surge, on the other hand the Ca 2þ chelator BAPTA inhibited the Ca 2þ

Transcription

1 ARTICLE Journal of Cellular Bochemstry 114: (2013) Journal of Cellular Bochemstry Interplay of Reactve Oxygen Speces, Intracellular Ca 2þ and Mtochondral Homeostass n the Apoptoss of Prostate Cancer Cells by Deoxypodophyllotoxn Kwang-Youn Km, 1 Hyo-Jn Cho, 2 Sun-Nyoung Yu, 1 Sang-Hun Km, 1 Hak-Sun Yu, 3 Yeong-Mn Park, 1 Nooshn Mrkhesht, 4 So Young Km, 4 Chung Seog Song, 4 Bandana Chatterjee, 4,5 * and Soon-Cheol Ahn 1,4,6 ** 1 Department of Mcrobology and Immunology, Pusan Natonal Unversty School of Medcne, Yangsan , Republc of Korea 2 Bran Dsease Research Center, Ajou Unversty School of Medcne, Suwon , Republc of Korea 3 Department of Parastology, Pusan Natonal Unversty School of Medcne, Yangsan , Republc of Korea 4 Department of Molecular Medcne and Insttute of Botechnology, The Unversty of Texas Health Scence Center at San Antono, San Antono, Texas South Texas Veterans Health Care System, San Antono, Texas Medcal Research Insttute, Pusan Natonal Unversty, Yangsan , Republc of Korea ABSTRACT The lmted treatment opton for recurrent prostate cancer and the eventual resstance to conventonal chemotherapy drugs has fueled contnued nterest n fndng new ant-neoplastc agents of natural product orgn. We prevously reported ant-prolferatve actvty of deoxypodophyllotoxn (DPT) on human prostate cancer cells. Usng the PC-3 cell model of human prostate cancer, the present study reveals that DPT nduced apoptoss va a caspase-3-dependent pathway that s actvated due to dysregulated mtochondral functon. DPT-treated cells showed accumulaton of the reactve oxygen speces (ROS), ntracellular Ca 2þ surge, ncreased mtochondral membrane potental (MMP, DC m ), Bax proten translocaton to mtochondra and cytochrome c release to the cytoplasm. Ths resulted n caspase-3 actvaton, whch n turn nduced apoptoss. The antoxdant N-acetylcystene (NAC) reduced ROS accumulaton, MMP and Ca 2þ surge, on the other hand the Ca 2þ chelator BAPTA nhbted the Ca 2þ overload and MMP wthout affectng the ncrease of ROS, ndcatng that the generaton of ROS occurred pror to Ca 2þ flux. Ths suggested that both ROS and Ca 2þ sgnalng play roles n the ncreased MMP va Ca 2þ -dependent and/or -ndependent mechansms, snce DC m elevaton was reversed by NAC and BAPTA. Ths study provdes the frst evdence for the nvolvement of both ROSand Ca 2þ -actvated sgnals n the dsrupton of mtochondral homeostass and the precedence of ROS producton over the falure of Ca 2þ flux homeostass. J. Cell. Bochem. 114: , ß 2012 Wley Perodcals, Inc. KEY WORDS: DEOXYPODOPHYLLOTOXIN (DPT); APOPTOSIS; MITOCHONDRIAL MEMBRANE POTENTIAL (MMP); REACTIVE OXYGEN SPECIES (ROS); Ca 2þ OVERLOAD Conflct of nterest: none. Kwang-Youn Km and Hyo-Jn Cho contrbuted equally to ths work. Grant sponsor: Natonal Research Foundaton of Korea (NRF); Grant number: 2012R1A1A ; Grant sponsor: US Department of Veterans Affars 1I01BX *Correspondence to: Bandana Chatterjee, Department of Molecular Medcne, Unversty of Texas Health Scence Center at San Antono, Lambda Drve, San Antono, TX E-mal: chatterjee@uthscsa.edu **Correspondence to: Soon-Cheol Ahn, Department of Mcrobology and Immunology, Pusan Natonal Unversty School of Medcne, Yangsan , Republc of Korea. E-mal: ahnsc@pusan.ac.kr Manuscrpt Receved: 18 July 2012; Manuscrpt Accepted: 5 November 2012 Accepted manuscrpt onlne n Wley Onlne Lbrary (wleyonlnelbrary.com): 28 November 2012 DOI /jcb ß 2012 Wley Perodcals, Inc. 1124

2 The lgnan deoxypodophyllotoxn (DPT) s the natural consttuent of a number of medcnal herb plants that s known to exert ant-prolferatve effects and apoptoss on varous cancer cell lnes [Jn et al., 2011]. The ant-nflammatory property of ths phytochemcal further underscores ts chemopreventve potental n cancer therapy, gven that nflammaton s recognzed as havng a crucal nvolvement n cancer etology [De Marzo et al., 2007]. Inhbton of ntracellular tubuln polymerzaton and nducton of cell cycle arrest by DPT have been extensvely reported. For example, HeLa uterne cancer cells accumulate at the G 2 /M phase [Yong et al., 2009; Shn et al., 2010] and A549 lung cancer cells were arrested n the G 1 phase upon treatment wth DPT. Synthetc dervatves of DPT were more potent n nhbtng cancer cell prolferaton than the parent molecule [Jn et al., 2011]. The molecular machnery drvng the death actvty of these agents aganst cancer cells s not known. Prostate cancer s a frequently dagnosed dsease and s one of the leadng causes of cancer deaths n men, especally n the Unted States and other ndustralzed natons. Pharmacologc nhbton of the androgen receptor (AR) pathway