Top Banner
Curcumin Induces G 2 /M Arrest and Apoptosis in Cisplatin- Resistant Human Ovarian Cancer Cells by Modulating Akt and p38 MAPK Nathan M. Weir 1,¥ , Karuppaiyah Selvendiran 1,¥ , Vijay Kumar Kutala 1 , Liyue Tong 1 , Shilpa Vishwanath 1 , Murugesan Rajaram 1 , Susheela Tridandapani 1 , Shrikant Anant 2 , and Periannan Kuppusamy 1,* 1 Davis Heart and Lung Research Institute and Comprehensive Cancer Center; Department of Internal Medicine; Ohio State University; Columbus, Ohio USA 2 University of Oklahoma; Norman, Oklahoma USA Abstract Curcumin, a major active component of turmeric, is known to induce apoptosis in several types of cancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the present study was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovarian cancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin- resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxide generation was observed in both CR and CS cells treated with curcumin. Curcumin induced G 2 /M phase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through the activation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylation of Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed that curcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the induction of superoxide generation, G 2 /M arrest, and apoptosis. Keywords ovarian cancer; curcumin; superoxide; cell cycle; apoptosis; Akt INTRODUCTION Ovarian cancer is the most commonly diagnosed and lethal gynecological malignancy in the United States and Europe. 1,2 The high mortality rate is attributed to the lack of early detection and also due to the development of chemoresistance. 3,4 Although platinum-based compounds such as cisplatin, in combination with taxanes are initially effective, the five-year survival rates are only about 50%. 5 Despite the fact that most of the ovarian tumors are sensitive to chemotherapy for the first time, the development of recurrent tumors that are resistant to cisplatin remains a major hurdle to successful therapy and is responsible for poor long-term overall survival. 5,6 Cisplatin resistance is associated with defects in the apoptotic pathway including p53 and Bcl-2 family members and death receptors. 7 Cisplatin resistance is also associated with the altered activation of signaling pathways which include PI3K/Akt, 8 MAPK 9 or JAK/STAT. 10 Another suggested mechanism for the drug resistance is the increase *Correspondence to: Periannan Kuppusamy; Ohio State University; 420 West 12th Ave, Room 114; Columbus, Ohio 43210 USA; Tel.: 614.292.8998; Fax: 614.292.8454; Email: [email protected]. These authors contributed equally to this paper. NIH Public Access Author Manuscript Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17. Published in final edited form as: Cancer Biol Ther. 2007 February ; 6(2): 178–184. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
16
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: nihms16826

Curcumin Induces G2/M Arrest and Apoptosis in Cisplatin-Resistant Human Ovarian Cancer Cells by Modulating Akt and p38MAPK

Nathan M. Weir1,¥, Karuppaiyah Selvendiran1,¥, Vijay Kumar Kutala1, Liyue Tong1, ShilpaVishwanath1, Murugesan Rajaram1, Susheela Tridandapani1, Shrikant Anant2, andPeriannan Kuppusamy1,*

1 Davis Heart and Lung Research Institute and Comprehensive Cancer Center; Department of InternalMedicine; Ohio State University; Columbus, Ohio USA

2 University of Oklahoma; Norman, Oklahoma USA

AbstractCurcumin, a major active component of turmeric, is known to induce apoptosis in several types ofcancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the presentstudy was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovariancancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin-resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxidegeneration was observed in both CR and CS cells treated with curcumin. Curcumin induced G2/Mphase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through theactivation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylationof Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed thatcurcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the inductionof superoxide generation, G2/M arrest, and apoptosis.

Keywordsovarian cancer; curcumin; superoxide; cell cycle; apoptosis; Akt

INTRODUCTIONOvarian cancer is the most commonly diagnosed and lethal gynecological malignancy in theUnited States and Europe.1,2 The high mortality rate is attributed to the lack of early detectionand also due to the development of chemoresistance.3,4 Although platinum-based compoundssuch as cisplatin, in combination with taxanes are initially effective, the five-year survival ratesare only about 50%.5 Despite the fact that most of the ovarian tumors are sensitive tochemotherapy for the first time, the development of recurrent tumors that are resistant tocisplatin remains a major hurdle to successful therapy and is responsible for poor long-termoverall survival.5,6 Cisplatin resistance is associated with defects in the apoptotic pathwayincluding p53 and Bcl-2 family members and death receptors.7 Cisplatin resistance is alsoassociated with the altered activation of signaling pathways which include PI3K/Akt,8MAPK9 or JAK/STAT.10 Another suggested mechanism for the drug resistance is the increase

*Correspondence to: Periannan Kuppusamy; Ohio State University; 420 West 12th Ave, Room 114; Columbus, Ohio 43210 USA; Tel.:614.292.8998; Fax: 614.292.8454; Email: [email protected].†These authors contributed equally to this paper.

NIH Public AccessAuthor ManuscriptCancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

Published in final edited form as:Cancer Biol Ther. 2007 February ; 6(2): 178–184.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 2: nihms16826

in intracellular thiols in the redox pathway, which may inactivate and remove the platinumcompounds.11,12 Several groups have targeted this redox pathway in an attempt to circumventthe thiol-induced resistance.13 Recently, we observed that a nitroaspirin derivative(NCX-4016, an NO donor) partially restored the cisplatin sensitivity by depleting thiols.14

