated withLaemmli buffer (pH 70) containing 5mM dithiothreitol andheated for 5 minutes at 100∘C High- and low-range pre-stained sodium dodecyl sulfate- (SDS-) polyacrylamide gelelectrophoresis standard proteins were used as molecularweight markers The samples were electrophoresed on 75SDS-polyacrylamide gels and then electrophoretically trans-ferred to nitrocellulosemembranesWestern blot analysiswascarried out as described by us previously [20]

Specific immunoreactivity was visualized as dark bandsagainst a clear background and themembranes were scannedwith an imaging densitometer (Bio-Rad Model GS-700 Bio-Rad Hercules CA) using a program (Analyst Bio-RadHercules CA) that corrects for background absorption Thedensitometric units were measured in the linear range ofimmunoreactivity forMMP-13 purified humanMMP-13 wasused as a positive control

210 Statistical Analysis Cytokine and MMP differences incell culture between groups were analyzed by Studentrsquos 119905-test with 119875 le 005 taken as statistically significant In therat animal studies when comparing two groups (normalversus untreated diabetic untreated diabetic versus diabetictreated with CMC 25) a studentrsquos 119905-test was used as well 119875values le 005 were considered statistically significant

3 Results

31 In Vitro MMP Inhibition Studies Each human chro-matographically pure MMP was incubated in vitro withthe collagenase-specific synthetic octapeptide substrate aspreviously described [20] For each compound tested as anMMP inhibitor (phenanthroline curcumin or CMC 25)the concentrations ranging from 1 5 20 to 100 120583M thatwas required to inhibit 50 of the proteolytic activity ofthe MMP (IC

50 see Table 1) was determined from a plot of

the extent () of inhibition versus the concentration of theinhibitor 110-Phenanthroline a zinc binding agent [11] wasused to quench the in vitroMMP assays Curcumin was usedas a positive control CMC 25 was found to inhibit both


Table 1 Potency of CMC 25 as an MMP inhibitor

Test compounds MMP-9 MMP-13IC50 (120583M) Maximum inhibition () IC50 (120583M) Maximum inhibition ()

110-Phenanthroline 9 100 4 100Curcumin 29 58 110 53CMC 25 16 72 15 69

0

200

400

600

800

1000

1200

1400

1600

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

TNF-120572

(pg

mL)

LPS

+5120583

m

LPS

+5120583

mlowast

lowast

lowastlowast

Figure 1 Inhibition of TNF-120572 levels by curcumin and CMC 25in PBMC cells PBMC cells (5 times 105 cellswell) were cultured inserum-free media (37∘C 95 air 5 CO

2) for 18 hours with LPS (P

gingivalis 50 ngmL) or vehicle alone Curcumin or CMC 25 wasadded at final concentrations of 2 or 5 120583M Conditioned mediumwas analysed for TNF-120572 by ELISA Each value represents the meanof 3 cultures plusmn the standard error of the mean (SEM) lowast119875 lt 005represents the significance of all groups of curcumin and CMC 25compared to LPS alone lowastlowast119875 lt 005 represents the significancebetween LPS + 5120583M curcumin and LPS + 5 120583MCMC 25

MMP-9 and MMP-13 activities in a dose-response fashion invitro (data not shown) and was two-seven times more potent(based on IC

50values) as an MMP inhibitor than its parent

compound curcumin (Table 1)

32 Cell Culture Studies As noted in Figure 1 in our cellculture studies control wells were incubated with monocytesin serum-free conditioned media (SFCM) (37∘C 95 air 5CO2) for 18 hourswith LPS (P gingivalis 50 ngmL) or vehicle

alone In the absence of CMC 25 and LPS about 200 pgmLof TNF-120572 was secreted by the monocytes which increasedto 1470 pgmL when LPS was added to the culture TNF-120572levels were reduced by 13 and 46 with either 2 or 5 120583Mcurcumin respectively however only the latter value wasstatistically significant (119875 lt 005) In contrast when CMC 25was added to the culture of the LPS-stimulated monocytesin final concentrations of 2 or 5120583M a greater effect wasseen at both concentrations the extracellular TNF-120572 levelswere decreased by 45 (119875 lt 005) and 79 (119875 lt 005)respectively In addition 5120583M CMC 25 was found to be72 more potent as an inhibitor of TNF-120572 secretion than5 120583M curcumin (119875 lt 005) Similarly monocytes secreted185 pgmL of IL-1120573 when LPS was added to the culture incontrast to the lt25 pgmL by control cells (Figure 2) IL-1120573

levels were reduced by 57 and 83 with 2 and 5 120583Mcurcumin respectively (119875 lt 005) When these cells wereincubated in the presence of CMC 25 at concentrations of2 and 5 120583M both concentrations of CMC 25 decreased IL-1120573 levels by more than 90 essentially back to the valuesseen in cells that were not treated with LPS (Figure 2) Allfour treatments were statistically significant compared toLPS alone In fact 2 120583M CMC 25 was 75 more effectivethan 2120583M curcumin (119875 lt 005) as an inhibitor of IL-1120573secretion although the 5 120583MCMC 25 which appeared tobe 50 more effective than 5 120583M curcumin did not differsignificantly from the effect of 5120583M curcumin (119875 gt 005)A similar pattern of change was observed for PGE