by depleton of crculatng androgen and by ant-ar drugs s a standard-of-care treatment, whch nduces apoptoss n androgen-dependent prostate cancer cells and brngs the dsease to remsson. However, the re-emergent cancer, whch occurs n many patents, nevtably progresses to therapy resstance followng response to conventonal chemotherapy for a lmted perod. Fndng new treatment optons for therapyresstant prostate cancer has been an overarchng challenge [Yap et al., 2011]. Markedly reduced prolferaton of the androgendependent LNCaP and androgen-ndependent PC-3 prostate cancer cells n response to DPT treatment was prevously reported [Jang et al., 2007; Cho et al., 2009]. A rgorous assessment of the therapeutc potental of DPT n prostate cancer requres mechanstc nsghts nto the DPT-medated nhbton of the cancer cell prolferaton. Some studes have reported that cancer chemopreventve agents nduce apoptoss n part through reactve oxygen speces (ROS) generaton and dsrupton of redox homeostass [Lng et al., 2003]. ROS are not only byproducts of mtochondral respraton but also key sgnalng molecules that regulate mtochondral dysfuncton and apoptotc events [Lu et al., 2004]. As a secondary messenger, ROS has the ablty to nfluence the mtochondral functon, medate the elevaton of ntracellular Ca 2þ, and lead to the actvaton of the caspase cascade [Parades et al., 2002]. Pro-apoptotc sgnals such as ROS producton can trgger the release of pro-apoptotc protens from the mtochondra ntermembrane space nto the cytosol, such as, cytochrome c, caspase-9, apoptoss nducng factor, and Smac/ DIABLO [Kroemer et al., 1998]. The mtochondron-medated ntrnsc apoptoss, employng a cytochrome c/apaf-1/caspase-9-dependent pathway, s the predomnant route to the elmnaton of tumor cells by the majorty of chemotherapeutc agents [Kaufmann and Earnshaw, 2000; Fulda and Debatn, 2006]. Loss of mtochondral membrane potental (MMP, DC m ), resultng from the openng of the permeablty transton pore, s an oblgatory feature of the mtochondral response to apoptotc stmul that precedes DNA fragmentaton and changes n the nuclear morphology [Mayer and Oberbauer, 2003]. Ca 2þ overload n the mtochondral matrx due to the dysregulaton of the homeostass n the ntracellular Ca 2þ flux, uncouplng of the electron transport chan and mtochondral generaton of ROS are some of the key events that lead to the permeablty transton and loss of MMP. Prevously we solated the boactve compound DPT from the Anthrscus sylvestrs root and demonstrated ts ant-prolferatve actvty aganst human prostate cancer cells [Cho et al., 2009]. In the present study we have examned the mechansm that underles prostate cancer cell death n the presence of DPT. Heren we report that DPT nduces G 2 /M cell cycle arrest and actvates the ntrnsc apoptotc pathway n the androgen-ndependent PC-3 prostate cancer cells. Also, we descrbe the role of ROS accumulaton, ntracellular Ca 2þ surge and mtochondral membrane hyperpolarzaton as part of the chan of events that led to apoptoss. Our results provde the frst evdence that ROS producton n response to DPT precedes the falure n Ca 2þ flux homeostass and that mtochondral hyperpolarzaton s nvolved n the DPT-nduced apoptotc loss of prostate cancer cells. MATERIALS AND METHODS REAGENTS AND ANTIBODIES DPT used n ths study was solated by us from A. sylvestrs roots and ts structural dentty was determned by nuclear magnetc resonance analyss as descrbed prevously [Cho et al., 2009]. The compound was confrmed to be >95% pure after hghperformance lqud chromatography. 3-(4,5-Dmethyl-thazol-2- yl)-2,5-dphenyl-tetrazolum bromde (MTT), N-acetylcystene (NAC), 4 0,6-damdno-2-phenylndole dhydrochlorde (DAPI), 3,3-dhexyloxacarbocyanne (DOC 6 ), dchlorodhydrofluorescen dacetate (DCFH-DA), 1,2-bs (2-amnophenoxy) ethane- N,N,N,N -tetraacetc acd tetraks (acetoxymethyl ester; BAPTA/ AM), and Fluo-3/AM were purchased from Sgma Chemcal Co. Annexn-V-Fluo Stanng Kts and Caspase-3 Colormetrc Assay Kts were purchased from BD Boscences and R&D Systems Inc., respectvely. The ECL Western Kt was purchased from Amersham. Antbodes for Bax, Bcl-2, and poly (ADP-rbose) polymerase-1 (PARP-1) were purchased from Santa Cruz Botechnology. Antbodes for cycln B1, cdk-1, caspase-3, and cytochrome c were purchased from Cell Sgnalng. CELL LINES AND CELL CULTURE Prostate cancer cell lnes, androgen-ndependent PC-3 cells, and androgen-dependent LNCaP cells, were obtaned from the Amercan Type Culture Collecton (ATCC) and Korean Cell Lne Bank (KCLB), respectvely. These cells were mantaned and cultured n Dulbecco s modfed Eagle s medum (DMEM; WelGENE Inc.) and RPMI 1640 (WelGENE Inc.), respectvely, supplemented wth 10% fetal bovne serum (FBS; WelGENE Inc.), 100 unts/ml of penclln and 100 mg/ml of streptomycn (WelGENE Inc.). Cells were cultured n a humdfed atmosphere wth 5% CO 2 at 378C. CELL VIABILITY ASSAY PC-3 cells and LNCaP cells were seeded n 48-well plates at a densty of /well and treated wth DPT of varous concentratons at JOURNAL OF CELLULAR BIOCHEMISTRY ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT 1125