Curcumin (diferuloylmethane) is a major constituent of turmeric powder which is extractedfrom the rhizomes of the plant Curcuma longa found in south and southeast tropical Asia. Ithas been widely used in Asian countries for centuries in daily cooking preparations.15 Themedicinal value of curcumin has been well recognized for its anti-inflammatory, antimicrobial,wound healing, and anti-tumor activities.16–19 Several studies have indicated that people insoutheastern Asian countries have a much lower risk of acquiring colon, gastrointestinal,prostate, breast, ovarian, and other cancers than Western populations.20,21 It is likely thatconstituents of their diets such as curcumin, garlic, ginger, chillies, etc., may play a role in theprevention of such cancers.21 Studies have shown the chemopreventive properties of curcuminfrom human malignances.22–24 Clinical evaluations of curcumin as a chemopreventive agentfor many cancers including breast, prostate, colon, and lung have been carried out.25,26 Theanti-carcinogenic properties of curcumin in animal models have been demonstrated.27,28However, the molecular mechanism underlying curcumin’s chemopreventive effect has notbeen fully elucidated, although several mechanisms have been proposed.29

Cell cycle inhibition and the induction of apoptosis are common mechanism(s) proposed forthe anticancer effects of curcumin.18,24,30,31 Recent studies have shown that curcumin is apotent inhibitor of tumor initiation in vivo.32,33 The antiproliferative and apoptotic effects ofcurcumin on tumor cells in vitro have also been reported.34 Curcumin has been shown to induceapoptosis of cancer cells via inhibition of NFκB and STAT3.32,33 Curcumin has also beenshown to suppress the expression of various NFκB-regulated genes, including Bcl-2, COX-2,cyclin D1 and adhesion molecules.35 The anticancer activity of curcumin has recently beenattributed to the modification of thioredoxin reductase, an enzyme that plays a key role inmodulating the redox pathway.36 Although the anti-cancer effects of curcumin have beenstudied in various cancer cells, it remains unknown whether curcumin possesses anticancereffects on drug-resistant cells such as cisplatin-resistant ovarian cancer cells. Therefore, theaim of this study was to determine whether curcumin shows cytotoxic effects in cisplatin-resistant human ovarian cancer cells and to elucidate the mechanism of its action. Our resultsshowed that (1) curcumin enhanced superoxide generation in ovarian cancer cells; (2) curcumininduced cell cycle arrest and apoptosis in cisplatin-resistant ovarian cancer cells; (3) thecurcumin-induced apoptosis in cisplatin-resistant cells was mediated through the inhibition ofAkt and an increase in the activation of p38 MAPK and p53.

MATERIALS AND METHODSReagents and culture materials

Curcumin ((1,7-bis[4-hy-droxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione), GSH, (L-glutamyl-L-cysteinylglycine), DMSO (dimethyl sulfoxide) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were obtained from Sigma (St Louis,MO). Cell culture medium (RPMI 1640), fetal bovine serum, antibiotics, sodium pyruvate,trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco, BRL (Grand Island,NY). Polyvinylidene fluoride membrane (PVDF) (Millipore), and molecular weight markerwere obtained from Bio-Rad, and Fluoromount-G from Southern Biotech. Lab-Tek II chamberslides were purchased from Nalge Nunc International (Naperville, IL). Antibodies against poly-adenosine diphosphate ribose polymerase (PARP), Bax, p38 MAPK, caspase-7 and cleavedcaspase-3 (19 kDa and 17 kDa) and cleaved caspase-7 (20 kDa) were purchased from CellSignaling Technology (Beverly, MA), and Akt (Ser473) and p53 from Santa CruzBiotechnology (Santa Cruz, CA). RNase was from Promega Corporation (Madison, WI).

Weir et al. Page 2

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 3: nihms16826

Enhanced chemiluminescence (ECL) reagents were obtained from Amersham PharmaciaBiotech (Buckinghamshire, UK). All other reagents and compounds were analytical grades.

Cells and culture conditionsCisplatin-resistant (CR) and cisplatin-sensitive (CS) human ovarian cancer cells were obtainedfrom Dr. Sridhar (Howard University Medical School, Washington DC). Cells were grown inRPMI 1640 medium supplemented with 10% FBS, 2% sodium pyruvate, 1% penicillin and1% streptomycin. Cells were grown in T-75 flasks to 80% confluence at 37°C in an atmosphereof 5% CO2 and humidified air. Cells were routinely trypsinized (0.05% trypsin/EDTA) andthe cell count was determined by using a NucleoCounter, automated cell counter, (NewBrunswick Scientific, Edison, NJ).

Cell proliferation assayCell proliferation was determined using the conversion of MTT to formazan via mitochondrialoxidation. Cells were grown in T-75 flasks to >80% confluence. They were then trypsinized,counted, and seeded in 96-well plates with an average of 7,000 cells/well. Cells were incubatedovernight and then treated in triplicate with 10 or 50 uM curcumin for 12 or 24 h. Allexperiments were repeated at least three times.

Superoxide determination by DHE fluorescenceCells were seeded in Chamber slides with 1 x 105 cells per chamber and incubated overnight.The following day, cells were preincubated with dihydroethidium (DHE, 10 μM) for 1 h. Thecells were then washed with PBS and treated with varying doses of curcumin for 4 h. Afterincubation, the cells were washed and fixed with 3% paraformaldehyde supplemented withMcIIvaine’s buffer for 15 min at 4°C. The paraformaldehyde was then washed off and a coverslip was fixed using Fluoromount-G. Red fluorescence, indicating superoxide formation, wasdetected on a Nikon Eclipse TE2000-U, using excitation/emission at 488/585 nm. Images (12bit) were acquired using a 400 ms exposure time and the fluorescence intensity was quantifiedby MetaMorph software. The intensity was calculated as an average of four areas with a similarnumber of cells.

