Cancer Letters 349 (2014) 15–25

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Inactivation of ATP citrate lyase by Cucurbitacin B: A bioactive compound from cucumber, inhibits prostate cancer growth Yajuan Gao a, Mohammad Shyful Islam a, Jiang Tian a, Vivian Wai Yan Lui b, Dong Xiao a,⇑ a Department of Urology, University of Pittsburgh Cancer Institute, University of Pittsburgh Medical College, University of Pittsburgh, Shadyside Medical Center, Suit G37, 5200 Centre Avenue, Pittsburgh, PA 15232, USA b Department of Pharmacology and Pharmacy, Li Ka Faculty of Medicine, University of Hong Kong, Hong Kong Special Administrative Region

a r t i c l e

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Article history: Received 15 January 2014 Received in revised form 12 March 2014 Accepted 13 March 2014

Keywords: Cucurbitacin B Prostate cancer Apoptosis ATP citrate lyase Xenografts

a b s t r a c t Prostate cancer, a leading cause of cancer-related deaths in males, is well recognized as having late disease on-set (mostly at age 60–70) and showing slow/latent disease development, and strategies to prevent cancer formation in late manhood may have significant health impacts. Cucurbitacin B (CuB) is a naturally occurring compound that is found abundantly in cucumbers and other vegetables, and it is known to exert anti-cancer activities (primarily via apoptosis-induction) in several human cancers. However, its chemopreventive potential for prostate cancer has not yet been investigated. Here, we reported that CuB significantly and specifically inhibited prostate cancer cell growth with low IC50 (0.3 lM; PC-3 and LNCaP), accompanied by marked apoptosis (Caspase 3/7 activation, PARP cleavage, increase of Annexin V-Alexa Fluor 488 (Alexa488)+ cells and accumulation of Sub-G0/G1 population), whereas normal human prostate epithelial cells (PrEC) were CuB-insensitive. Using a chemopreventive model, pretreatment of mice with CuB (2 weeks before PC-3 prostate cancer cell implantation) significantly reduced the rate of in vivo tumor-formation. A 79% reduction in tumor size (accompanied by marked in situ apoptosis) was observed in the CuB-treated group (with no noticeable toxicity) vs. controls at day 31. Strikingly, mechanistic investigations demonstrated that CuB drove dose-dependent inhibition of ATP citrate lyase phosphorylation (ACLY; an important enzyme for cancer metabolism) both in vitro and in the CuB-chemopreventive mouse model. Importantly, ACLY over-expression abrogated CuB’s apoptotic effects in prostate cancer cells, confirming ACLY as a direct target of CuB. Thus, CuB harbors potent chemopreventive activity for prostate cancer, and we revealed a novel anti-tumor mechanism of CuB via inhibition of ACYL signaling in human cancer. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Introduction Prostate cancer is the first leading cause of cancer-related deaths among males in the UK and the second in the US. Despite recent advances in surgery, radiation, medical management and screening, prostate cancer patients suffer high morbidity and mortality and significant treatment-associated complications [1]. Chemoprevention has the potential to reduce cancer formation, and decrease the morbidity and mortality of many types of cancer including prostate cancer [1–3]. Among all chemopreventive agents, natural products and naturally occurring compounds have

Abbreviations: CUs, Cucurbitacins; CuB, Cucurbitacin B; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline; PrEC, normal human prostate epithelial cell line; ACLY, ATP citrate lyase. ⇑ Corresponding author. Tel.: +1 412 623 3914; fax: +1 412 623 3909. E-mail address: [email protected] (D. Xiao). http://dx.doi.org/10.1016/j.canlet.2014.03.015 0304-3835/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