3 dfferent tme ponts. After ncubaton, these cells were treated wth 0.5 mg/ml of the MTT soluton for further 4 h ncubaton and the precptates were dssolved n dmethyl sulfoxde. A colormetrc analyss was performed at 570 nm wth an ELISA reader (VERSA max mcroplate reader, Molecular Devces) [Jarrett et al., 2006]. Cell vablty at a gven tme pont was determned from the optcal densty of the treated cells relatve to the untreated cells. varous concentratons of DPT for 24 h. And the cells were then fxed n 70% ethanol, washed wth PBS twce and fnally ncubated n RNase A (200 mg/ml) at 378C for 30 mn. DNA content per cell was evaluated n flow cytometer (Becton Dcknson Co.) after stanng cells wth 1 mg/ml propdum odde soluton. Data collecton and analyss of the cell cycle dstrbuton were performed usng Cell Quest and Modft software (Becton Dcknson Co.). MORPHOLOGICAL CHANGES PC-3 cells plated on chamber sldes were ether untreated or treated wth 800 nm DPT for 24 h. Next, cells were fxed wth 4% paraformaldehyde for 30 mn, washed wth phosphate buffered salne (PBS) twce and staned wth DAPI soluton (1 mg/ml) at 48C for 15 mn. Staned nucle were vsualzed by laser scan confocal mcroscopy (FV-1000, Olympus) and the DAPI stanng was used to analyze the apoptotc morphologcal change of cells. CELL CYCLE ANALYSIS PC-3 cells were plated n 6-well plates at a densty of /well for 24 h after serum starvaton overnght. The culture medum was refreshed wth new medum and then the cells were treated wth ANNEXIN-V/PROPIDIUM IODIDE (PI) ASSAY Apoptotc cells were detected by usng an Annexn-V-Fluo stanng kt. PC-3 cells, exposed to 8, 80, and 800 nm of DPT for 24 h n 6-well plates at a densty of cells/well, were harvested, rnsed wth PBS twce, mxed n 100 mlof1 bndng buffer and ncubated wth an Annexn-V/PI double stanng soluton at room temperature for 15 mn. Staned cells were analyzed by flow cytometry. The percentage of apoptotc cells was calculated usng Cell Quest software (Becton Dcknson Co.). ASSESSMENT OF CASPASE-3 ACTIVITY For detecton of caspase-3 actvaton, a colormetrc assay kt (R&D Systems Inc.) was used accordng to the manufacturer s protocol. Fg. 1. Growth nhbton of prostate cancer cells by DPT. A: Chemcal structure of DPT. B D: Cell vablty measurements. PC-3 cells ( cells/ml) were treated wth varous concentratons of DPT (8, 80, and 800 nm) at dfferent tme ponts (12, 24, 36, and 48 h) (B), or at 24 h (C) and LNCaP cells (D) were at 24 h. Cell vablty was determned by a 3-(4,5-dmethyl-thazol-2-yl)-2,5-dphenyl-tetrazolum bromde (MTT) assay. Data are mean SD (n ¼ 3 n each group). P < 0.05 versus control group ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT JOURNAL OF CELLULAR BIOCHEMISTRY

4 Equal amounts of proten (220 mg) were resuspended n reacton buffer contanng substrate (Ac-DEVD-pNA) and then ncubated at 378C for 4 h n the dark. The absorbance of the released pna was measured at 405 nm usng an ELISA reader. MEASUREMENT OF INTRACELLULAR REACTIVE OXYGEN SPECIES (ROS) Intracellular ROS was measured usng the DCFH-DA fluorescent dye. PC-3 cells were treated wth 80 nm DPT for ndcated tmes and then ncubated wth 10 mm DCFH-DA at 378C for 30 mn and then washed twce wth PBS. For each experment, the cells were analyzed for DCFH-DA fluorescence by flow cytometry. ANALYSIS OF INTRACELLULAR Ca 2R CONCENTRATION Intracellular Ca 2þ levels were determned usng the Ca 2þ -senstve fluorescence dye Fluo-3/AM. After exposure to DPT at varous concentratons for 8 and 24 h, cells were centrfuged and washed twce wth PBS and then ncubated wth 5 mm Fluo-3/AM at 378C for 30 mn. Then the cells were washed and subjected to flow cytometry. STATISTICAL ANALYSIS Experments were repeated at least three tmes wth consstent results. Unless otherwse stated, data are expressed as the mean SD. ANOVA was used to compare the expermental groups wth the control, whereas comparsons among multple groups were performed usng a Tukey s multple comparson test. Results were statstcally sgnfcant at P < RESULTS SUSCEPTIBILITY OF PROSTATE CANCER CELLS TO DEOXYPODOPHYLLOTOXIN (DPT) DPT, a polycyclc aromatc molecule (Fg. 1A), reduced the vablty of PC-3 cells n a dose- and tme-dependent manner (Fg. 1B). Comparson of the prolferaton rates