nutrients Article Inhibition of Cell Survival by Curcumin Is Associated with Downregulation of Cell Division Cycle 20 (Cdc20) in Pancreatic Cancer Cells Yu Zhang 1,†, Ying-bo Xue 1,†, Hang Li 1, Dong Qiu 1, Zhi-wei Wang 2,* and Shi-sheng Tan 1,* 1 Department of Oncology, Guizhou People’s Hospital, Guizhou 550002, China; skyline_zyu@163.com (Y.Z.); xueyingbo0619@163.com (Y.-b.X.); lihang.sy@163.com (H.L.); qiudcds@126.com (D.Q.) 2 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA * Correspondence: zwang6@bidmc.harvard.edu (Z.-w.W.); tssh18018@126.com (S.-s.T.) † These authors contributed equally to this work. Received: 22 November 2016; Accepted: 25 January 2017; Published: 4 February 2017 Abstract: Pancreatic cancer is one of the most aggressive human tumors in the United States. Curcumin, a polyphenol derived from the Curcuma longa plant, has been reported to exert its antitumor activity in pancreatic cancer. However, the molecular mechanisms of curcumin-mediated tumor suppressive function have not been fully elucidated. In the current study, we explore whether curcumin exhibits its anti-cancer function through inhibition of oncoprotein cell division cycle 20 (Cdc20) in pancreatic cancer cells. We found that curcumin inhibited cell growth, enhanced apoptosis, induced cell cycle arrest and retarded cell invasion in pancreatic cancer cells. Moreover, we observed that curcumin significantly inhibited the expression of Cdc20 in pancreatic cancer cells. Furthermore, our results demonstrated that overexpression of Cdc20 enhanced cell proliferation and invasion, and abrogated the cytotoxic effects induced by curcumin in pancreatic cancer cells. Consistently, downregulation of Cdc20 promoted curcumin-mediated anti-tumor activity. Therefore, our findings indicated that inhibition of Cdc20 by curcumin could be useful for the treatment of pancreatic cancer patients. Keywords: curcumin; Cdc20; pancreatic cancer; growth; invasion 1. Introduction Pancreatic cancer (PC) is one of the most highly aggressive malignancies in humans. According to the American Cancer Society, there will be 53,670 estimated new cases and 43,090 deaths due to PC in the United States in 2017 [1]. The routine treatments include surgery, radiation and chemotherapy. Although multiple-treatment approaches have been improved, the outcomes of PC patients are still poor. Because PC patients are diagnosed at a late stage and exhibit acquired drug resistance during chemotherapeutic treatment, the 5-year relative survival of PC is currently 7% [1]. Therefore, it is essential to find new agents to improve the treatment outcome in patients with PC. Emerging evidence has supported that curcumin, a golden pigment extracted from turmeric, exhibits anti-tumor properties in various types of human cancers including PC [2–5]. For example, one study showed that curcumin inhibited interleukin 8 production in PC cells [6]. Moreover, it has been reported that curcumin suppressed cell proliferation and induced apoptosis through inhibition of nuclear factor-kappa B (NF-κB) and IkappaB (IκB) kinase in PC cells [7,8]. Further study found that curcumin synergistically potentiated the growth inhibitory and pro-apoptotic effects of celecoxib in PC cells [9]. Consistently, curcumin potentiates anticancer activity of gemcitabine via inhibition of proliferation, angiogenesis, and suppression of the NF-κB pathway in an orthotopic model of Nutrients 2017, 9, 109; doi:10.3390/nu9020109 www.mdpi.com/journal/nutrients Nutrients 2017, 9, 109 2 of 15 PC [10]. Notably, curcumin inhibited PC cell proliferation via downregulation of cyclooxygenase-2 (COX-2), epidermal growth factor receptor (EGFR) and extracellular signal regulated kinase (ERK1/2) signaling pathways [11]. Sahu et al. found that curcumin activated ataxia telangiectasia mutated (ATM)/check point kinase-1 (Chk1), leading to cell cycle arrest and apoptosis in human PC cells [12]. One study identified that Wilms’ tumor gene 1 (WT1) is one target of curcumin in PC cells [13]. Another study validated that curcumin inhibited