received increasing attention in recent years for the discovery and development of novel chemopreventive or anti-cancer agents [2,3]. ATP citrate lyase (ACLY) is a key enzyme recently shown to be crucial for cancer cell metabolism. ACLY is a metabolic enzyme responsible for the conversion of mitochondria-derived citrate into acetyl CoA, a precursor for the synthesis of both fatty acids and mevalonate [4–6]. Both fatty acid synthesis and mevalonate synthesis are associated with cancer cell growth and transformation [4–9]. Activated ACLY signaling is associated with many human cancer types including prostate cancer, lung adenocarcinoma, leukemia, glioblastomas, ovarian cancer, and liver cancer [4–16]. It is believed that specific blockade of ACLY signaling may have therapeutic potential for human cancers [4–16]. However, such anti-ACLY agents deemed suitable for and applicable to humans have yet to be developed. The cucumber (Cucumis sativus L) is a member of the Cucurbitaceae family, which includes the melons, squash and pumpkins.

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Cucumbers are commercially cultivated as a seasonal vegetable crop worldwide [17,18]. Cucurbitacins (CUs) are one of the main bioactive compounds found in cucumber. CUs are a class of highly oxidized tetracyclic triterpenoids and have been used for their potential antidiabetic, lipid-lowering and antioxidant activities since ancient times [17,18]. Structurally, CUs are characterized by a tetracyclic cucurbitane nucleus skeleton, namely, 9bmethyl-19-nor lanosta-5-enea, which is traditionally divided arbitrarily into twelve categories, incorporating CUs A-T [17,18]. In recent years, several types of CUs have been shown to inhibit proliferation and induce apoptosis and autophagy in human cancer using in vitro and in vivo models [19–36]. Cucurbitacin B (CuB) is one of the most abundant forms of CU and one of the best studied members of the CU family of compounds [19–34]. CuB-induced apoptotic cell death has been reported in human breast cancer cells [19,20,26,28,30,31,33], pancreatic cancer cells [21,22], hepatocellular carcinoma cells [23,24], lung cancer cells [25], cervical cancer cells [26], primary glioblastoma cells [26], skin cancer cells [26], renal carcinoma cells [29], laryngeal squamous cell carcinoma cells [32] and colon cancer cells [26,30]. The mechanism behind the anticancer effect of CuB is not fully understood, but several signaling molecules have been shown to be targets for the anticancer activity of CuB, including the signal transducer and activator of transcription (STAT), cyclooxygenase-2, BRCA1, and the p34CDC2/ Cyclin B1 complex [19–34]. Among all CUs, only Cucurbitacin E [35] and 23,25-dihydrocucurbitacin F [36] have been shown to harbor anti-proliferative activity against human prostate cancer cells, and the potential anti-tumor and chemopreventive activity of CuB in human prostate cancer has not been investigated. Here, we identified for the first time the potent anti-cancer activity of CuB against human prostate cancer cells, and, more importantly, its potent chemopreventive activity in a prostate cancer in vivo model. Further mechanistic investigations reveal a novel anticancer mechanism of CuB via the inhibition of ATP citrate lyase signaling in prostate cancer models. Materials and methods Reagents CuB (purity, P98%) was purchased from Sigma–Aldrich (St. Louis, MO). Reagents for cell culture, including media, penicillin and streptomycin antibiotic mixture, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). The Caspase-GloÒ3/7 activity assay kit, CellTiter-GloÒ luminescent cell viability assay kit and RNase were procured from Promega (Madison, MI). The antibodies against Cleaved-poly(ADP-ribose) polymerase (PARP), Cleaved-Caspase 3, ACLY and Phospho-ACLY, Mcl-1, Bcl-xL, Survivin, Bim and Bax were purchased from Cell Signaling (Danvers, MA), the antibody against Bcl-2 was from Santa Cruz Biotechnology (Dallas, TX), and the antibody against b-Tubulin was from Sigma–Aldrich (St. Louis, MO). Negative nonspecific control-siRNA was from QIAGEN (Valencia, CA). ACLY-targeted siRNA and Alexa FluorÒ488 Annexin V/Dead cell Apoptosis Kit were from Life Technologies (Grand Island, NY). Oligofectamine 2000 was from Invitrogen (Grand Island, NY). pCMV6 and pCMV6-ACLY were from OriGene Technologies, Inc (Rockville, MD). Hydroxycitrate tribasic (HT) and N-acetyl-L-cysteine (NAC) were from Sigma–Aldrich (St. Louis, MO). MitoSOX Red and propidium iodide were from Molecular Probes (Eugene, OR). Protease inhibitor cocktail tablets and phosphatase inhibitors cocktail tables were from Roche (Indianapolis, IN).