of the LNCaP and PC-3 cells n the presence of DPT revealed a hgher susceptblty of the latter cell type (Fg. 1C vs. D). For PC-3 cells, 24 h ncubaton wth DPT at 80 and 800 nm caused 50% and 60% reducton n cell vablty, respectvely, whereas the vablty of LNCaP cells was reduced by 25% and 40%, respectvely, under the same condtons. Thus, DPT MEASUREMENT OF MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) MMPs (DC m ) were determned from the retenton of the dye DOC 6, whch s a mtochondral membrane potental senstve dye. PC-3 cells were collected at 8 and 24 h after treatment wth an 8, 80, and 800 nm of DPT and washed twce wth PBS, and ncubated wth 100 nm DOC 6 at 378C for 30 mn. The washed cells were analyzed by flow cytometry for DOC 6 fluorescence. WESTERN BLOTTING Cell extracts were prepared by ncubatng the cells n lyss buffer [150 mm NaCl, 10 mm Trs (ph 7.4), 5 mm EDTA (ph 8.0), 1% Trton X-100, 1 mm PMSF, 20 mg/ml aprotnn, 50 mg/ml leupeptn, 1 mm benzdne, and 1 mg/ml pepstatn]. Forty mcrograms of protens were electrophoretcally separated usng sodum dodecyl sulfatepolyacrylamde gel electrophoress (SDS PAGE) on a 12 15% gel and transferred to a polyvnyldenefluorde (PVDF) membrane (Amersham). After blockng wth TBS-T buffer [20 mm Trs (ph 7.4), 150 mm NaCl, 0.1% Tween 20] contanng 5% skm mlk, the membranes were ncubated wth prmary and secondary antbodes. The membranes were then washed wth TBS-T buffer and vsualzed wth ECL Western blottng detecton reagents (Amersham). The densty of each band was determned wth a fluorescence scanner (LAS 3000, Fuj Flm) and analyzed wth Mult Gauge V3.0 software. MITOCHONDRIAL AND CYTOSOLIC FRACTIONS Release of pro-apoptotc factors from the mtochondra to the cytosol was detected by Western blottng usng a Qproteome TM Mtochondra solaton Kt (Qagen) accordng to the manufacturer s nstructons. Western blottng was carred out accordng to the protocol descrbed n the prevous secton. Fg. 2. Effect of DPT on PC-3 cell cycle progresson. A: G 2 /M cell cycle arrest, revealed from flow cytometry. B: Quantfcaton of cell populatons at varous phases of cell cycle. After treatment wth DPT for 24 h, the cells were fxed wth ce-cold 70% ethanol at 208C and then staned wth propdum odde (1 mg/ml), followed by flow cytometery. Data are presented as mean SD (n ¼ 3, each group). P < 0.05 versus control group. JOURNAL OF CELLULAR BIOCHEMISTRY ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT 1127

5 may be more effcent n ablatng the hghly aggressve and chemotherapy-resstant PC-3 cancer cells than the hormoneresponsve, poorly metastatc LNCaP cells. We focused on the PC- 3 cell model to examne the molecular events assocated wth the DPT-nduced cell loss. reported for HeLa cells, whch were prevented by DPT from cell cycle progresson due to a blockade at G 2 /M [Yong et al., 2009; Shn et al., 2010]. The levels of cycln B1 and cdk1 dd not change n the cells treated wth DPT for 24 h, although at 48 h, the treated cells showed reduced levels of these protens (data not shown). G 2 /M PHASE CELL CYCLE ARREST BY DPT Cell cycle progresson and the expresson of a number of cell cyclerelated protens n PC-3 cells were examned n the presence and absence of DPT. PC-3 cells treated wth 8, 80, and 800 nm of DPT for 24 were analyzed by flow cytometry usng propdum odde (PI) stanng. A representatve cell cycle profle s shown n Fg. 2A. The cell populaton n the G 2 /M phase ncreased from 26.11% n control, untreated cells to 30.34% and 52.02% when the cells were treated wth 8 and 80 nm of DPT, respectvely (Fg. 2B). These results show that DPT nduces G 2 /M arrest of PC-3 cells, smlar to what was APOPTOSIS OF DPT-TREATED PC-3 CELLS Stanng of DPT-treated PC-3 cells wth DAPI revealed a hgh percentage of non-adherent cells and a clear ndcaton of nuclear condensaton, whch s a hallmark feature of apoptotc cells. Reducton n the cell volume was also observed. Ths contrasted the vehcle-treated control cells, whch appeared normal wth round nucle and all of these cells adhered to the culture plate (Fg. 3A). Annexn-V-FITC stanng of the cells provded further evdence for DPT-nduced apoptoss (Fg. 3B). Thus, flow cytometry analyss shows that after treatment wth DPT for 24 h, the early and late Fg. 3. DPT-nduced apoptoss n PC-3 cells. A: Cytomorphologcal changes. After treatment wth DPT for 24 h, nuclear fragmentaton was observed by lght and laser scannng confocal mcroscopy. Magnfcaton, 1,800. B: Flow cytometrc analyss. PC-3 cells were treated wth varous concentratons of DPT for 24 h and staned wth Annexn-V/ propdum odde (PI). The dual parameter dot plots combnng Annexn-V-fluorescen sothocyanate (FITC) and PI fluorescence show the vable cell populaton n the lower left quadrant (Annexn-V PI ), the apoptotc cells n the lower rght quadrant (Annexn-V þ PI ) together wth the upper rght quadrant (Annexn-V þ PI þ ), and necrotc cells n the upper left quadrant (Annexn-V PI þ ). Data are presented as mean SD (n ¼ 3 n each group). P < 0.05 versus control group ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT JOURNAL OF CELLULAR BIOCHEMISTRY