GSH assayIntracellular levels of GSH were determined by spectrophotometry using DTNB (5,5'-dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, Cayman Chemical Co., Ann Arbor, Michigan) whichproduces a yellow color with GSH to yield TNBA (5-thio-2- nitrobenzoic acid). Cell extractswere treated with phosphoric acid, the precipitated proteins were centrifuged, and thesupernatants were treated with triethanolamine to bring to neutral pH and then the DTNBreagent was added and the resulting solution was measured using a 96-well plate ELISA reader(Beckmann Coulter, AD 340) at 405 nm. All experiments were run in at least four parallelsand repeated thrice. The concentration of GSH was determined from a standard curve preparedwith known concentrations of GSH under similar conditions.

Cell cycle analysis by flow cytometryCurcumin-treated cells were harvested, fixed overnight with 75% ethanol at −20°C, washedthree times with PBS, treated with RNase A (1 μg/ml), and then stained with propidium iodide.After propidium iodide staining, the cells were analyzed by flow cytometry (Becton Dickinson,Franklin Lakes, NJ). The percentage of cells in the G2/M and sub-G1 apoptotic cell populationwas determined using CELLQuest software (Becton Dickinson).

Weir et al. Page 3

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 4: nihms16826

Immunoblot analysisCells were lysed in RIPA buffer, phenyl-methylsulphonyl fluoride (PMSF, 0.1 mM), sodiumorthovanadate (1 mM), and aprotinin and leupeptin (2 μg/ml). The lysate was centrifuged at12000 x g for 20 min at 4°C and the supernatant was removed. The protein concentration wasmeasured using a Bio-Rad protein assay kit. After boiling for 10 min in the presence of 2-mercaptoethanol, samples containing cell lysate protein were separated on a 10 or 15% sodiumdodecyl sulfate-polyacrylamide (SDS) gel and then transferred onto equilibrated PVDFmembranes. After skimmed milk blocking, the membranes were incubated with primaryantibodies individually with caspase-7, PARP, cleaved caspase-3 (19 kDa and 17 kDa),phosphorylated Akt (Ser473), p38 MAPK, or p53 (1:1000 dilution). The bound antibodieswere detected with horseradish peroxidase-labelled sheep anti-mouse IgG or horseradishperoxidase-labelled donkey anti-rabbit IgG (GE Health Care Corp, Piscataway, NJ). Theimmunoblots were then developed with enhanced chemiluminescence reagents according tomanufacturer’s recommendations.

Statistical analysisAll data were expressed as mean ± SE. Comparisons among groups were performed byStudent’s t-test. The significance level was set at p < 0.05.

RESULTSCisplatin-sensitive (CS) and cisplatin-resistant (CR) human ovarian cancer cells

The cisplatin sensitivity of the two ovarian cancer cell lines was confirmed by a cell-proliferation assay (MTT) after treating the cells with cisplatin for 24 h (Fig. 1A). The CS andCR cells exposed to cisplatin (5 μg/ml) for 24 h showed a viability of 42 and 82%, respectively,confirming their differential sensitivity to cisplatin.

Curcumin inhibits the proliferation of CS and CR cellsThe sensitivity of CR ovarian cancer cells to curcumin is unknown. To determine whethercurcumin inhibits the proliferation of CR cell lines, cells were grown in the presence of varyingconcentrations of curcumin (0–50 μM) for 48 h and the cell proliferation was measured byMTT assay. As shown in Figure 1B, curcumin inhibited both CS and CR cell proliferation ina dose-dependent manner. The IC50 dose of curcumin for proliferation of CS and CR cells was20 μM. Under similar conditions Chinese hamster ovary (CHO) cells (noncancer) exhibitedan IC50 of 26 μM.

Curcumin induces superoxide generation in ovarian cancer cellsTo determine whether curcumin induces superoxide generation in CS and CR cell lines, cellswere grown on chamber slides for 24 h. The cells were then preincubated with DHE (10 μM)for 1 h, followed by curcumin (10 μM) for 3 h. The nonfluorescent DHE is converted bysuperoxide into fluorescent hydroethidine which can be measured by fluorescence microscopy.Greater fluorescence intensity would indicate more superoxide was present. Both CS and CRcells showed intense fluorescence (Fig. 2), suggesting the generation of superoxide duringcurcumin treatment. CS cells treated with curcumin showed a significantly higher fluorescenceintensity compared to the CR cells (p < 0.01) (Fig. 2). Cells pretreated with N-acetylcysteine(NAC, 10 mM), an antioxidant, exhibited significantly lower levels of superoxide comparedto the cells not treated with NAC. The superoxide generation in CHO cells treated withcurcumin was significantly lower compared to the cancer cells and NAC completely abolishedthe fluorescence intensity.

Weir et al. Page 4

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 5: nihms16826

Curcumin increases glutathione levels in ovarian cancer cellsThe effect of curcumin on glutathione synthesis in CS and CR cells was determined bymeasuring the levels of intracellular GSH by spectrophotometry. Glutathione levels in theuntreated CR cells were significantly higher (p < 0.01) compared to CS cells (Fig. 3). Both CSand CR cells incubated with curcumin (10 or 50 μM) for 24 h showed increased glutathionelevels. Compared to controls, there were a 214 and 271% increase in glutathione levels in CScells and a 168 and 235% increase in CR cells treated with 10 and 50 μM of curcumin,respectively.

Curcumin causes G2/M Phase cell cycle arrest and induces apoptosis in CR cellsTo determine whether curcumin inhibits the cell cycle progression of CR cells, cells weregrown to 70% confluence and the cell cycle distribution was analyzed by flow cytometry aftera 12- and 24-h exposure to curcumin (50 μM). It was observed that the percentage of cells inG2/M phase with curcumin treatment was 51.5% after 12 h of incubation and decreased to20.1% after 24 h. The percentage of cells in sub-G0/G1 phase was 3.2% and 35.8% after 12and 24 h of incubation, respectively. In control cells the percentage of cells in G2/M and Sub-G0/G1 phase was 20.5% and 1.2% respectively.