Cell culture and cell survival assays Monolayer cultures of LNCaP and PC-3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 medium supplemented with 10% (v/v) FBS and antibiotics. The normal prostate epithelial cell line PrEC (Clonetics, Walkersville, MD) was maintained in PrEBM (Cambrex, Walkersville, MD). Each cell line was maintained in an atmosphere of 95% air and 5% CO2 at 37 °C. The effect of CuB on cell viability was determined by the following assays as described in our previous publications [37–40]: (a) clonogenic survival assay [37]; (b) trypan blue dye exclusion assays [38,39]; and (c) CellTiter-GloÒ luminescent cell viability assays [40]. For the clonogenic survival assay, cells (1.5  105) were plated in 6-well-plates for incubation overnight and then were treated with 0.1% DMSO (control group) or the desired concentration of CuB for 24 h. The treated

cells were re-seeded in 6-well plates (500 cells/well) in complete medium without drug. The media were changed every two days. After 10 days in culture, the cells were fixed and stained with 0.5% crystal violet in 20% MeOH for colony counting. Detection of apoptosis Apoptosis induction was assessed by the assays described in our previous publications [40–44]: (a) analysis of Caspase 3/7 activity by a Caspase-GloÒ3/7 activity assay kit [40], (b) flow cytometric analysis of cells with sub-G0/G1 DNA content following staining with propidium iodide [41,42], (c) immunoblotting analysis of cleavage of PARP and Caspase 3 [43,44], and (d) Flow cytometric analysis of apoptosis with Alexa FluorÒ488-annexin V binding assay using Alexa FluorÒ488 Annexin V/Dead Cell Apoptosis Detection Kit (Life Technologies, Grand Island, NY, USA). Briefly, cells (5  105 cells) were seeded in 100-mm dishes and cultured overnight in normal medium. The next day, cells were treated with vehicle (0.1% DMSO, control) or with CuB 0.2 or 0.4 lM for 24 h, and then harvested in cold PBS and collected by centrifugation for 2 min at 500g. Cells were then resuspended and stained simultaneously with Alexa FluorÒ488-labeled annexin V and propidium iodide (PI; Pharmingen, San Diego, CA, USA). Apoptosis was verified based on loss of plasma membrane integrity. Viable cells excluded these dyes, whereas apoptotic cells allowed moderate staining. Cells were analyzed using an Accuri C6 flow cytometer (Becton Dickinson, San Jose, CA, USA). Experiments were repeated three times with similar results. Immunoblotting The cells and the frozen tumor tissue were lysed as described previously by us [37,38]. The lysate proteins were resolved by 6–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto membranes. Immunoblotting was performed as described in our previous studies [43,44]. The blots were stripped and re-probed with an anti-b-tubulin antibody to correct for differences in protein loading. Changes in protein level were determined by densitometric scanning of the immunoreactive band and corrected for the b-tubulin loading control. Immunoblotting for each protein was performed at least twice using independently prepared lysates to ensure reproducibility of the results. Xenograft study Male athymic mice (5 weeks old) were purchased from Taconic (Germantown, NY) and housed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. The use of mice for studies described herein was approved by the IACUC (IACUC Protocol No: 13071950). The nude mice were randomized into 2 groups of 4 mice per group: PBS (control) and 0.1 lmol CuB in corn oil per day. The groups of mice were orally gavaged with 0.1 ml of vehicle (PBS in corn oil, control group) or CuB in 0.1 ml of corn oil five times (from Monday to Friday) per week for 2 weeks. Then, exponentially growing PC-3 cells were mixed in a 1:1 ratio with Matrigel (Becton Dickinson, Bedford, MA) and a 0.1-ml suspension containing 3  106 cells was injected subcutaneously on both the left and right flanks of each mouse (n = 8). Experimental mice were treated by oral gavage with 0.1 ml of vehicle (PBS in corn oil, control group) or CuB in 0.1 ml of corn oil 5 times (from Monday to Friday) per week. Tumor growth was monitored 3 times (Monday, Wednesday and Friday) per week as described previously by us [43] using external caliper measurement (tumor size = [length  width  height]  0.52). The body weights of the control and drug-treated mice were recorded prior to the start of the experiment and every week thereafter. The mice from each group were also monitored for other symptoms of side effects including food and water withdrawal and impaired posture or movement. The mice in both the control and CuB-treated groups were sacrificed at the 31 days after the tumor cell implantation. The end-point of the study was set for when the tumors reached approximately 1000 mm3 in size. At the termination of the experiment, in accordance with the IACUC guidelines, the mice of each group were sacrificed by CO2 inhalation. Tumors from each mouse from every group were carefully dissected out at the time of sacrifice, weighed, and frozen in liquid nitrogen, stored at 80 °C, and used for immunoblotting of various proteins. RNA interference against ACLY The cells (1  105) were seeded in six-well plates and allowed to attach during overnight incubation. The cells were transfected with 200 nmol/L of control nonspecific siRNA or ACLY-targeted siRNA using Oligofectamine as described in our previous studies [36,37]. Twenty-four hours after transfection, the cells were treated with DMSO (control) or 0.2 or 0.4 lmol/L CuB for 24 h. The cells were collected, washed with phosphate-buffered saline (PBS), and processed for immunoblotting or analysis of Caspase 3/7 activity as described in our previous studies [37–40]. Transient transfection PC-3 and LNCaP cells were transiently transfected with pCMV6 vector or pCMV6-ACLY using the reagents supplied and following the protocol provided by the manufacturer, OriGene Technologies Inc. Briefly, the cells were plated at a