6 changes negatvely affected the growth of PC-3 cells, whch subsequently underwent apoptoss. Fg. 4. Regulaton of apoptoss-related proten levels n DPT-treated PC-3 cells. A: Immunoblots of Bax, Bcl-2, procaspase-3 and poly (ADP-rbose) polymerase (PARP-1) levels. Cells were exposed to 8, 80, and 800 nm of DPT for 24 h. B: Caspase-3 actvty. The actvty was determned by colormetrc assay usng a specfc substrate (Ac-DEVD-pNA). Data are presented as mean SD (n ¼ 3 n each group). P < 0.05 versus control group. apoptotc PC-3 cells ncreased from 9.79% n untreated cells to 21.40% and 26.39% at 8 and 80 nm, respectvely (Fg. 3B). Apoptoss was further augmented when the DPT concentraton ncreased to 800 nm. These results ndcate that the DPT-nduced morphologcal CHANGES IN THE PRO-APOPTOTIC AND ANTI-APOPTOTIC BCL-2 FAMILY PROTEINS AND ACTIVATION OF CASPASE-3 Inducton of the PC-3 cell apoptoss n the presence of DPT prompted us to examne the expresson dynamcs of the apoptoss- and survval-related protens n the Bcl-2 famly and the converson of the pro-caspase-3 zymogen to actve caspase n the total cell lysate usng Western blottng. The pro-apoptotc Bax proten level was nduced by DPT after ncubaton for 24 h at as low as 8 nm concentraton and the nducton ncreased steadly wth hgher concentratons of DPT 80 and 800 nm, whereas the Bcl-2 ant-apoptotc proten dd not show any sgnfcant change wthn the total cell lysate at any of the DPT concentratons (Fg. 4A, upper panels). In a coordnate and dose-dependent manner, DPT-medated events led to the reducton of the pro-caspase-3 zymogen level, ndcatng the generaton of caspase-3, whch n turn caused cleavage of PARP-1, a well-characterzed caspase-3 substrate (Fg. 4A, lower panels). Inducton of the caspase-3 actvty n DPT-treated PC-3 cells was further confrmed usng a colormetrc assay for caspase-3 actvty (Fg. 4B). These results establsh a role of DPT n the nducton of caspase-3-dependent Fg. 5. ROS producton n DPT-treated PC-3 cells. A: Intracellular ROS levels. Cells were treated wth 80 nm DPT for the ndcated tmes n the presence or absence of a pror 1 h ncubaton wth 10 mm N-acetylcystene (NAC). The dchlorodhydrofluorescen (DCF) fluorescence ntensty n the cells was detected by flow cytometry. B: Cell vablty determned by MTT assay. Cells were treated wth 8, 80, and 800 nm DPT for 24 h n the presence or absence of pror 1 h ncubaton wth 10 mm N-acetylcystene (NAC). Data are mean SD (n ¼ 3 n each group). P < 0.05 versus control group. JOURNAL OF CELLULAR BIOCHEMISTRY ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT 1129

7 apoptoss n PC-3 cells that follows nducton of the pro-apoptotc proten Bax. ACCUMULATION OF REACTIVE OXYGEN SPECIES (ROS) It has been reported that the ROS level n cells correlates wth the nducton of mtochondral permeablty dysfunctons, and cancer chemotherapy drugs medate apoptoss n part by promotng ROS producton n a varety of tumor cell types [Ouyang et al., 2002; Lng et al., 2003]. The ntracellular ROS level, measured from the fluorescence ntensty, ncreased wthn 1 h of exposure to 80 nm DPT and the hghest level of ROS (55.17% from the basal level) was observed from the DPT exposure for 8 h (Fg. 5A, lower panel). ROS producton was prevented when the cells were treated wth the antoxdant N-acetylcystene (NAC) for 1 h pror to DPT exposure (Fg. 5A, upper panel). In the presence of 10 mm of NAC, we observed 24.26% recovery of the number of DCF-postve PC-3 cells that were treated wth 80 nm DPT for 8 h (Fg. 5A, lower panel). The vablty of DPT-treated PC-3 cells also ncreased n the presence of NAC, showng an ncrease from 62.16% to 80.08% at 8 nm DPT and 51.40% to 61.21% after 24 h ncubaton of 80 nm DPT (Fg. 5B). However, the recovery of cell vablty was not complete at any of the condtons that we examned. It s possble that a complete recovery requres a hgher concentraton of NAC, whch we could not test snce NAC by tself also exerted a cytotoxc effect on these cells. Nevertheless, the results n Fgure 6 ndcate that ROS producton plays an mportant role n the DPT-nduced apoptoss n PC-3 cells. MITOCHONDRIAL MEMBRANE HYPERPOLARIZATION, BAX TRANSLOCATION AND CYTOCHROME c RELEASE Flow cytometry analyss of DOC 6 fluorescence dye