Immunoblotting of CR cells treated with curcumin showed increased caspase-7 activity andcleavages of caspase-3 (19 and 17 kDa), caspase-7 (20 kDa), and PARP (89 kDa) after 12 hand were even higher after 24 h of incubation (Fig. 5). Quantification of cleaved caspase-3 andcleaved PARP, done by measuring the relative band intensities, showed that cleaved caspase-3and PARP levels were significantly higher in CR cells incubated with curcumin for 12 (p <0.01) and 24 h (p < 0.001) of incubation (Fig. 5B and C). This data indicated that curcumininduced cell cycle arrest within 12-h of incubation and apoptosis after 24 h of incubation.Preincubation of CR cells with NAC significantly attenuated the curcumin-induced increaseof cleaved caspase-3 and cleaved PARP. Thus, the data clearly showed that curcumin inducedG2/M phase cell-cycle arrest and apoptosis.

Curcumin inhibits Akt and enhances p38 MAPK activationTo investigate the effect of curcumin on Akt activity in CR cells, we examined the regulationof Akt phosphorylation by curcumin. The results showed increased phosphorylation of Akt inCR cells, which was inhibited by curcumin (50 μM) after 12 and 24 h (p < 0.001) of incubation(Fig. 6). A marked increase in the p38 MAPK and p53 phosphorylation (p < 0.001) wasobserved following curcumin exposure for 12 and 24 h (Fig. 6). Pretreatment of the cells withNAC attenuated the curcumin-induced inhibition of Akt, and activation of p38 MAPK andp53.

DISCUSSIONThe present study demonstrated that curcumin induced cytotoxicity in both cisplatin-sensitive(CS) and cisplatin-resistant (CR) human ovarian cancer cells. Both cells exhibited similarresponses to curcumin. We also observed that curcumin induced G2/M cell cycle arrest leadingto apoptosis, possibly by down-regulating anti- apoptotic Akt signaling and/or also byactivating the pro-apoptotic p38 MAPK in parallel with the generation of superoxide radicals.

Reactive oxygen species (ROS) such as superoxide radicals are implicated as importantmediators of apoptotic cell death. MAPK is considered as one of the most important signalingmolecules in ROS-mediated apoptosis in cancer cells.37,38 The results of the present studyindicate that curcumin induces superoxide generation in both CS and CR cells but not in CHOcells. The amount of superoxide generation by curcumin in CR cells was significantly less thanin CS cells. This could be due to the higher levels of endogenous thiol in CR cells as observed

Weir et al. Page 5

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 6: nihms16826

in the current study and also in our earlier study.14 On the other hand, curcumin was lesseffective in CHO cells wherein the superoxide production was observed to be significantlyless. Curcumin-induced ROS generation has been reported in tumor cells such as rathistiocytoma (AK-5), human renal carcinoma cell line (Caki cells), human submandibulargland caracinoma (HSG) was others.39,40 The accumulation of intercellular superoxide maylead to the disruption of the mitochondrial membrane potential, release of cyctochrome c intothe cytosol, with subsequent activation of the caspase cascade, and apoptosis.41 Therefore, thecurcumin-induced superoxide generation may be critical for the modulation of the apoptoticsignaling pathways.

Our study demonstrated that curcumin inhibited the cisplatin-resistant ovarian cancer cellproliferation by inducing G2/M cell cycle arrest leading to apoptotic cell death. Recent studiessuggested that caspase-3 plays an important role in several key events causing DNAfragmentation during apoptosis.42,43 It has been suggested that the cleavage of procaspase-3is an early event in apoptosis induced by chemotherapeutic agents.44 The activation ofcaspase-3 leads to the cleavage of PARP, which serves as a “death substrate”.45 In our study,cleavages of caspase-7, caspase-3, and PARP were observed in CR cells treated with curcumin.These results are in agreement with the published reports, which indicate that curcumin inducesapoptosis in cancer cells.18,31 Pretreatment with NAC attenuated the curcumin-inducedcleavage of caspase-3 and 7, and PARP. In colon carcinoma cells, curcumin induced apoptoticcell death by cell cycle arrest in the S and G2/M phases, whereas in the MCF-7 breast cancercell line this occurred at the G2 or M phases.30,46 Previous studies have shown that curcuminblocks the proliferation of various cancer cells by downregulating the expression of the cyclinD1 protein.47 In vitro studies have shown that curcumin has multiple biological andbiochemical targets which may be related to its anticancer and antitumor activities.18,48 Thesefunctions include inhibition of PI3 kinase activities and induction of G2/M cell cycle arrestalong with the down-regulation of vascular endothelial growth factor (VEGF).48 Curcumininduces caspase-3-independent apoptosis in human multidrug-resistant cells such as humanlymphoblastic leukemia cell line and the human colon-carcinoma cell line.49

The PI3-kinase/Akt pathway contributes to the tumor formation by elevating the activity ofthe anti-apoptotic action of Akt. Akt inhibits apoptosis through phosphorylation of Bad, GSK3,and caspase-9 and activation of transcriptional factors such as Forkhead (FOXO1) and NFκB.50,51 The present study showed increased phosphorylation of Akt in CR cells and curcumininhibited Akt phosphorylation. The phosphorylation of Akt is routinely used as readout for theAkt activation. The inhibition of Akt phosphorylation by curcumin is an important mechanismof action in CR ovarian cancer cells. Similar results were also observed by others in humanmantle cell lymphoma (MCL).32 Suppression of Akt activation could lead to p53 activation,which in turn may lead to the activation of pro-apoptotic signaling pathways.52 The regulationof p53 by Akt is a critical determinant of cisplatin-induced chemoresistance in ovarian cancercells.53 In the current study, the observation of an increase in the phosphorylation of p53 inCR cells treated with curcumin is supported by the PI3K-Akt pathway.