2levels

(Figure 3) CMC 25 at concentrations of 2 and 5120583M reducedextracellular PGE

2levels by 23 (119875 gt 005) and 51 (119875 lt

005) respectively compared to LPS alone In addition theextracellular levels of MCP-1 IL-6 and MMP-9 (the lattera major MMP secreted by monocytes) in the SFCM fromthese monocyte cultures in the presence or absence of 5120583MCMC 25 were analyzed by ELISA and gelatin zymographyrespectively Similar to the results observed for IL-1120573 theextracellular levels of these bioactive proteins were reducedby more than 90 essentially down to untreated cell values(Figures 4 and 5) by 5120583MCMC 25 and these effects were allstatistically significant (119875 lt 005) In contrast curcumin at5 120583M concentration did not have a significant effect onMCP-1 IL-6 (data not shown) or MMP-9 levels (Figure 5)

To begin to explore the underlyingmechanisms of actionthe levels of phosphorylation of NF120581B (p65S536) in thepresence of CMC 25 were analyzed by calculating thepercent phosphorylation relative to total NF120581B protein levels(Figure 6) In these cultures control wells were incubatedwith monocytes in serum-free conditioned media (SFCM37∘C 95 air 5 CO


50 ngmL) or vehicle alone The phosphorylation of NF120581Bwas increased 3-fold in the presence of LPS (Figure 6) WhenCMC 25 was added to the culture of the LPS-stimulatedmonocytes in a final concentration of 5120583M the phosphory-lation of NF-120581B was decreased by 354 (119875 le 005)

Thus in these cell culture studies LPS from gram-negative bacteria P gingivalis increased the secretionof TNF-120572 PGE

2 IL-1120573 IL-6 MCP-1 and MMP-9 and

increased the phosphorylation (activation) of NF-120581B inhuman mononuclear cells All of these effects were largelynormalized by CMC 25 Therefore the in vivo therapeuticpotential of this chemically modified curcumin (CMC 25)was further evaluated in the diabetesperiodontal inflamma-tion rat model as described below

33 In Vivo Studies The diabetic condition was induced inthe rats by STZ as described above CMC 25 (100mgkg) was


0

50

100

150

200

250

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

LPS

+5120583

m

LPS

+5120583

m

lowast

lowast

lowast

lowastlowast

IL-1120573

(pg

mL)

Figure 2 Inhibition of IL-1120573 levels by curcumin and CMC 25in PBMC cells PBMC cells (5 times 105 cellswell) were cultured inserum-free media (37∘C 95 air 5 CO

2) for 18 hours with LPS

(P gingivalis 50 ngmL) or vehicle alone Curcumin or CMC 25 wasadded at final concentrations of 2 or 5 120583MConditionedmediumwasanalysed for IL-1120573 by ELISA Each value represents the mean of 3cultures plusmn SEM lowast119875 lt 005 represents the significance of all groupsof curcumin and CMC 25 compared to LPS alone lowastlowast119875 lt 005represents the significance betweenLPS+ 2 120583Mcurcumin andLPS +2120583MCMC 25

0

20

40

60

80

100

120

Vehi

cle

LPS

alon

e

CMC

25

CMC

25

LPS

+2120583

m

LPS

+5120583

m

PGE 2

(pg

mL)

lowast

Figure 3 Inhibition of PGE2levels by CMC 25 in PBMC cells

PBMC cells (5 times 105 cellswell) were cultured in serum-free media(37∘C 95 air 5 CO


50 ngmL) or vehicle alone CMC 25 was added at final concen-trations of 2 or 5120583M Conditioned medium was analysed for PGE

2

by ELISA Each value represents the mean of 3 cultures plusmn SEMlowast119875 lt 005

administered orally for 3 weeks beginning after the diabeticcondition was established As expected we found that thediabetic condition markedly increased the activity of MMP-9 in both plasma and gingiva (Figures 7 and 8) In additionMMP-13 which in the rats is analogous toMMP-1 in humans[24] was also increased in the plasma of the diabetic rats(Figure 7) whereas MMP-13 was not detected in the pooledgingival tissues In the plasma CMC25 reduced the excessiveMMP-9 and MMP-13 levels to near normal levels Howeverthe effects of both diabetes and CMC 25 treatment on plasmaMMP-2were not statistically significant although the pattern

0

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450

MCP-1 IL-6

pgm

L

VehicleLPS aloneLPS + 5120583m CMC 25

lowast

lowast

(a)

0

02

04

06

08

1

12

14

16

Vehicle LPS alone

ngm

L

MMP-9

LPS + 5120583m CMC 25

lowast

(b)

Figure 4 Inhibition of MCP-1 IL-6 and MMP-9 levels by CMC25 in PBMC cells PBMC cells (5 times 105 cellswell) were cultured inserum-free media (37∘C 95 air 5 CO


(P gingivalis 50 ngmL) or vehicle alone CMC 25 was added at afinal concentration of 5120583M Conditioned medium was analysed forMCP-1 or IL-6 orMMP-9 by ELISA Each value represents themeanof 3 cultures plusmn SEM lowast119875 lt 005

of change for this 72 kDa gelatinase paralleled the changesseen for the other MMPs (Figure 7) It should be notedthat the elevated blood glucose (Figure 9) and HbA1c (datanot shown) levels in the diabetic rats were not affected byCMC 25 treatment In the gingiva the dominant gelatinasein this periodontal tissue in the nondiabetic control ratsis MMP-2 present both as the 72 kDa proform and as thelower molecular weight activated form Inducing diabetesand severe hyperglycemia results in the induction of MMP-9 (92 kDa gelatinase) in the gingival tissues but this effect isldquonormalizedrdquo by CMC 25 treatment in spite of there beingno effect on the severity of the hyperglycemia (Figure 9) Notethat the levelsactivity ofMMP-2 in the gingiva is not affectedeither by diabetes or by treatment with CMC 25 Moreoverthe diabetic rats exhibited approximately 200 higher IL-1120573levels in the gingiva compared to the normal rats but the