constitutive signal transducer and activator of transcription 3 (STAT3) phosphorylation and down-regulated survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) expression in PC cells [14]. Sun et al. reported that curcumin reversed the epithelial-mesenchymal transition through inhibition of the Hedgehog signaling pathway in PC cells [15]. Although these reports identified the molecular basis of curcumin-triggered tumor suppression, the underlying molecular mechanisms are still not clear. Emerging evidence has revealed that cell division cycle 20 (Cdc20), an activator of the ligase anaphase-promoting complex/C (APC/C), plays an oncogenic role in tumorigenesis [16]. Higher expression of Cdc20 has been observed in a variety of human cancers and is correlated with poor prognosis [17–19]. For instance, patients with glioblastomas exhibited upregulation of Cdc20, while low-grade glioma patients have downregulation of Cdc20 [20]. Kim et al. found that Cdc20 was upregulated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the uterine cervix [17]. Similarly, increased Cdc20 expression is associated with development and progression of hepatocellular carcinoma [21]. Cdc20 and securin overexpression predicted short-team breast cancer survival [22]. Consistently, high Cdc20 expression is associated with poor prognosis in oral squamous cell carcinoma [18], gastric cancer [23], urothelial bladder cancer [24], colorectal cancer [19], non-small cell lung cancer [25], and pancreatic cancer [26]. In the current study, we explored whether Cdc20 exerts its oncogenic effects on cell growth, apoptosis, migration and invasion in PC cells. Moreover, we defined whether curcumin exerts its antitumor activity via downregulation of Cdc20 in PC cells. We found that curcumin inhibited the Cdc20 expression, leading to anti-cancer activity in PC cells. These results suggest that curcumin could be useful to inhibit the expression of Cdc20 in PC cells. 2. Materials and Methods 2.1. Cell Culture and Reagents Human pancreatic cancer cell lines Patu8988 and Panc-1 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin. The anti-Cdc20 antibody was purchased from Abcam Company (Cambridge, MA, USA). The anti-p21, anti-B-cell lymphoma 2 (Bcl-2), anti-B-cell lymphoma-extra large (Bcl-xL), anti-Bax, anti-caspase-3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). All secondary antibodies were purchased from Thermo Scientific (Waltham, MA, USA). The anti-β-actin, anti-Tubulin, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inhibitor of APC/C (Apcin), a Cdc20 inhibitor, was purchased from BostonBiochem (Boston, MA, USA). Lipofectamine 2000 was purchased from Invitrogen (Waltham, MA, USA). 2.2. MTT Assay The transfected PC cells were seeded at 5 × 103 cells/well in 96-well plate for 24 h and treated with different concentrations of curcumin (chemical abstract service (CAS) number 458-37-7, 99.5% curcumin). Curcumin was dissolved in dimethyl sulfoxide (DMSO) to make a 30-mM stock solution and was directly added to the medium at different concentrations. Cells were treated with 0.1% DMSO as the control group. After 48 h and 72 h, MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37 ◦C. Then, 100 µL DMSO was added to dissolve the MTT-formazan crystals after the supernatant was absorbed. The absorption was measured by the microplate at 490 nm. Nutrients 2017, 9, 109 3 of 15 2.3. Cell Apoptosis Analysis The transfected cells were cultured in six-well plate overnight and treated with curcumin for 48 h. Then, cells were harvested and washed with phosphate buffered saline (PBS), resuspended in 500 µL binding buffer with 5 µL propidium iodide (PI) and 5 µL fluorescein isothiocyanate (FITC)-conjugated anti-annexin V antibody. Apoptosis was measured with a FACScalibur flow cytometer (Becton, Dickinson Company, Franklin Lakes, NJ, USA). The Cell Death Detection ELISA Kit was also used for measuring apoptosis in PC cells treated with curcumin and Cdc20 short hairpin RNA (shRNA) according to the manufacturer’s protocol. Briefly, the cells were lysed after treatments, and the cell lysates were incubated in microtiter plate modules coated with antihistone antibody, and subsequently incubated with anti-DNA peroxidase followed by color development with 2,2 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS)™ substrate. The optical densities of the samples were determined by using the Ultra Multifunctional Microplate Reader at 405 nanometers (nm). 