The p38 MAPK pathway is implicated in cancer cell apoptosis and is induced by severalchemotherapeutic drugs.54 Oxidative stress has been reported to play a role in p38 MAPKactivation.38 We found a marked increase in the phosphorylation of p38 MAPK followingcurcumin treatment in CR cells. The increased super-oxide production induced by curcuminsubsequently activated p38 MAPK resulting in the activation of caspases, PARP cleavage,followed by cell death. These findings imply that the ROS trigger is an upstream signal whichinitiates the series of apoptotic events induced by curcumin. Pretreatment with NAC attenuatedthe curcumin-induced p38 MAPK activation. These results suggest that the activation of thep38 MAPK pathway plays a causal role in the curcumin-induced apoptosis in CR cells. Thus,the increased superoxide generation by curcumin may contribute to the enhanced p38 MAPK

Weir et al. Page 6

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 7: nihms16826

activity in resistant cells. It was also reported that the loss of the capacity to activate p38 MAPKin response to cisplatin treatment may be one of the mechanisms of chemoresistance.55 Thus,activation of p38 MAPK and an increase in the caspase-3 activities appears to contribute tothe proapoptotic effect of curcumin in CR cells. Taken together, our results indicate thatcurcumin produces a profound effect on chemoresistant ovarian cancer cell proliferation. Theinhibition of Akt, the activation of p38 MAPK and p53 and the increase in caspase-3 activityappear to contribute to the proapoptotic effects of curcumin.

The chemoresistance of ovarian cancer was also linked to increased cellular glutathionecontent.14,56–58 Depletion of cellular glutathione has been shown to sensitize the resistantcancer cells to cisplatin and other anticancer drugs.14,59,60 In the present study we observedthat curcumin significantly enhanced the intracellular GSH levels in both CR and CS cells andbut not in CHO cells. However, the anti-proliferative efficacy of curcumin on CR cells wasnot significantly different from that of CS cells. These results indicate that in CR cells, curcumininduces apoptosis through non-glutathione dependent pathways. The exact mechanism of theenhanced glutathione synthesis in resistant cells is not yet known and warrants furtherinvestigation. In a similar fashion, Piwocka et al, demonstrated the Jurkat cells treated withcurcumin showed increased intracellular GSH levels.49 Curcumin has also been shown toincrease the levels of GSH in kidney cells,61 and rat hepatocytes.62

In summary, the current study demonstrated that curcumin induces G2/M arrest and apoptosisin cisplatin-resistant human ovarian cancer cells. Further, we showed that curcumin inducessuperoxide generation and enhances p38 MAPK and p53 phosphorylation. The increase in Aktactivity in cisplatin-resistant ovarian cancer cells was attenuated by curcumin. Our resultshighlight the importance of Akt and p53 signaling pathways as the new targets for thedevelopment of therapeutic strategies against drug-resistant cancer cells, in general, andcisplatin-resistant cancer cells, in particular. Our results indicate that curcumin may be apromising compound, especially for the treatment of chemo-resistant human ovarian cancer.

Acknowledgements

We acknowledge the financial support from the NIH grant CA 102264.

References1. Cancer Facts and Figures. Atlanta: American Cancer Society (ACS); 2005.2. Smith LH, Morris CR, Yasmeen S, Parikh-Patel A, Cress RD, Romano PS. Ovarian cancer: Can we

make the clinical diagnosis earlier? Cancer 2005;104:1398–407. [PubMed: 16116591]3. McClay EF, Albright KD, Jones JA, Eastman A, Christen R, Howell SB. Modulation of cisplatin

resistance in human malignant melanoma cells. Cancer Res 1992;52:6790–6. [PubMed: 1458467]4. McClay EF, Albright KD, Jones JA, Christen RD, Howell SB. Tamoxifen modulation of cisplatin

sensitivity in human malignant melanoma cells. Cancer Res 1993;53:1571–6. [PubMed: 8453625]5. Cannistra SA. Cancer of the ovary. N Engl J Med 2004;351:2519–29. [PubMed: 15590954]6. Kelland LR. Emerging drugs for ovarian cancer. Expert Opin Emerg Drugs 2005;10:413–24. [PubMed:

15934876]7. Arts HJ, Willemse PH, Tinga DJ, de Vries EG, van der Zee AG. Laparoscopic placement of PAP

catheters for intraperitoneal chemotherapy in ovarian carcinoma. Gynecol Oncol 1998;69:32–5.[PubMed: 9570995]

8. Lee S, Choi EJ, Jin C, Kim DH. Activation of PI3K/Akt pathway by PTEN reduction and PIK3CAmRNA amplification contributes to cisplatin resistance in an ovarian cancer cell line. Gynecol Oncol2005;97:26–34. [PubMed: 15790433]

9. Choi KC, Auersperg N, Leung PC. Mitogen-activated protein kinases in normal and (pre)neoplasticovarian surface epithelium. Reprod Biol Endocrinol 2003;1:71. [PubMed: 14577832]

Weir et al. Page 7

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 8: nihms16826

10. Ikuta K, Takemura K, Kihara M, Nishimura M, Ueda N, Naito S, Lee E, Shimizu E, Yamauchi A.Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human nonsmall cell lung cancer cells. Oncol Rep 2005;13:217–22. [PubMed: 15643501]

11. Jansen BA, Brouwer J, Reedijk J. Glutathione induces cellular resistance against cationic dinuclearplatinum anticancer drugs. J Inorg Biochem 2002;89:197–202. [PubMed: 12062123]

12. Henderson CJ, McLaren AW, Moffat GJ, Bacon EJ, Wolf CR. Pi-class glutathione S-transferase:Regulation and function. Chem Biol Interact 1998;111–112:69–82.