Vehicle LPS alone

MMP-9

curcuminLPS + 2120583m

CMC 25LPS + 5120583m


Figure 5 Inhibition of MMP-9 levels by CMC 25 in PBMC cells PBMC cells (5 times 105 cellswell) were cultured in serum-free media (37∘C95 air 5 CO

2) for 18 hours with LPS (P gingivalis 50 ngmL) or vehicle alone CMC 25 was added at a final concentration of 5120583M

Conditioned medium was analyzed for MMP-9 by gelatin zymography

0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast

Figure 6 Inhibition of NF120581B phosphorylation by CMC 25 inPBMC cells PBMC cells (5 times 104 cellswell) were cultured in serum-free media (37∘C 95 air 5 CO


gingivalis 50 ngmL) or vehicle alone CMC 25 was added at afinal concentration of 5120583M Following the incubation cells werefixed and phosphorylation of NF120581B was analyzed by CASE cellularactivation of signaling ELISA kit lowast119875 le 005

oral administration of CMC 25 reduced this level in pooledgingival tissue by 26 (Figure 8)

4 Discussion

Curcumin is considered a safemolecule and has been used forthousands of years as a food additive inAsia [25] Structurallystudies have shown that curcumin contains a 120573-diketonezinc binding site [25] similar to that in the tetracycline-based MMP inhibitors [26] In the current study CMC25 demonstrated greater therapeutic activity compared tocurcumin based on its improved inhibitory activity againstMMPs and proinflammatory cytokines in in vitro and incell culture it also showed efficacy in an animal model ofdiabetes-enhanced periodontal inflammation (see below) Toverify that the inhibition of the MMPs (MMP-9 and MMP-13) and proinflammatory mediators (TNF-120572 IL-1120573 MCP-1IL-6 and PGE

2) by CMC 25 was not the result of cell tox-

icity the Cell Proliferation Assay using a novel tetrazoliumcompound to determine cell cytotoxicity (MTS assay) wasalso performed (data not shown) These data demonstratedthat CMC 25 at doses used in our assay is not toxic to thehuman mononuclear cells in culture In additional studies(not shown) no adverse effects were observed in the diabeticrats orally administered doses as high as 500mgkg bodyweight once per day over a 3-week protocol (much higherthan the oral dose used in the current study 100mgkg)

0

50

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250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)

MMP levels in plasma (by gelatin zymography)

NormalDiabeticCMC 25

(a)

0

05

1

15

2

Normal Diabetic D + CMC 25

MM

P-13

(AU

)

lowast

(b)

Figure 7The effect of diabetes and orally administered CMC 25 onplasma MMPs Male Sprague-Dawley rats (119899 = 6) were injected ivwith streptozotocin (STZ) 70mgkg to induce diabetes as describedby us previously STZ-diabetic rats were daily administered by oralgavage CMC25 (100mgkg) for 3 weeks At the end of the treatmentprotocol rats were sacrificed by exsanguination blood sampleswere collected and analyzed for MMP-9 by gelatin zymography andMMP-13 by Western blot and were scanned densitometrically toquantify the levels of MMP-9 and MMP-13 lowast119875 lt 005

[17] In fact oral administration of 100mgkg CMC 25 tothe diabetic rats attenuated the complications caused by theseverely hyperglycemic condition such as bleeding under thenails severely inflamed sclera and impaired wound healingwithout any detectable effect on blood glucose or HbA1clevelsThis indicates that CMC 25 can reduce the destructive


MMP-9

MMP-2

Normal CMC 25diabetic

(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540

NDC UD D + CMC 25(100mgkg)

IL-1120573

(pg

mL)

(c)

Figure 8 The effect of diabetes and orally administered CMC 25 on gingival MMPs (-2 -9) and IL-1120573 Gingival tissue from diabetic ratstreated with vehicle or CMC 25 was obtained and pooled by group as described in Section 2 since insufficient gingival tissue is usuallyavailable for individual analysis Gingival tissues were then extracted and aliquots of each gingival extract were measured for MMP-2 andMMP-9 by gelatin zymography and were scanned densitometrically to quantify gelatinase activity and IL-1120573 was measured by ELISA NDCnondiabetic control UD untreated diabetic

0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)

Figure 9 The effect of diabetes and orally administered CMC25 on blood glucose levels Male Sprague-Dawley rats (119899 = 6)were injected iv with streptozotocin (STZ) 70mgkg to inducediabetes as described by us previously STZ-diabetic rats weredaily administered by oral gavage CMC 25 (100mgkg) for 3weeks At the end of the treatment protocol rats were sacrificed byexsanguination blood samples were then collected and analyzed forblood glucose levels by the blood glucose monitoring system NDCnondiabetic control UD untreated diabetic

effects of the inflammatory mediators (cytokines andMMPs)during local (periodontal disease) and systemic (diabetes)conditions without exhibiting toxicity

To begin to understand the underlying mechanisms ofthe inhibitory effects of CMC 25 on inflammatory mediators

secreted by humanmononuclear cells the phosphorylation ofNF-120581B was evaluated Since NF-120581B controls transcription ofmany genes involved in inflammation it is found chronicallyactivated in many inflammatory diseases such as arthritis[27] The upregulation of proinflammatory cytokines andMMPs may be mediated through the NF-120581B as well as otherintracellular signaling transduction pathways including P38MAP kinase [28] In the current study we have shown thatLPS stimulation of human monocytes increased by 3-foldthe phosphorylation of NF120581B and that CMC 25 can sig-nificantly reduce this excessive NF120581B activation Thereforethe increased production of these inflammatory mediatorsmay be regulated at least partially through the NF-120581B cellsignaling transduction pathways and CMC 25 treatment canattenuate this effect