Y. Gao et al. / Cancer Letters 349 (2014) 15–25 density of 4  105 cells/ml and allowed to attach overnight. The cells were transfected with expression constructs encoding the constitutively active ACLY or with empty vector. After 6 h, the medium was replaced with fresh complete medium, and after 24 h the cells were treated with 0.2 or 0.4 lM of CuB or DMSO (control) for 24 h. The cells were collected and processed for the apoptosis assay or for immunoblotting. Measurement of mitochondrial ROS production The mitochondrial ROS generation was assessed by flow cytometry after staining with MitoSOX as described in our previous study [41]. Briefly, cells were plated, allowed to attach overnight, and treated with DMSO (control) or CuB at 0.2 or 0.4 lmol/L for 1 h. Control and treated cells were rinsed with Hank’s balanced salt solution supplemented with magnesium and calcium and treated with 5 lM MitoSOX Red for 30 min at 37 °C. The cells were collected by trypsinization, washed with phosphate-buffered saline (PBS), resuspended in Hank’s solution containing 1% bovine serum albumin (BSA), and used for flow cytometric analysis.

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whether CuB treatment can increase the Caspase 3/7 activity in the cancer cells. The CuB treatment resulted in a dose-dependent increase in Caspase 3/7 activity in both PC-3 and LNCaP cells (Fig. 2C). We also investigated whether CuB treatment increased in Sub-G0/ G1 phase in both cancer cell lines. A significant increase in SubG0/G1 phase cells was observed in CuB-treated PC-3 and LNCaP cells (Fig. 2D). Furthermore, flow cytometric analysis of apoptosis by the Alexa FluorÒ488-annexin V binding assay was used to measure the apoptosis induction effect of CuB. The present study showed that the LNCaP and PC-3 cells exhibited a statistically significant apoptotic cell death by CuB treatment (Fig. S1). Consistent with our cell viability data (Fig. 1D), the same CuB treatment of PrEC caused no apoptotic cell death (Fig. 2E). Taken together, the present results clearly indicate that the anticancer effect of CuB against prostate cancer cells was associated with the induction of apoptosis (Fig. 2).