13. Rudin CM, Yang Z, Schumaker LM, VanderWeele DJ, Newkirk K, Egorin MJ, Zuhowski EG, CullenKJ. Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res2003;63:312–8. [PubMed: 12543781]

14. Bratasz A, Weir NM, Parinandi NL, Zweier JL, Sridhar R, Ignarro LJ, Kuppusamy P. Reversal tocisplatin sensitivity in recurrent human ovarian cancer cells by NCX-4016, a nitro derivative ofaspirin. Proc Natl Acad Sci USA 2006;103:3914–9. [PubMed: 16497833]

15. Ammon HP, Wahl MA. Pharmacology of curcuma longa. Planta Med 1991;57:1–7. [PubMed:2062949]

16. Manju V, Nalini N. Chemopreventive efficacy of ginger, a naturally occurring anticarcinogen duringthe initiation, post-initiation stages of 1,2 dimethylhydrazine-induced colon cancer. Clin Chim Acta2005;358:60–7. [PubMed: 16018877]

17. Lodha R, Bagga A. Traditional Indian systems of medicine. Ann Acad Med Singapore 2000;29:37–41. [PubMed: 10748962]

18. Mukhopadhyay A, Bueso-Ramos C, Chatterjee D, Pantazis P, Aggarwal BB. Curcumindownregulates cell survival mechanisms in human prostate cancer cell lines. Oncogene2001;20:7597–609. [PubMed: 11753638]

19. Jagetia GC, Rajanikant GK. Role of curcumin, a naturally occurring phenolic compound of turmericin accelerating the repair of excision wound, in mice whole-body exposed to various doses of gamma-radiation. J Surg Res 2004;120:127–38. [PubMed: 15172199]

20. Dorai T, Aggarwal BB. Role of chemopreventive agents in cancer therapy. Cancer Lett 2004;215:129–40. [PubMed: 15488631]

21. Sinha R, Anderson DE, McDonald SS, Greenwald P. Cancer risk and diet in India. J Postgrad Med2003;49:222–8. [PubMed: 14597785]

22. Pal S, Choudhuri T, Chattopadhyay S, Bhattacharya A, Datta GK, Das T, Sa G. Mechanisms ofcurcumin-induced apoptosis of Ehrlich’s ascites carcinoma cells. Biochem Biophys Res Commun2001;288:658–65. [PubMed: 11676493]

23. Rinaldi AL, Morse MA, Fields HW, Rothas DA, Pei P, Rodrigo KA, Renner RJ, Mallery SR.Curcumin activates the aryl hydrocarbon receptor yet significantly inhibits (−)-benzo(a)pyrene-7R-trans-7,8-dihydrodiol bioactivation in oral squamous cell carcinoma cells and oral mucosa. CancerRes 2002;62:5451–6. [PubMed: 12359752]

24. Choudhuri T, Pal S, Agwarwal ML, Das T, Sa G. Curcumin induces apoptosis in human breast cancercells through p53-dependent Bax induction. FEBS Lett 2002;512:334–40. [PubMed: 11852106]

25. Kelloff GJ, Crowell JA, Steele VE, Lubet RA, Malone WA, Boone CW, Kopelovich L, Hawk ET,Lieberman R, Lawrence JA, Ali I, Viner JL, Sigman CC. Progress in cancer chemoprevention:Development of diet-derived chemopreventive agents. J Nutr 2000;130:467S–71S. [PubMed:10721931]

26. Boone CW, Kelloff GJ. Biomarker end-points in cancer chemoprevention trials. IARC Sci Publ1997:273–80. [PubMed: 9354926]

27. Liu JY, Lin SJ, Lin JK. Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells. Carcinogenesis 1993;14:857–61. [PubMed:8504477]

28. Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV, Reddy BS. Chemopreventiveeffect of curcumin, a naturally occurring anti-inflammatory agent, during the promotion/progressionstages of colon cancer. Cancer Res 1999;59:597–601. [PubMed: 9973206]

29. Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: Preclinical and clinical studies.Anticancer Res 2003;23:363–98. [PubMed: 12680238]

Weir et al. Page 8

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 9: nihms16826

30. Chen H, Zhang ZS, Zhang YL, Zhou DY. Curcumin inhibits cell proliferation by interfering with thecell cycle and inducing apoptosis in colon carcinoma cells. Anticancer Res 1999;19:3675–80.[PubMed: 10625938]

31. Anto RJ, Mukhopadhyay A, Denning K, Aggarwal BB. Curcumin (diferuloylmethane) inducesapoptosis through activation of caspase-8, BID cleavage and cytochrome c release: Its suppressionby ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 2002;23:143–50. [PubMed: 11756235]

32. Shishodia S, Amin HM, Lai R, Aggarwal BB. Curcumin (diferuloylmethane) inhibits constitutiveNF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantlecell lymphoma. Biochem Pharmacol 2005;70:700–13. [PubMed: 16023083]

33. Rajasingh J, Raikwar HP, Muthian G, Johnson C, Bright JJ. Curcumin induces growth-arrest andapoptosis in association with the inhibition of constitutively active JAK-STAT pathway in T cellleukemia. Biochem Biophys Res Commun 2006;340:359–68. [PubMed: 16364242]