Diabetes is recognized as an important risk factor forchronic periodontitis based on human clinical trials [29]Thecurrent view is that the hyper- (or prolonged-) inflammatoryresponse during diabetes is mainly caused by the long-termexposure of various proteins to elevated glucose levels Thisresults in the formation of advanced glycation end-products(AGEs) which promote the secretion of proinflammatorymediators (eg TNF-120572 IL-1120573 and IL-6) and alters theinnate immune response [2 14] as well as increasing the


production and activity of tissue and bone-destructiveMMPs[15 30] These abnormalities progress to periodontal tissuedestruction that is initiated by bacterial factors such as LPSOn the other hand periodontitis can be more than justa localized chronic inflammation This oral disease mayalso have profound effects on the systemic health of thediabetic patient During the pathogenesis of periodontaldisease the host immunoinflammatory response to plaquebacteria produces destructive cytokines such as TNF-120572 IL-1120573 and MMPs [2] Initially this response is protective innature and designed to control the bacterial infection thiscan be observed clinically as gingival inflammation with noalveolar bone loss However when the above inflammatoryprocess is not well controlled it results in excessive levelsof the inflammatory mediators and MMPs as seen in dia-betes These inflammatory mediators can eventually enterinto the circulation stimulating a systemic inflammatoryresponsewhich then increases the risk for developing diabeticcomplications including an increased risk for cardiovasculardisease [2] In this regard the interrelationships betweendiabetes and periodontal disease may represent a ldquotwo-hitrdquomodel in which diabetes predisposes the patient to oral tissuedestruction and the oral infection exacerbates the abnormalglucose metabolism and its complications in the patient withdiabetes [30] Thus effective management of both local andsystemic inflammation is critical to attenuate these severecomplications

Studies have shown that greater gingival inflammationand periodontal tissue destruction including alveolar boneloss can be observed in diabetic rat models [13ndash16] Specif-ically in our in vivo rat model we have previously demon-strated [14 15] that inducing diabetes with streptozotocin(STZ) increases both systemically (plasma) and locally (gin-giva) the levels of cytokines and MMPs which are associatedwith the collagen and bone destruction that characterizeperiodontal disease Alveolar bone loss has been found tobe increased in this animal model of diabetes as well [14ndash16] Importantly a number of studies have not found signifi-cant differences in periodontal pathogens in the subgingivalbiofilm in comparing the nondiabetic controls and diabetichumans or rats indicating that host-response differencesbetween these two groups are largely responsible for theexcessively severe periodontal disease in poorly controlleddiabetic patients [31] In the current study cytokines andMMPs levels were increased and oral administration of CMC25 to the diabetic rats significantly reduced the MMP-9and MMP-13 levels in plasma and decreased both MMP-9 and IL-1120573 levels in rat gingival tissue with no detectableeffect on blood glucose orHbA1c levels However statisticallysignificant elevated periodontal bone loss in the diabeticswas not observed But preliminary data in a previous studyindicated that CMC 25 administration to diabetic rats didsignificantly reduce alveolar bone loss (119875 lt 005) [17] Theinconsistency regarding diabetes-induced alveolar bone lossbetween the earlier and current studies may be due to theduration of each study Changes in inflammatory biomarkersin gingiva presumably occur at an earlier stage while alveolarbone loss takes more time therefore the latter occurs at alater stage of the disease In addition any differences in the

age of the rats in the different studies may also play a rolein the alveolar bone response to the diabetic condition anddrug treatment Longer term studies to examine the effectsof CMC 25 and related congeners on alveolar bone loss inthe diabetic rats are currently underway In addition we arealso working on a rat model of experimental periodontitisthat is LPS injection into the gingiva and have preliminarydata showing thatCMC25 reduces bothMMPs in the gingivaand alveolar bone loss in defleshed jaws in this model Thesewill be discussed in future reports Of specific interest thecurrent study indicates that CMC 25 reduces excessive levelsof inflammatory cytokines and MMPs in the gingiva andplasma of the diabetic rats at the 3-week time period Thissuggests that before the clinical signs of progressive periodon-titis are observed CMC 25 can prevent the progression toclinically evident periodontitis (characterized by bone loss)by reducing these inflammatory mediators both locally andsystemically at an early stage of the disease This could alsoreduce the risk of developing diabetic complications overlonger periods of hyperglycemia

In addition the in vitro MMP inhibition assays werecarried out with MMP-9 (92 kDa gelatinase) and MMP-13(collagenase-3) both known to be associated with periodon-tal disease and other conditions of connective tissue loss [1932] Our study indicates that CMC 25 can inhibit bothMMP-9 and MMP-13 activities directly and that CMC 25 is morepotent as an MMP inhibitor than the parent compound cur-cumin Moreover the cell culture studies indicate that CMC25 can significantly reduce the MMP-9 and cytokine levels(TNF-120572 IL-1120573 MCP-1 IL-6 and PGE