2.4. Cell Scratch Assays The transfected cells were cultured in 6-well plate. After cells reached almost 100%, the supernatant was absorbed. Yellow pipette tips were used to scratch the cell surface. The cells were washed by PBS and treated with curcumin. The scratched area was photographed with a microscope at 0 h and 20 h, respectively. The experiment was repeated twice. The quantification of the results represents three times for cell scratch assays. 2.5. Cell Invasion Assay Cell invasion assay was performed to explore the invasive activity of PC cells treated with curcumin or Cdc20 transfection or combination [27]. Briefly, transfected cells were seeded in the upper chamber with serum-free medium and 500 µL of complete medium was added in the under chamber with the same concentration of curcumin. After incubation for 24 h, the membrane of the chamber was strained with Giemsa and photographed with a microscope. 2.6. Transfection Cells were seeded into 6-well plates and transfected with Cdc20 cDNA or Cdc20 shRNA or empty vector by lipofectamine 2000 following the instruction’s protocol. The transfected cells were subjected to further analysis as described under the results section. 2.7. Quantitative Real-Time Reverse Transcription-PCR (RT-PCR) Analysis The total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) and reversed-transcribed into cDNA by RevertAid First Strand cDNA Synthesis Kit. PCR were conducted by Power SYBR Green PCR Master Mix. The primers used in the PCR reaction were: Cdc20, forward primer (5 -GACCACTCCTAGCAAACCTGG-3 ) and reverse primer (5 -GGGCGTCTGGCTGTTTT CA-3 ); Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward primer (5 -ACCCAGAAG ACTGTGGATGG-3 ) and reverse primer (5 -CAGTGAGCTTCCCGTTCA G-3 ). 2.8. Western Blotting Analysis The harvested cells were washed by PBS and treated with protein lysis buffer. The BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was used to measure the concentrations of the proteins. Protein samples were separated by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane, and then incubated with primary antibody at 4 ◦C overnight. Then, cells were washed with Tris-buffered saline and Tween 20 (TBST) for three times and incubated with second antibody at Nutrients 2017, 9, 109 4 of 15 room teNmutprieenrtas t2u01r7e, f9o, 1r019  h. Then the expression of protein was determined by enhanced luminal-ba4s oefd 15  chemiluromoimne  stceemnpte(rEaCtuLr)e asfsoar y.1  h.  Then  the  expression  of  protein  was  determined  by  enhanced  2.9. Staltuismticinaal lA‐bnaasleydsi schemiluminescent (ECL) assay.  Al2l.9st. aSttiasttiisctaiclala Ananlaylysseis were measured by GraphPad Prism 5.0 (Graph Pad Software, La Jolla, San Diego, AClAl s,taUtiSsAtic)a. lA aNnaOlyVseAs wasesraey mweaassuuresded byto GervapalhuPaatde Pstraistmis t5ic.0a l(Gsirganpihfi cPaandc Seo. ftRweasruel,t sLaw Jeorllea,  presentSeadna  Ds imegeoa,n  Cs A±, SUDS.Ap).<  A0N.05OVwAas  acsosnaysi dwearse duasesds ttaot isetviaclaullaytes isgtnatiifisctiacnalt .significance.  Results  were  3. Resuplrtsesented as means ± SD. p < 0.05 was considered as statistically significant.  