staned cells was performed to assess mtochondral membrane potental (MMP, Dc m ) before and after treatment wth DPT for 24 h. After treatment wth 80 nm DPT at 8 h, DPT nduced unque mtochondral Fg. 6. Effects of DPT on mtochondral membrane potental (MMP). A: Tme course of MMP. PC-3 cells were treated wth 80 nm DPT for ndcated tme. Changes n MMP (Dc m ) were determned from the shft n DOC 6 fluorescence and analyzed by flow cytometry. B: Effect of NAC on MMP. PC-3 cells were treated wth 80 nm DPT for 24 h n the presence or absence of pror 1 h ncubaton wth 10 mm NAC. C: Mtochondral (M) and cytoplasmc (C) levels of apoptoss-related protens. D: Levels of Bax and Bcl-2 n the mtochondral fracton. PC-3 cells were treated wth 8, 80, and 800 nm DPT for 24 h. The levels of Bax and cytochrome c n the cytosol and mtochondral fractons were determned by Western blottng. Data are mean SD (n ¼ 3 n each group). P < 0.05 versus control group ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT JOURNAL OF CELLULAR BIOCHEMISTRY

8 membrane depolarzaton (Fg. 6A). However, after longer treatment for 24 h, the depolarzaton peak was shfted to hyperpolarzaton n dose-dependent manner (Fg. 6A,B). The mtochondral membrane hyperpolarzaton was partally prevented when the cells were pretreated wth 10 mm NAC for 24 h (Fg. 6B). These data ndcated that the MMP dysfuncton occurred at the downstream of ROS producton. Furthermore, the MMP dysfuncton was lnked to the translocaton of Bax from the cytoplasm to the mtochondron and mtochondral cytochrome c release to the cytosol (Fg. 6C). Thus, n Western blot assay, the mtochondral fracton of the treated cells showed progressvely hgher levels of Bax at ncreasng doses of DPT wth a parallel reducton n the cytosolc Bax level (Fg. 6C). DPT also provoked elevaton n the cytosolc level of cytochrome c along wth ts concomtantly reduced level n the mtochondra (Fg. 6C). In addton, the ncreased Bax level n the mtochondral fracton of the treated cells was accompaned by a coordnated decrease n the antapoptotc survval proten Bcl-2 (Fg. 6D). These results ndcate that DPT ntates a cascade of events that result n the redstrbuton of crtcal components of the caspase-3-dependent apoptotc pathway between the cytosol and mtochondra. Ca 2R FLUX IN DPT-TREATED CELLS Dsrupton of ntracellular calcum homeostass s one of the characterstc events assocated wth mtochondral membrane dsrupton and apoptoss. We used Fluo-3/AM, a Ca 2þ -senstve fluorescence probe, to montor changes n the ntracellular Ca 2þ level. The fluorescence emtted from 80 nm DPT treated cells was shfted to a hgher ntensty n tme-dependent manner, ndcatng a DPT-nduced surge n the ntracellular Ca 2þ concentraton (Fg. 7A). But the Ca 2þ chelator BAPTA/AM sgnfcantly reduced the Ca 2þ surge (Fg. 7B). The rse n the Ca 2þ concentraton was partally blocked when the cells were pretreated wth NAC and ths blockade was statstcally sgnfcant (Fg. 7C), ndcatng that the ROS producton and the resultng oxdant stress are nvolved n the elevaton of the Ca 2þ level n the treated cells wth 80 nm DPT. Fg. 7. Intracellular Ca 2þ concentraton n DPT-treated PC-3 cells. A: Intracellular Ca 2þ flux. Cells were treated wth 80 nm DPT for varous tmes. B: Effect of BAPTA on Ca 2þ level. Cells were treated wth 80 nm DPT for 24 h n the presence or absence of pror 1 h ncubaton wth 2 mm BAPTA. C: The effect of NAC on the Ca 2þ level. Cells were treated wth 80 and 800 nm DPT for 24 h n the presence or absence of pror 1 h ncubaton wth 10 mm NAC. The Ca 2þ concentraton was determned by flow cytometry usng Fluo-3/AM fluorescence. Data are mean SD (n ¼ 3 n each group). P < 0.05 versus control group. JOURNAL OF CELLULAR BIOCHEMISTRY ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT 1131

9 DEPENDENCE OF Ca 2R SURGE ON ROS PRODUCTION AND MMP AND CELL VIABILITY By the pretreatment wth BAPTA/AM, a cell-permeable Ca 2þ chelator, ROS nducton n the presence of DPT was not prevented (Fg. 8A) but the ncreased MMP by DPT was sgnfcantly attenuated (Fg. 8B). The hyperpolarzaton decreased by 4.75%, that s, from 19.49% to 14.74%. Inhbton of the Ca 2þ surge by NAC (Fg. 7C) but contnued ROS accumulaton despte the chelaton of Ca 2þ (Fg. 8A). Also, after the pretreatment wth BAPTA/AM, cell vablty and apoptoss of PC-3 cells n the presence of DPT were sgnfcantly recovered (Fg. 8C,D), when taken together, led us to conclude that the ROS producton n the treated cells precedes the release of ntracellular Ca 2þ. Furthermore, snce both NAC and BAPTA/AM attenuated mtochondral hyperpolarzaton (Fgs. 6B and 8B), we conclude that both Ca 2þ overload-dependent and -ndependent mechansms are nvolved