2) produced by chronic

inflammatory cells in response to the microbial endotoxinLPS In addition to monocytes human polymorphonuclearleukocytes (neutrophils PMNs) also form an essential partof the innate immune system and play a critical role inacute inflammation Our studies now underway have shownsignificant impairment of PMN function in diabetic ratsspecifically reduced PMN chemotaxis and abnormal PMNaccumulation in peritoneal exudates from these animalsMoreover oral administration of a CMC reduced the severityof these abnormalities These findings will be detailed infuture reports [33]

The findings presented in this paper support the hypoth-esis that a chemically modified curcumin (CMC 25) is apleiotropic compound having both intracellular and extra-cellular effects which collectively ameliorate local and sys-temic inflammation and prevent hyperglycemia-associatedtissue destruction Safety and toxicity studies on CMC 25on two animal species for example rats and dogs will beneeded to enable proof-of-concept preliminary clinical trialsin patients with periodontal disease Our ultimate goal is tocomplete additional animal studies and advance to humanclinical trials Future studies using this or related novelCMCs could also test the safety and efficacy of these com-pounds on animal models of other inflammatory diseasessuch as rheumatoid arthritis and diabetes-induced impairedwound healing In conclusion CMC 25 a methoxycar-bonyl curcumin demonstrated therapeutic potential in treat-ing inflammatory and connective tissue-destructive diseases


such as periodontal disease and may also reduce the risks ofother complications of diabetes

Conflict of Interests

Lorne M Golub is listed as an inventor on several relatedpatents and these have been fully assigned to his institu-tion Stony Brook University and to Chem-Master Intl IncFrancis Johnson declares that he has no conflict of interestsfinancial or otherwise with regard to the publication of thispaper He is listed as a coinventor on several related patentswhich have been fully assigned to Stony Brook Universityand to Chem-Master Int Inc on a shared basis All otherauthors declare that there is no conflict of interests regardingthe publication of this paper

Acknowledgments

This study was supported by a Grant no A43273 from theNew York State Office of Science Technology and AcademicResearch (NYSTAR) throughNYSTARrsquos Center of AdvancedTechnology Stony Brook University and Helsinki UniversityCentral Hospital Research Foundation

References

[1] T E Van Dyke and A J Van Winkelhoff ldquoInfection andinflammatory mechanismsrdquo Journal of Periodontology vol 84supplement 14 no 4 pp S1ndashS7 2013

[2] Y Gu and M E Ryan ldquoOverview of periodontal diseasescauses pathogenesis and characteristicsrdquo in Periodontal Dis-eases andOverall Health A Clinicianrsquos Guide RWilliams and RGenco Eds pp 5ndash23 Professional Audience CommunicationsYardley Pa USA 2009

[3] B B Aggarwal and K B Harikumar ldquoPotential therapeuticeffects of curcumin the anti-inflammatory agent against neu-rodegenerative cardiovascular pulmonary metabolic autoim-mune and neoplastic diseasesrdquo International Journal of Bio-chemistry and Cell Biology vol 41 no 1 pp 40ndash59 2009

[4] E Sikora A Bielak-Zmijewska G Mosieniak and K PiwockaldquoThe promise of slow down ageing may come from curcuminrdquoCurrent Pharmaceutical Design vol 16 no 7 pp 884ndash892 2010

[5] B L Queen and T O Tollefsbol ldquoPolyphenols and agingrdquoCurrent Aging Science vol 3 no 1 pp 34ndash42 2010

[6] B B Aggarwal A Kumar and A C Bharti ldquoAnticancer poten-tial of curcumin preclinical and clinical studiesrdquo AnticancerResearch vol 23 no 1A pp 363ndash398 2003

[7] O P Sharma ldquoAntioxidant activity of curcumin and relatedcompoundsrdquo Biochemical Pharmacology vol 25 no 15 pp1811ndash1812 1976

[8] A J Ruby G Kuttan K Dinesh Babu K N Rajasekharanand R Kuttan ldquoAnti-tumour and antioxidant activity of naturalcurcuminoidsrdquo Cancer Letters vol 94 no 1 pp 79ndash83 1995

[9] A N BegumM R Jones G P Lim et al ldquoCurcumin structure-function bioavailability and efficacy inmodels of neuroinflam-mation and Alzheimerrsquos diseaserdquo Journal of Pharmacology andExperimental Therapeutics vol 326 no 1 pp 196ndash208 2008

[10] PAnandA BKunnumakkara RANewman andB BAggar-wal ldquoBioavailability of curcumin problems and promisesrdquoMolecular Pharmaceutics vol 4 no 6 pp 807ndash818 2007

[11] Y Zhang L M Golub F Johnson and A Wishnia ldquopKazinc- and serum albumin-binding of curcumin and two novelbiologically-active chemically-modified curcuminsrdquo CurrentMedicinal Chemistry vol 19 no 25 pp 4367ndash4375 2012

[12] Y Zhang Y Gu H M Lee et al ldquoDesign synthesis andbiological activity of new polyenolic inhibitors of matrix met-alloproteinases a focus on chemically-modified curcuminsrdquoCurrent Medicinal Chemistry vol 19 no 25 pp 4348ndash43582012

[13] C Tesseromatis A Kotsiou H Parara E Vairaktaris and MTsamouri ldquoMorphological changes of gingiva in streptozotocindiabetic ratsrdquo International Journal of Dentistry vol 2009Article ID 725628 4 pages 2009

[14] M E Ryan N S Ramamurthy T Sorsa and L M GolubldquoMMP-mediated events in diabetesrdquo Annals of the New YorkAcademy of Sciences vol 878 pp 311ndash334 1999