Quantitation of ACLY by ELISA

CuB inhibited the growth of PC-3 xenografts in athymic mice The level of ACLY in human prostate cancer cells was determined with Human ATP-Citrate Synthase (ACLY) Elisa Kit (Cusabio Biotech Co., LTD, Wuhan, China) following the manufacturer’s instructions. Statistical analysis Statistical significance of difference in the measured variables between control and treated groups was determined by t-test or one-way ANOVA. Differences were considered significant at P < 0.05.

Results CuB inhibited the viability of human prostate cancer cells Initially, we investigated the effect of CuB on the proliferation of human prostate cancer PC-3 and LNCaP cells. The cell proliferation of both PC-3 and LNCaP cells was significantly inhibited in a concentration-dependent manner with an IC50 of CuB 0.3 lmol/L for 24 h treatment, as determined by CellTiter-GloÒ luminescent cell viability assays (Fig. 1A). To confirm the growth inhibitory effect of CuB, we used the trypan blue dye exclusion assay. The results indicated that treatment with CuB for 24 h resulted in a significant reduction in cell viability in both cancer cell lines (Fig. 1B). Furthermore, the clonogenic assay was used to determine the effect of CuB on cell viability. Using the colony formation assay procedure, the cells were cultured for 10 days after 24 h exposure to CuB and the colony formation (>50 cells/colony) was determined. These data suggested that CuB treatment resulted in a remarkable inhibition of cancer cell growth (Fig. 1C). To determine whether the cell death induced by CuB was selective for cancer cells, we investigated the effect of CuB on the proliferation of the normal human prostate epithelial cell line PrEC. It is noteworthy that PrEC was significantly more resistant to growth inhibition by CuB than were the prostate cancer cells (Fig. 1D). For instance, 0.2 lM CuB, which inhibited the viability of PC-3 and LNCaP cells by approximately 40% (Fig. 1A), had a minimal effect on PrEC cell viability (Fig. 1D). These data indicated that human prostate cancer cells, but not the normal human prostate epithelial cell line PrEC, were sensitive to inhibition of cell viability by CuB. CuB-mediated inhibition of cancer cell growth involved in apoptosis inducing CuB and other CUs have been shown to induce apoptotic cell death in some types of cancer cells [19–34]. Next, we determined whether CuB-caused prostate cancer cell death is related to the induction of apoptosis. Immunoreactive band corresponding to cleaved PARP and cleaved Caspase 3 were observed in both cancer cell lines (Fig. 2A and B). To confirm the results of CuB-induced apoptosis, we used a Caspase-GloÒ3/7 activity assay kit to determine