34. Bush JA, Cheung KJ Jr, Li G. Curcumin induces apoptosis in human melanoma cells through a Fasreceptor/caspase-8 pathway independent of p53. Exp Cell Res 2001;271:305–14. [PubMed:11716543]

35. Li L, Aggarwal BB, Shishodia S, Abbruzzese J, Kurzrock R. Nuclear factor-kappaB and IkappaBkinase are constitutively active in human pancreatic cells, and their down-regulation by curcumin(diferuloylmethane) is associated with the suppression of proliferation and the induction of apoptosis.Cancer 2004;101:2351–62. [PubMed: 15476283]

36. Fang J, Lu J, Holmgren A. Thioredoxin reductase is irreversibly modified by curcumin: A novelmolecular mechanism for its anticancer activity. J Biol Chem 2005;280:25284–90. [PubMed:15879598]

37. Cobb MH. MAP kinases pathways. Prog Biophys Mol Biol 1999;71:479–500. [PubMed: 10354710]38. Park SJ, Kim IS. The role of p38 MAPK activation in auranofin-induced apoptosis of human

promyelocytic leukaemia HL-60 cells. Br J Pharmacol 2005;146:506–13. [PubMed: 16086031]39. Bhaumik S, Anjum R, Rangaraj N, Pardhasaradhi BV, Khar A. Curcumin mediated apoptosis in

AK-5 tumor cells involves the production of reactive oxygen intermediates. FEBS Lett1999;456:311–4. [PubMed: 10456330]

40. Woo JH, Kim YH, Choi YJ, Kim DG, Lee KS, Bae JH, Min do S, Chang JS, Jeong YJ, Lee YH, ParkJW, Kwon TK. Molecular mechanisms of curcumin-induced cytotoxicity: Induction of apoptosisthrough generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release ofcytochrome c and inhibition of Akt. Carcinogenesis 2003;24:1199–208. [PubMed: 12807727]

41. Singh SV, Srivastava SK, Choi S, Lew KL, Antosiewicz J, Xiao D, Zeng Y, Watkins SC, JohnsonCS, Trump DL, Lee YJ, Xiao H, Herman-Antosiewicz A. Sulforaphane-induced cell death in humanprostate cancer cells is initiated by reactive oxygen species. J Biol Chem 2005;280:19911–24.[PubMed: 15764812]

42. Blanc C, Deveraux QL, Krajewski S, Janicke RU, Porter AG, Reed JC, Jaggi R, Marti A. Caspase-3is essential for procaspase-9 processing and cisplatin-induced apoptosis of MCF-7 breast cancer cells.Cancer Res 2000;60:4386–90. [PubMed: 10969782]

43. Taylor JK, Zhang QQ, Monia BP, Marcusson EG, Dean NM. Inhibition of Bcl-xL expressionsensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli. Oncogene1999;18:4495–504. [PubMed: 10442640]

44. Henkels KM, Turchi JJ. Cisplatin-induced apoptosis proceeds by caspase-3-dependent and -independent pathways in cisplatin-resistant and -sensitive human ovarian cancer cell lines. CancerRes 1999;59:3077–83. [PubMed: 10397248]

45. Rheaume E, Cohen LY, Uhlmann F, Lazure C, Alam A, Hurwitz J, Sekaly RP, Denis F. The largesubunit of replication factor C is a substrate for caspase-3 in vitro and is cleaved by a caspase-3-likeprotease during Fas-mediated apoptosis. Embo J 1997;16:6346–54. [PubMed: 9351817]

46. Simon A, Allais DP, Duroux JL, Basly JP, Durand-Fontanier S, Delage C. Inhibitory effect ofcurcuminoids on MCF-7 cell proliferation and structure-activity relationships. Cancer Lett1998;129:111–6. [PubMed: 9714342]

47. Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M, Aggarwal BB. Curcumin-inducedsuppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-

Weir et al. Page 9

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 10: nihms16826

mediated retinoblastoma protein phosphorylation. Oncogene 2002;21:8852–61. [PubMed:12483537]

48. Shao ZM, Shen ZZ, Liu CH, Sartippour MR, Go VL, Heber D, Nguyen M. Curcumin exerts multiplesuppressive effects on human breast carcinoma cells. Int J Cancer 2002;98:234–40. [PubMed:11857414]

49. Piwocka K, Bielak-Mijewska A, Sikora E. Curcumin induces caspase-3-independent apoptosis inhuman multidrug-resistant cells. Ann NY Acad Sci 2002;973:250–4. [PubMed: 12485871]

50. Bharti AC, Donato N, Singh S, Aggarwal BB. Curcumin (diferuloylmethane) down-regulates theconstitutive activation of nuclear factor-kappa B and IkappaBalpha kinase in human multiplemyeloma cells, leading to suppression of proliferation and induction of apoptosis. Blood2003;101:1053–62. [PubMed: 12393461]

51. Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular function ofphosphoinositide 3-kinases: Implications for development, homeostasis, and cancer. Annu Rev CellDev Biol 2001;17:615–75. [PubMed: 11687500]

52. Fraser M, Leung BM, Yan X, Dan HC, Cheng JQ, Tsang BK. p53 is a determinant of X-linkedinhibitor of apoptosis protein/Akt-mediated chemoresistance in human ovarian cancer cells. CancerRes 2003;63:7081–8. [PubMed: 14612499]

53. Yang X, Fraser M, Moll UM, Basak A, Tsang BK. Akt-mediated cisplatin resistance in ovarian cancer:Modulation of p53 action on caspase-dependent mitochondrial death pathway. Cancer Res2006;66:3126–36. [PubMed: 16540663]

54. Bulavin DV, Fornace AJ Jr. p38 MAP kinase’s emerging role as a tumor suppressor. Adv Cancer Res2004;92:95–118. [PubMed: 15530558]