[15] K M Chang M E Ryan L M Golub N S Ramamurthyand T F McNamara ldquoLocal and systemic factors in periodontaldisease increase matrix-degrading enzyme activities in rat gin-giva effect of micocycline therapyrdquo Research Communicationsin Molecular Pathology and Pharmacology vol 91 no 3 pp303ndash318 1996

[16] H Toker H Ozdemir H Balcı and H Ozer ldquoN-acetylcysteinedecreases alveolar bone loss on experimental periodontitis instreptozotocin-induced diabetic ratsrdquo Journal of PeriodontalResearch vol 47 no 6 pp 793ndash799 2012

[17] M Elburki A Goren H Lee et al ldquoChemically-modifiedcurcumins and alveolar bone loss in diabetic ratsrdquo Journal ofDental Research vol 90 abstract 2295 2011

[18] L Tornatore A K Thotakura J Bennett M Moretti andG Franzoso ldquoThe nuclear factor kappa B signaling pathwayintegrating metabolism with inflammationrdquo Trends in CellBiology vol 22 no 11 pp 557ndash566 2012

[19] H-M Lee S G Ciancio G Tuter M E Ryan E Komaroffand L M Golub ldquoSubantimicrobial dose doxycycline efficacyas a matrix metalloproteinase inhibitor in chronic periodontitispatients is enhanced when combined with a non-steriodal anti-inflammatory drugrdquo Journal of Periodontology vol 75 no 3 pp453ndash463 2004

[20] L M Golub H-M Lee J A Stoner et al ldquoSubantimicrobial-dose doxycycline modulates gingival crevicular fluid biomark-ers of periodontitis in postmenopausal osteopenic womenrdquoJournal of Periodontology vol 79 no 8 pp 1409ndash1418 2008

[21] Y GuH-M Lee T Sorsa S R Simon and LMGolub ldquoDoxy-cyline inhibits mononuclear cell-mediated connective tissuebreakdownrdquo FEMS Immunology and Medical Microbiology vol58 no 2 pp 218ndash225 2010

[22] D L Brown K K Desai B A Vakili C Nouneh H-MLee and L M Golub ldquoClinical and biochemical results ofthe metalloproteinase inhibition with subantimicrobial dosesof doxycycline to prevent acute coronary syndromes (MIDAS)pilot trialrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 24 no 4 pp 733ndash738 2004

[23] A Naderi A E Teschendorff J Beigel et al ldquoBEX2 is over-expressed in a subset of primary breast cancers and mediatesnerve growth factornuclear factor-120581B inhibition of apoptosisin breast cancer cell linesrdquo Cancer Research vol 67 no 14 pp6725ndash6736 2007

[24] P G Mitchell H A Magna L M Reeves et al ldquoCloningexpression and type II collagenolytic activity of matrixmetalloproteinase-13 from human osteoarthritic cartilagerdquoJournal of Clinical Investigation vol 97 no 3 pp 761ndash768 1996


[25] P Anand S GThomas A B Kunnumakkara et al ldquoBiologicalactivities of curcumin and its analogues (Congeners) made byman and Mother Naturerdquo Biochemical Pharmacology vol 76no 11 pp 1590ndash1611 2008

[26] L M Golub H M Lee M E Ryan W V Giannobile J Payneand T Sorsa ldquoTetracyclines inhibit connective tissue break-down by multiple non-antimicrobial mechanismsrdquoAdvances inDental Research vol 12 no 2 pp 12ndash26 1998

[27] C Monaco E Andreakos S Kiriakidis et al ldquoCanonical path-way of nuclear factor 120581B activation selectively regulates proin-flammatory and prothrombotic responses in human atheroscle-rosisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 101 no 15 pp 5634ndash5639 2004

[28] Y A Ivanenkov K V Balakin and S E Tkachenko ldquoNewapproaches to the treatment of inflammatory disease focuson small-molecule inhibitors of signal transduction pathwaysrdquoDrugs in R amp D vol 9 no 6 pp 397ndash434 2008

[29] V R Santos J A Lima T S Miranda et al ldquoFull-mouth dis-infection as a therapeutic protocol for type-2 diabetic subjectswith chronic periodontitis twelve-month clinical outcomesa randomized controlled clinical trialrdquo Journal of ClinicalPeriodontology vol 40 no 2 pp 155ndash162 2013

[30] L M Golub J B Payne R A Reinhardt and G Nieman ldquoCansystemic diseases co-induce (not just exacerbate) periodontitisa hypothetical ldquotwo-hitrdquo modelrdquo Journal of Dental Research vol85 no 2 pp 102ndash105 2006

[31] M Claudino G Gennaro T M Cestari et al ldquoSponta-neous periodontitis development in diabetic rats involves anunrestricted expression of inflammatory cytokines and tissuedestructive factors in the absence of major changes in commen-sal oral microbiotardquo Experimental Diabetes Research vol 2012Article ID 356841 10 pages 2012

[32] V-J Uitto K Airola M Vaalamo et al ldquoCollagenase-3 (matrixmetalloproteinase-13) expression is induced in oral mucosalepithelium during chronic inflammationrdquo American Journal ofPathology vol 152 no 6 pp 1489ndash1499 1998

[33] Y Gu H-M Lee H Callen et al ldquoA novel chemically-modified-curcumin ldquoNormalizesrdquo impaired leukocyte compe-tence in diabetic ratsrdquo Journal of Dental Research In press