n the regulaton of the mtochondral dysfuncton by the oxdant stress from ROS accumulaton and by the Bcl-2 famly from redstrbuton n mtochondra, respectvely. DISCUSSION Deoxypodophyllotoxn (DPT), a natural product of several medcnal plants, potently nhbts prostate cancer cell prolferaton and nduces apoptoss. Our data show that the androgen-ndependent PC-3 human prostate cancer cells are more susceptble to DPTmedated growth nhbton than the androgen-dependent LNCaP human prostate cancer cells. The present study was conducted to elucdate the molecular bass for the DPT-nduced apoptoss n the PC-3 cell model. We show that DPT nduced the followng (1) cell cycle arrest at G 2 /M; (2) ntracellular reactve oxygen speces (ROS) accumulaton; (3) ntracellular Ca 2þ surge; (4) hyperpolarzaton of the mtochondral membrane; (5) translocaton of the pro-apoptotc Bcl-2 famly member Bax to mtochondra along wth reducton n the mtochondral level of the ant-apoptotc proten Bcl-2 and hence, a declne n the mtochondral rato of Bcl-2 to Bax; (6) cytoplasmc release of cytochrome c from the mtochondral ntermembrane space; and (7) caspase-3 actvaton leadng to PARP-1 cleavage and nuclear condensaton that culmnated n the apoptotc cell death (Fg. 9). Prevously we had reported that the antbotc salnomycn nduces apoptoss n several human prostate cancer cells, ncludng PC-3 cells, by elevatng the ntracellular ROS level, whch was accompaned by decreased MMP. Unlke DPT, salnomycn-treated PC-3 cells dd not show any change n the ntracellular Ca 2þ concentraton [Km et al., 2011], ndcatng that dsparate mechansms are at play for cancer cell apoptoss by structurally unrelated natural products. We observed G 2 /M arrest of DPTtreated PC-3 cells, smlar to what was reported for HeLa cells [Shn et al., 2010]. DPT nduced p53 expresson n HeLa cells that resulted Fg. 8. Effect of BAPTA on ROS producton, MMP and cell vablty n PC-3 cells. A: ROS producton. B: MMP measurement. C: Cell vablty. D: Apoptotc cell death. For (A,B), PC-3 cells were treated wth 80 nm DPT for 24 h n the presence or absence of pror 1 h ncubaton wth 2 mm BAPTA. For (C,D), DPT treatment at ndcated concentratons was for 24 h. Intenstes of dchlorodhydrofluorescen and DOC 6 and Annexn-V/PI fluorescence were detected by flow cytometry analyss, respectvely. Data are mean SD (n ¼ 3 n each group). P < 0.05 versus control ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT JOURNAL OF CELLULAR BIOCHEMISTRY

10 Fg. 9. An ntegrated depcton of the results from the current study that show the role of ROS and Ca 2þ sgnalng n the DPT-nduced dsrupton of mtochondral homeostass, caspase-3 actvaton and apoptoss n PC-3 prostate cancer cells. n a p53-dependent DNA damage response, revealed from the actvaton of the DNA damage-sensng knases such as ATM and Chk2 [Shn et al., 2010]. It wll be mportant to determne whether PC-3 cell apoptoss by DPT also assocates wth a DNA damage response. In a number of expermental models of apoptoss, hyperpolarzaton of the mtochondral membrane has been detected as an early event, leadng to nducton of apoptoss [L et al., 1999; Govannn et al., 2002; Km et al., 2003; Cao et al., 2007]. The events leadng to mtochondral hyperpolarzaton are not well-understood; t has been speculated that the cell type, cellular envronment and duraton of apoptotc stmul may dctate the underlyng mechansm for hyperpolarzaton [Km et al., 2003]. Opposte to publshed reports, our results show a bphasc phenomenon that a drop n MMP occurred as an early event wthn 8 h post-dpt treatment and MMP was shfted to ncreased Dc m at 24 h and furthermore even at 72 h post-dpt treatment the hyperpolarzaton was sustaned (data not shown). In case of the methyl protodoscn-nduced apoptoss of K562 leukema cells, elevated Dc m was detected for up to 60 h posttreatment and thereafter, loss of MMP ensued [Km et al., 2003]. Tme course analyss utlzng early as well as late tme ponts and utlzaton of a synchronzed cell populaton wll be needed to thoroughly address ths apparent anomaly. We conclude that ROS s produced pror to the endoplasmc retculum stress and that Ca 2þ release s under ROS regulaton, snce the antoxdant N-acetylcystene (NAC) reduced both ntracellular ROS accumulaton and Ca 2þ overload, whereas the Ca 2þ chelator BAPTA nterfered wth only the Ca 2þ surge wthout affectng ROS accumulaton. Hyperpolarzaton of the mtochondral membrane potental (MMP) was observed n PC-3 cells at the 24 h tme pont post DPT treatment, and the rse of MMP was attenuated by NAC as well as BAPTA. Ths suggests that both ROS and the Ca 2þ overload contrbuted to the ncreased MMP, and the mtochondral dysfuncton by the