55. Brozovic A, Fritz G, Christmann M, Zisowsky J, Jaehde U, Osmak M, Kaina B. Long-term activationof SAPK/JNK, p38 kinase and fas-L expression by cisplatin is attenuated in human carcinoma cellsthat acquired drug resistance. Int J Cancer 2004;112:974–85. [PubMed: 15386344]

56. Yao KS, Godwin AK, Johnson SW, Ozols RF, O’Dwyer PJ, Hamilton TC. Evidence for alteredregulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive andcisplatin-resistant human ovarian cancer cell lines. Cancer Res 1995;55:4367–74. [PubMed:7671249]

57. Pan B, Yao KS, Monia BP, Dean NM, McKay RA, Hamilton TC, O’Dwyer PJ. Reversal of cisplatinresistance in human ovarian cancer cell lines by a c-jun antisense oligodeoxy-nucleotide (ISIS10582): Evidence for the role of transcription factor overexpression in determining resistantphenotype. Biochem Pharmacol 2002;63:1699–707. [PubMed: 12007573]

58. Okuno S, Sato H, Kuriyama-Matsumura K, Tamba M, Wang H, Sohda S, Hamada H, Yoshikawa H,Kondo T, Bannai S. Role of cystine transport in intracellular glutathione level and cisplatin resistancein human ovarian cancer cell lines. Br J Cancer 2003;88:951–6. [PubMed: 12644836]

59. Kachadourian R, Day BJ. Flavonoid-induced glutathione depletion: Potential implications for cancertreatment. Free Radic Biol Med 2006;41:65–76. [PubMed: 16781454]

60. Benlloch M, Ortega A, Ferrer P, Segarra R, Obrador E, Asensi M, Carretero J, Estrela JM.Acceleration of glutathione efflux and inhibition of gamma-glutamyltranspeptidase sensitizemetastatic B16 melanoma cells to endothelium-induced cytotoxicity. J Biol Chem 2005;280:6950–9. [PubMed: 15561710]

61. Venkatesan N, Punithavathi D, Arumugam V. Curcumin prevents adriamycin nephrotoxicity in rats.Br J Pharmacol 2000;129:231–4. [PubMed: 10694226]

62. White EL, Ross LJ, Schmid SM, Kelloff GJ, Steele VE, Hill DL. Screening of potential cancerpreventing chemicals for induction of glutathione in rat liver cells. Oncol Rep 1998;5:507–12.[PubMed: 9468590]

Weir et al. Page 10

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 11: nihms16826

Figure 1.Curcumin inhibits the growth of cisplatin-sensitive (CS) and cisplatin-resistant (CR) ovariancancer cells. (A) To confirm cisplatin resistance, CS and CR cells were treated with cisplatin(5 μg/ml) and incubated for 24 h. Viability was determined using the MTT assay. (B) CS, CRand CHO cells were treated with varying doses of curcumin for 24 h. Cell viability wasdetermined by MTT assay. Values are expressed as mean ± SE (n = 3). *p<0.01 vs control.

Weir et al. Page 11

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 12: nihms16826

Figure 2.Curcumin induces superoxide generation in CS and CR cells. CS, CR or CHO cells were grownin chamber slides and were pretreated with dihydroethidium (10 μM) for 30 min and thenexposed to curcumin (10 μM) for 3 h. All experiments were performed with and without NAC(10 mM). The florescence was monitored using a Nikon fluorescence microscope equippedwith a rhodamine filter. Top, Representative micrographs from triplicate experiments areshown. Bottom. Fluorescence intensity in curcumin treated CR, CS and CHO cells with andwithout NAC are shown. Values are expressed as mean ± SE (n = 3). *p<0.01 vs curcumin.

Weir et al. Page 12

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 13: nihms16826

Figure 3.Curcumin induces glutathione synthesis in CS and CR cells. CS and CR were treated withcurcumin (10 and 50 μM) and incubated for 24 h. Values are expressed as mean ± SE (n = 3).*p < 0.01 vs CS cells; **p < 0.05 vs control; #p < 0.01 vs control.

Weir et al. Page 13

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 14: nihms16826

Figure 4.Curcumin causes G2/M arrest of CR cells. Flow cytometric analysis of the PI staining of CRcells after 24 h of growth in the presence of DMSO (control) vehicle or curcumin (50 μM).Representative values of three experiments are shown. Top: The distribution of total cells inM1-M4 gate in the flow cytometry plot. Bottom: The percentage of distribution of total cellsin sub-G0-G1, G0/G1, S, and G2/M phase.

Weir et al. Page 14

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 15: nihms16826

Figure 5.Curcumin activates procaspases and PARP degradation in CR cells. CR cells were treated withcurcumin (50 μM) for 12 and 24 h and then Western blot analysis was performed for cleavedcaspase-3, caspase-7, and PARP. Top: Representative blot from 3 independent experiments.Bottom: Quantification of band intensities. Values are expressed mean ± SE (n=3).

Weir et al. Page 15

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

Page 16: nihms16826

Figure 6.Curcumin inhibits Akt phosphorylation and enhances the p38 MAPK and p53 phosphorylation.Cells were treated with curcumin (50 μM) for 24 h and then Western blot analysis wasperformed for phospho-Akt (Ser473), phospho-p38 MAPK and phospho-p53. Top: Arepresentative blot from 3 independent experiments is shown. Bottom: Quantification of bandintensities. Values are expressed mean ± SE (n = 3). *p < 0.001 vs control; **p < 0.001 vscurcumin.

Weir et al. Page 16

Cancer Biol Ther. Author manuscript; available in PMC 2007 April 17.

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript

NIH

-PA Author Manuscript