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


studied in both in vitro and in vivo model systems [6ndash8]Despite these many beneficial effects curcumin has majorlimitations including poor solubility a lack of systemicbioavailability and rapid metabolic disposition [9] Thusextremely high oral doses of the compound are neededand even then it results in only very low levels in thesystemic circulation of both animals and humans Thishas severely limited its clinical application [10] Recentlyour laboratory has developed a series of novel chemicallymodified curcumins with a carbonyl substituent at theC-4 position [11 12] Such analogues have an additionalelectron-withdrawing group which enhances their anti-inflammatory therapeutic effects One such compound(CMC 25) contains a methoxycarbonyl group at C4 showsan improved solubility better serum albumin-bindingactivity and greater acidity and enhanced zinc-bindingcharacteristics This modification has been found to enhancethe MMP-inhibitory properties of this novel compoundversus curcumin [11 12]

In the current report we investigate the effect of thisnovel substance 4-methoxycarbonylcurcumin (CMC 25)on proinflammatory cytokines and MMPs in an in vivodiabetes-enhanced periodontal inflammation rat model andin a relevant cell culture model Rats with experimentaldiabetes mellitus manifest increased gingival inflammationand periodontal tissue destruction including alveolar boneloss [13ndash16] This animal model of STZ-induced diabetes asan enhancer of periodontal disease is well established inour laboratory [14 15] and has been described by othersas well [16] It was used in preclinical studies during thedevelopment of Periostat the only host modulation andMMP inhibitory therapy for periodontitis approved by theFDA This unique animal model different from traditionalrat models of experimental periodontitis using ligatures ororal pathogen infection will allow us to study the possibleassociation between this local inflammatory disease andrelevant systemic conditions We have previously demon-strated that the diabetic condition increases the levels ofcytokines and MMPs locally in the gingival tissues as wellas systemically in plasma [14 15] In addition levels ofMMP-8 in skin and both local and systemic bone loss wereincreased in this animal model [17] Therefore this willallow us to study not only the periodontal disease but alsothe systemic factors associated with this local inflammatorycondition

In addition a periodontal disease-relevant cell culturesystem involving human mononuclear cells challenged withLPS (derived from the periodontal pathogen P gingivalis)was also used to evaluate the effect of CMC25 on the inducedexcessive levels of proinflammatory MMPs and cytokinesNuclear factor-kappaB (NF-120581B) is a transcription factorinvolved in the cell signaling transduction pathway associatedwith inflammation and plays a key role in regulating the cellu-lar immune response to stimuli such as stress cytokines andbacterial or viral antigens [18] Dysregulation of NF-120581B hasbeen linked to cancer and inflammatory diseases [18] There-fore the effect of CMC 25 on the activationphosphorylationof NF-120581B was evaluated as well

2 Materials and Methods

21 Chemical Reagents All chemical reagents LPS from Pgingivalis and curcumin were purchased fromSigma-AldrichCo (St Louis MO) All cell culture reagents were purchasedfrom GibcoInvitrogen Corp (Carlsbad CA) CMC 25 wassynthesized purified (995 pure) and provided by ChemMaster Intl Inc Stony Brook NY

22 InVitroMMP InhibitionAssay (IC50) Human chromato-

graphically pure MMP-9 was purchased from CalbiochemEMD Biosciences Inc (La Jolla CA) MMP-13 was pur-chased from RampD Systems Inc (Minneapolis MN) and thesynthetic octapeptide MMP substrate (DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-dArg) was purchased from Bachem (Kingof Prussia PA) Curcumin and CMC 25 (1ndash500 120583M) wereincubated in 1mM CaCl

2 02M NaCl and 50mM TrisHCl

buffer (pH = 76) with MMP-9 (gelatinase B) or MMP-13 (collagenase-3) at 37∘C for 4 hours as described by uspreviously [11] The reaction mixture was quenched with110-phenanthroline (a zinc chelator that binds this cationin the MMP molecule) and the tripeptide degradation frag-ments of the synthetic octapeptide substrate after incubation(37∘C lmMCa2+) with each MMP were measured by high-performance liquid chromatography (HPLC) using a reverse-phase C18 column (46 times 75mm 35 120583Mmacroporous spher-ical support)The eluate was monitored at 375 nm to quantifythe DNP-labeled peptides The IC

50for each compound was

calculated from the plot of the percentage of inhibition ofenzyme activity versus the concentration of inhibitor [19 20]

23 Cell Culture Assay Human peripheral blood mononu-clear cells (PBMC) were isolated and purified from Leuko-cyte Concentrate (Long Island Blood Bank Melville NY)by density gradient centrifugation and adherence using amethod described by us previously [21] PBMC cells werethen cultured for 18 hours in serum-free macrophage media(Invitrogen Corp Carlsbad CA) at 37∘C (95 air 5 CO

2)

with either LPS derived from P gingivalis (50 ngmL) orvehicle alone Curcumin or CMC 25 was added at final con-centrations of 2 or 5 120583M Conditioned media were analyzedfor the cytokines and proinflammatory mediators TNF-120572IL-1120573 IL-6 MCP-1 and PGE

2by ELISA (see below) and for

MMP-9 by ELISA and by gelatin zymography (see below) asdescribed previously [21 22]

24 Gelatin Zymography The gelatin zymography systemand SDS-PAGE gels containing polyacrylamide copolymer-ized with gelatin at a final concentration of 1mgmL werepurchased from Invitrogen Corp (Carlsbad CA) After elec-trophoresis (120V) the gels were washed with 25 TritonX-100 incubated at 37∘C overnight in calcium assay buffer(40mM TrisHCl 200mMNaCl 10mM CaCl