Next, we used the PC-3 xenograft mouse model to test the in vivo relevance of these in vitro founding. We tested the effect of CuB administration by oral gavage (0.1 lmol, once a day through Monday–Friday) on PC-3 xenograft growth in athymic mice. The CuB concentration used in the present study was based on the previous in vivo studies [24,27]. As shown in Fig. 3A, the average tumor volume in mice treated with 0.1 lmol CuB was significantly lower than that in vehicle-treated control mice throughout the experimental protocol. For example, 31 days after treatment commenced, the average tumor volume in the CuB-treated mice (179 ± 41 mm3) was approximately 21% of the average tumor volume in the control mice (863 ± 59 mm3, Fig. 3A). The CuB treatment resulted in a significant reduction of the tumor weight compared with the control group (Fig. 3B). The body weights of the control and experimental groups were recorded periodically to determine whether CuB administration caused weight loss. As shown in Fig. 3C, the average body weight of the control and CuB-treated mice did not differ significantly throughout the experimental protocol. Moreover, the CuB treated mice appeared healthy and did not exhibit impaired movement and posture, indigestion, or areas of redness or swelling. Our results strongly suggest that CuB administration significantly inhibited PC-3 xenograft growth without causing any side effects to the mice. Oral administration of CuB increased apoptosis in tumors Because CuB induced apoptotic cell death in human prostate cancer PC-3 and LNCaP cells, we therefore examined the tumor tissues from control and CuB-treated mice to determine whether CuB-mediated inhibition of PC-3 xenograft growth in vivo is due to increased apoptosis. As shown in Fig. 3D, CuB treatment caused a statistically significant increase in cleaved PARP and cleaved Caspase 3 immunoreactive bands compared with the control group. The present study indicated that CuB can induce apoptosis in human prostate cancer both in vitro and in vivo. CuB administration inactivated the protein of ACLY in tumors Activated ACLY signaling is associated with many human cancer types including prostate cancer [4–16]. Inhibition of ACLY signaling can suppress tumor cell growth and induce apoptosis [8–16]. We showed that CuB has the potential to inhibit prostate cancer growth both in vitro and in vivo (Figs. 1–3). Therefore, we speculated about whether CuB treatment would result in a down-regulation of ACLY protein in the PC-3 xenografts. The levels of ACLY and phospho-ACLY proteins in tumors from control and CuB-treated mice were determined by immunoblotting. Representative immunoblots for ACLY and phospho-ACLY proteins using tumor

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Fig. 1. Effect of CuB on survival of PC-3 and LNCaP cells determined by CellTiter-GloÒ luminescent cell viability assays (A), the trypan blue dye exclusion assay (B) and the colonogenic survival assay (C). (D) Effect of CuB on survival of PrEC cells determined by CellTiter-GloÒ luminescent cell viability assays. Cells were treated with different concentrations of CuB for 24 h. Columns, mean of three determinations; bars, SE. ⁄Significantly different (P < 0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown.

supernatants from the control and CuB-treated mice are shown in Fig. 3D. Changes in protein levels were quantified by densitometric scanning of the immunoreactive bands and corrected for b-tubulin loading control. As shown in Fig. 3D, the protein level of phosphoACLY was significantly lower, in tumors from CuB-treated mice than in control tumors. The protein level of total ACLY was comparable to that in the tumors of the CuB-treated and control mice (Fig. 3D). These results indicated that CuB-mediated suppression of PC-3 xenograft growth in vivo was accompanied by inactivation of ACLY. CuB down-regulated the protein expression of ACLY in prostate cancer cells Next, we tested whether CuB can decrease the level of ACLY protein in human prostate cancer cells. As shown in Fig. 4A and B, treatment with CuB at 0.2 or 0.4 lM resulted in a dose-dependent decrease of the protein levels of ACLY and phospho-ACLY in human prostate cancer PC-3 and LNCaP cells compared to those in DMSO-treated (control) cells. For example, the phospho-ACLY levels in PC-3 and LNCaP cells treated for 24 h with 0.2 lM of CuB were decreased by approximately 70% and 60%, respectively, compared to the levels in the control group (Fig. 4A and B). These observations clearly indicated that CuB treatment resulted in the inhibition of ACLY signaling in human prostate cancer cells. Induction of apoptosis by pharmacologic inhibition of ACLY HT is a competitive inhibitor of ACLY [16]. To confirm that the inhibition of ACLY is involved in the inhibition of cell growth and induction of apoptosis in human prostate cancer cells, we tested the effect of HT on the proliferation and induction of apoptosis of human prostate cancer PC-3 and LNCaP cells. HT treatment at 20 mM resulted in significant cell growth inhibition (Fig. 4C) and apoptotic cell death (Fig. 4D) in both cancer cell lines. Increased immunoreactivity to cleaved PARP and cleaved Caspase 3 was observed in the