3.1. Cur3c. uRmeisnulItnsh  ibited the Expression of Cdc20 M3u.l1t.i pCluercsutumdinie Isnhhiabviteds thhoe wExnptrhesastiocnu orfc Cudmc2in0  inhibited cell growth in PC cells. Since Cdc20 has been considMerueldtiptolep sltauydaiens ihmavpeo rsthaonwt no ntchoagt ecnuirccuromlieni ninphaibnicterdea cteicll tgurmoworthig ienn ePsCis c, ewlles. tSeisntceed Cwdhce2t0h hears  curcumbienecno cuolndsisdueprpedre tsos pthlaey eaxnp irmespsoiortnanotf oCndcoc2g0eninic ProClec ienll ps.aRnceraela-ttiicm teum(RoTr)i-gPeCneRs)isw, wase pteesrtfeodr mwhedethtoer  measurceurthcuemminR cNoAuldle svueplporfesCsd thc2e 0exinprPesCsicoenl losf tCredact2e0d inw PitCh cceullrsc. uRmeailn‐t.imOeu (rRRTT)‐-PPCCRR) wreassu pltesrfsohromweded to  that curmcueamsuinret rtehaet mmeRnNt Asi glenvifielc aonf tClydcd2e0c rinea PseCd cCeldlsc 2tr0eamteRdN wAitlhe vcuelrcinumbointh. OPuart uR8T9‐8P8CaRn drePsualntsc -s1hcoewllesd  (Figuret1hAat) .cTuorcduemteinrm  trienaetmwehnett hsiegrnciufirccaunmtlyi ndceocrueladsedde cCredacs2e0 tmheRCNdAc 2le0vperl oitne ibnoltehv  Pela,twu8e9s8te8r  annbdl oPtatinncg‐1  analysicsewllsa s(Fciognudreu c1tAed). Ttoo mdetaesrumreinteh we Chedtch2e0r pcurorctueimnienx cporuesldsi odnecirneaPsCe tcheell sCadfct2e0r cpurorcteuimn ilnevterle, awtmesetnertn.   We foubnldotthinagt acnuarclyusmis iwnarse mcoanrdkuacbtelyd rteod muecaesdurthe ethCe dCcd2c020p rportoetieninl eevxeplreinssPioCn cine lPlsC( Fceigllus raeft1erB c,Cur)c. uItmisin  knownttrheaattmBeinmt. aWnde fpo2u1ndar tehtawt coudrcouwmnisnt rreeammartkaarbgleyt sreodfuCceddc 2th0e.  ICnddce2e0d p, rwoteeoinb lseevrevle idn tPhCa tcceullsr c(uFmiguinre  treatme1nBt,Cle)d. Itt oisu kpnroewgun ltahtaiot nBiomf  Banimd pa2n1d apre2 1twino bdootwhnPsCtrecaemlls ta(Frgigetusr oef1 CBd,Cc2)0. .T Ihnedseeedfi,n wdein ogbsserervveeda ltehdat  that curccuurmcuimniinn htribeaittemdeCntd  cle2d0  etox purpesresigounlaitnioPnC  ocf eBllism.   and  p21  in  both  PC  cells  (Figure  1B,C).  These  findings revealed that curcumin inhibited Cdc20 expression in PC cells.  3.2. Overexpression of Cdc20 Decreased Curcumin-Induced Cell Growth Inhibition 3.2. Overexpression of Cdc20 Decreased Curcumin‐Induced Cell Growth Inhibition  To explore whether curcumin-mediated cell growth inhibition is through suppression of Cdc20 in PC cells, TPoa teux8p9l8o8rea wndhePthaenrc c-1urcceullms iwn‐emreedtriaantesdf eccetlel dgrwowiththC indhci2b0itcioDnN isA thorroeumghp stuypvpercetsosrioans ocfo Cntdrco2l0  gbcerolotlhus p(PF.CiibcgOgneorcu loutPlehrusrCle  plPM (2c.lFC ieAOnTi lgcleu)Tsue.sr,l rIr lP(meMe Fla sipitTnu2guoTeAlu8tsr )s9r rt.(ea8e Fss8nIhi2um gtaoAlluntywps)r,do .eeso rC Phdv2taoaoAetnnwnrh)tec.slea ‐xyCid1tsp, o  toctrenheovenlsvaselitsstersl i erytowoeex,vnnxecpeptruorrleryeerf extc,sC sruspcasiudrminooercsnsnci2fsnue 0oicmooifrtnnfeeiC  nhdsoCdc i fiwbucd nCi2ehicttd0d2eihb0cds ci2C itec0gerdle ednlscls iig2lccgfi0rugenco lercialwofdD nigwc tNrtahclotnyAehwstleu lltoi ynhnpr gh e pierPnnaormahn ewPtapscuatsneth8tiycduo 9 evn8s8dce9ue8 bc8cpltya8leopl ngralcr d ruengaosdrrsPswco  ciauPwootnamhnntnch tii-cr bnn1io‐ny1l     treatmecnutrcinumPiCn ctreellastm(Feingtu irne P2CA )c.eOllsu r(Fdigautares u2Agg).e Ostutrh datatcau srucugmgeisnt tehxaetr ctsuritcsuimnihni beixteiortns oitfs cienlhl igbriotiwont hof  via dowcenlrl eggruowlatthio vniao fdCowdcn2r0egiunlaPtCionce ollfs C. dc20 in PC cells.  Figure 1. Cont.   Nutrients 2017, 9, 109 Nutrients 2017, 9, 109  Nutrients 2017, 9, 109  5 of 15 5 of 15  5 of 15    (FPAiCg)F(PucTAiCreghe)l  uelcTdF(Ps1erAiChCe.elgtlt)e r dseC u1c eT Cccet.rau htre2ldCetelre 0esdcac1u  dC2utt .mru er0dcCmedws Rumcaiu nim2wNtinretRg0chidiA  NutndmWh cmw eA edRucecxii eusNrrtnepchtcerx erAcur adpecreusmn euaresemce srsxbdirsecinipelsodunoacir.n m.otese ct**nseleiwi nsldpn lpwilg d a.o < cd< *asnia e v0isp nlmv0w. li 0ma si<.ds5e0ali yieo0,ais5o vas. vns,0nmisiusss5vu c. icer, isroy ceynav.eoncdsc dspnlu lc.ece  batorbcy rney2no2yocdc00n tllrrre ;r teb ee((o r(Caa2CayBoll0lt; dl)d‐-ri;  tct( ce(Tci(iB C2amBcm2h0l)ad0)e‐e)ne tTc)  T ieecr2mrhhexeex0exveeprpe)v  p e r(reeerePererxexxrssesCvpspespssee) irirsrto oerecreitsnsnosaersess aln nso iliatnossiofrtoc a ns naaCrRntc nf iodrtapNRseofitcctp r NACfi2rR otci0Cd iApNnua,o ct‐drBnnAiP2caocdi-0uCm nn2P a,m pRd0‐CBn,P r, aidi(RopCnmBRn t rpRdeti(T,omr Rri  a‐ ent(poPTRnea, t2Cil-dteTa1enmPi R‐nv npwCPle)ed 2e ClnRlai1evsntspR)v .;ew    2)e(il nCsl1aisn.s).     wasddeteetQcetucetaden dutisutianstiginv Wge rWeessteuesrlttnes r banlroebt ltioilnltugtsi ntargnaataelnydsa filsoy risn ips painannelpc rBae.na *ct irpce