2 and pH 75)

and then stained with Coomassie Brilliant Blue R-250 Asdescribed by us earlier [19] clear zones of lysis against a bluebackground indicate gelatinolytic activity and were scanneddensitometrically to assess gelatinase activity MMP-2 andMMP-9 standards were purchased from RampD Systems Inc(Minneapolis MN)











2buffer (pH 76


2buffer





3 Results








0

200

400

600

800

1000

1200

1400

1600

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

TNF-120572

(pg

mL)

LPS

+5120583

m

LPS

+5120583

mlowast

lowast

lowastlowast










2levels












0

50

100

150

200

250

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

LPS

+5120583

m

LPS

+5120583

m

lowast

lowast

lowast

lowastlowast

IL-1120573

(pg

mL)




0

20

40

60

80

100

120

Vehi

cle

LPS

alon

e

CMC

25

CMC

25

LPS

+2120583

m

LPS

+5120583

m

PGE 2

(pg

mL)

lowast





2



0

50

100

150

200

250

300

350

400

450

MCP-1 IL-6

pgm

L


lowast

lowast

(a)

0

02

04

06

08

1

12

14

16

Vehicle LPS alone

ngm

L

MMP-9

LPS + 5120583m CMC 25

lowast

(b)






Vehicle LPS alone

MMP-9


CMC 25LPS + 5120583m





0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast





4 Discussion




0

50

100

150

200

250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)



(a)

0

05

1

15

2


MM

P-13

(AU

)

lowast

(b)




MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2buffer (pH 76


2buffer





3 Results








0

200

400

600

800

1000

1200

1400

1600

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

TNF-120572

(pg

mL)

LPS

+5120583

m

LPS

+5120583

mlowast

lowast

lowastlowast










2levels












0

50

100

150

200

250

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

LPS

+5120583

m

LPS

+5120583

m

lowast

lowast

lowast

lowastlowast

IL-1120573

(pg

mL)




0

20

40

60

80

100

120

Vehi

cle

LPS

alon

e

CMC

25

CMC

25

LPS

+2120583

m

LPS

+5120583

m

PGE 2

(pg

mL)

lowast





2



0

50

100

150

200

250

300

350

400

450

MCP-1 IL-6

pgm

L


lowast

lowast

(a)

0

02

04

06

08

1

12

14

16

Vehicle LPS alone

ngm

L

MMP-9

LPS + 5120583m CMC 25

lowast

(b)






Vehicle LPS alone

MMP-9


CMC 25LPS + 5120583m





0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast





4 Discussion




0

50

100

150

200

250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)



(a)

0

05

1

15

2


MM

P-13

(AU

)

lowast

(b)




MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

200

400

600

800

1000

1200

1400

1600

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

TNF-120572

(pg

mL)

LPS

+5120583

m

LPS

+5120583

mlowast

lowast

lowastlowast










2levels












0

50

100

150

200

250

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

LPS

+5120583

m

LPS

+5120583

m

lowast

lowast

lowast

lowastlowast

IL-1120573

(pg

mL)




0

20

40

60

80

100

120

Vehi

cle

LPS

alon

e

CMC

25

CMC

25

LPS

+2120583

m

LPS

+5120583

m

PGE 2

(pg

mL)

lowast





2



0

50

100

150

200

250

300

350

400

450

MCP-1 IL-6

pgm

L


lowast

lowast

(a)

0

02

04

06

08

1

12

14

16

Vehicle LPS alone

ngm

L

MMP-9

LPS + 5120583m CMC 25

lowast

(b)






Vehicle LPS alone

MMP-9


CMC 25LPS + 5120583m





0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast





4 Discussion




0

50

100

150

200

250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)



(a)

0

05

1

15

2


MM

P-13

(AU

)

lowast

(b)




MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

Vehi

cle

LPS

alon

e

curc

umin

curc

umin

CMC

25

CMC

25

LPS

+2120583

m

LPS

+2120583

m

LPS

+5120583

m

LPS

+5120583

m

lowast

lowast

lowast

lowastlowast

IL-1120573

(pg

mL)




0

20

40

60

80

100

120

Vehi

cle

LPS

alon

e

CMC

25

CMC

25

LPS

+2120583

m

LPS

+5120583

m

PGE 2

(pg

mL)

lowast





2



0

50

100

150

200

250

300

350

400

450

MCP-1 IL-6

pgm

L


lowast

lowast

(a)

0

02

04

06

08

1

12

14

16

Vehicle LPS alone

ngm

L

MMP-9

LPS + 5120583m CMC 25

lowast

(b)






Vehicle LPS alone

MMP-9


CMC 25LPS + 5120583m





0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast





4 Discussion




0

50

100

150

200

250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)



(a)

0

05

1

15

2


MM

P-13

(AU

)

lowast

(b)




MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Vehicle LPS alone

MMP-9


CMC 25LPS + 5120583m





0

2010

3040506070

Vehicle LPS alone

Phos

phor

ylat

ion

()

LPS + 5120583m CMC 25

NF120581B

lowast





4 Discussion




0

50

100

150

200

250

300

350

400

MMP-9 MMP-2

Scan

ning

uni

t (AU

)



(a)

0

05

1

15

2


MM

P-13

(AU

)

lowast

(b)




MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMP-9

MMP-2


(a)

0100200300400500600700800900

1000

NDC UD

Scan

ning

uni

ts

MMP-9MMP-2

D + CMC 25(100mgkg)

(b)

05

10152025303540


IL-1120573

(pg

mL)

(c)


0100200300400500600700

NDC UD D + CMC 25

Glu

cose

(mg

dL)


















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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