oxdant stress from ROS accumulaton may nvolve Ca 2þ overload-dependent as well as -ndependent mechansms. In concluson, the present study provdes new nsghts nto the role of ROS- and Ca 2þ -actvated sgnals n the DPT-nduced dsrupton of mtochondral homeostass n prostate cancer cells. Addtonal nsghts nto the DPT-nduced cross talks among multple sgnalng networks that are nvolved n the dsrupton of mtochondral homeostass and nducton of apoptoss n cancer cell lnes, n prmary tumor cells and n anmal models wll help assess the potental utlty of DPT and ts dervatves n the management of therapy-resstant advanced prostate cancer. ACKNOWLEDGMENTS Ths research was supported by Basc Scence Research Program through the Natonal Research Foundaton of Korea (NRF) funded by the Mnstry of Educaton, Scence and Technology (2012R1A1A ) and by a Mert Revew grant (1I01BX000280) from the US Department of Veterans Affars. B.C. s a VA Senor Research Career Scentst. JOURNAL OF CELLULAR BIOCHEMISTRY ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT 1133

11 REFERENCES Cao J, Lu Y, Ja L, Zhou HM, Kong Y, Yang G, Jang LP, L QJ, Zhong LF Curcumn nduces apoptoss through mtochondral hyperpolarzaton and mtdna damage n human hepatoma G2 cells. Free Radc Bol Med 43: Cho HJ, Yu SN, Km KY, Sohn JH, Oh H, Ahn SC Screenng and purfcaton of an ant-prostate cancer compound, deoxypodophyllotoxn, from Anthrscus sylvestrs Hoffm, J. Lfe Sc 19:9 14. De Marzo AM, Platz EA, Sutclffe S, Xu J, Gronberg H, Drake CG, Naka Y, Isaacs WB, Nelson WG Inflammaton n prostate carcnogeness. Nat Rev Cancer 7: Fulda S, Debatn KM Extrnsc versus ntrnsc apoptoss pathways n antcancer chemotherapy. Oncogene 25: Govannn C, Matarrese P, Scazzoccho B, Sanchez M, Masella R, Malorn W Mtochondra hyperpolarzaton s an early event n oxdzed lowdensty lpoproten-nduced apoptoss n Caco-2 ntestnal cells. FEBS Lett 523: Jarrett SG, Albon J, Boulton M The contrbuton of DNA repar and antoxdants n determnng cell type-specfc resstance to oxdatve stress. Free Radc Res 40: Jang RW, Zhou JR, Hon PM, L SL, Zhou Y, L LL, Ye WC, Xu HX, Shaw PC, But PP Lgnans from Dysosma verspells wth nhbtory effects on prostate cancer cell lnes. J Nat Prod 70: Jn Y, Lu J, Huang WT, Chen SW, Hu L Synthess and bologcal evaluaton of dervatves of 4-deoxypodophyllotoxn as anttumor agents. Eur J Med Chem 46: Kaufmann SH, Earnshaw WC Inducton of apoptoss by cancer chemotherapy. Exp Cell Res 256: Km JM, Bae HR, Park BS, Lee JM, Ahn HB, Rho JH, Yoo KW, Park WC, Rho SH, Yoon HS, Yoo YH Early mtochondral hyperpolarzaton and ntracellular alkalnzaton n lactacystn-nduced apoptoss of retnal pgment epthelal cells. J Pharmacol Exp Ther 305: Km KY, Yu SN, Lee SY, Chun SS, Cho YL, Park YM, Song CS, Chatterjee B, Ahn SC Salnomycn-nduced apoptoss of human prostate cancer cells due to accumulated reactve oxygen speces and mtochondral membrane depolarzaton. Bochem Bophys Res Commun 413: Kroemer G, Dallaporta B, Resche-Rgon M The mtochondral death/lfe regulator n apoptoss and necross. Annu Rev Physol 60: L PF, Detz R, von Harsdorf R p53 regulates mtochondral membrane potental through reactve oxygen speces and nduces cytochrome c-ndependent apoptoss blocked by Bcl-2. EMBO J 18: Lng YH, Lebes L, Zou Y, Perez-Soler R Reactve oxygen speces generaton and mtochondral dysfuncton n the apoptotc response to Bortezomb, a novel proteasome nhbtor, n human H460 non-small cell lung cancer cells. J Bol Chem 278: Lu MJ, Wang Z, L HX, Wu RC, Lu YZ, Wu QY Mtochondral dysfuncton as an early event n the process of apoptoss nduced by woodfordn I n human leukema K562 cells. Toxcol Appl Pharmacol 194: Mayer B, Oberbauer R Mtochondral regulaton of apoptoss. News Physol Sc 18: Ouyang YB, Carredo SG, Gffard RG Effect of Bcl-x(L) overexpresson on reactve oxygen speces, ntracellular calcum, and mtochondral membrane potental followng njury n astrocytes. Free Radc Bol Med 33: Parades G, Petrosllo G, Pstolese M, Ruggero FM Reactve oxygen speces affect mtochondral electron transport complex I actvty through oxdatve cardolpn damage. Gene 286: Shn SY, Yong Y, Km CG, Lee YH, Lm Y Deoxypodophyllotoxn nduces G2/M cell cycle arrest and apoptoss n HeLa cells. Cancer Lett 287: Yap TA, Zv A, Omln A, de Bono JS The changng therapeutc landscape of castraton-resstant prostate cancer. Nat Rev Cln Oncol 8: Yong Y, Shn SY, Lee YH, Lm Y Anttumor actvty of deoxypodophyllotoxn solated from Anthrscus sylvestrs: Inducton of G2/M cell cycle arrest and caspase-dependent apoptoss. Boorg Med Chem Lett 19: ROS PRODUCTION, Ca 2þ SURGE AND MITOCHONDRIAL DYSFUNCTION SIGNALS BY DPT JOURNAL OF CELLULAR BIOCHEMISTRY

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