HT-treated cancer cell line PC-3 (Fig. 4E) and in LNCaP cells (data not shown). As shown in Fig. 4E, HT treatment caused a remarkable down-regulation of the ACLY protein. These data indicate that pharmacologic inhibition of ACLY signaling can inhibit cell growth and cause apoptosis in human prostate cancer cells. CuB-induced apoptosis was enhanced by RNA interference against ACLY To directly test the contribution of ACLY to the regulation of CuB-induced apoptosis in human prostate cancer cells, we used siRNA technology. The levels of ACLY and phospho-ACLY protein were decreased by 97% by upon the transient transfection of PC-3 (Fig. 5A) and LNCaP (data not shown) cells with ACLY-targeted siRNA compared with cells transfected with a nonspecific control siRNA. Cell growth was inhibited by approximate 25% in the ACLY-targeted siRNA-transfected PC-3 and LNCaP cells when compared with the control nonspecific siRNA-transfected cells (data not shown). As shown in Fig. 5B and C, more than 20% of ACLY-targeted siRNA-transfected PC-3 and LNCaP cells advanced to apoptosis compared with the cells transfected with the control nonspecific siRNA, as determined by analysis of Caspase 3/7 activity with a Caspase-GloÒ3/7 activity assay. Similar to CuB’s effect on untransfected cells (Fig. 4A and B), the CuB treatment (0.2 and 0.4 lM, 24 h) caused decrease in the levels of ACLY and phosphoACLY in both PC-3 (Fig. 5A) and LNCaP (data not shown) cells transfected with the nonspecific control-siRNA. The 24-h exposure of PC-3 and LNCaP cells transfected with the nonspecific control-siRNA to 0.2 lM of CuB resulted in approximately 1.5-fold increase in Caspase 3/7 activity compared with the DMSO-treated controls (Fig. 5B and C). Remarkable increases in apoptotic cell death were observed in both ACLY-depleted cells treated with CuB, compared to cells transfected with control-siRNA and treated with CuB (Fig. 5B and C). Collectively, these results indicated that ACLY plays an important role in CuB-induced apoptosis in human prostate cancer cells.

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Fig. 2. Immunoblotting analysis of cleavage of PARP and Caspase 3 was performed using lysates from CuB-treated or DMSO-treated PC-3 (A) and LNCaP (B) cells. The blot was stripped and reprobed with-anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in levels relative to DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates. CuB induced apoptosis in PC-3 and LNCaP cells, but not in PrEC, determined by analysis of Caspase 3/7 activity by a Caspase-GloÒ3/7 activity assay kit (C and E) and flow cytometric analysis of cells with sub-G0/G1 DNA content following staining with propidium iodide (D). Cells were treated with the indicated concentrations of CuB or DMSO (control) for 24 h. Results are expressed as enrichment factor relative to cells treated with DMSO (control). Results are mean ± SE (n = 3). ⁄Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Dunnett’s test. Similar results were observed in at least two independent experiments. Representative data from a single experiment are shown.

CuB-induced apoptosis was inhibited by the ectopic expression of ACLY To further provide experimental evidence for the critical role of ACLY in the CuB-induced apoptosis, we proceeded to investigate whether the ectopic expression of ACLY could inhibit the CuB-induced apoptotic cell death in the prostate cancer cells. To address this question, we transiently transfected PC-3 and LNCaP cells with pCMV6-ACLY and the pCMV6 vector. The protein levels of ACLY and phospho-ACLY were significantly increased in both PC-3 (data not shown) and LNCaP (Fig. 5D) cells upon transient transfection with pCMV6-ACLY when compared with cells transfected with empty pCMV6 vector. Furthermore, exposure to CuB at 0.2 or 0.4 lM for 24 h resulted in statistically significant increases in Caspase 3/7 activity in the PC-3 and LNCaP transfected with empty pCMV6 vector, but showed significant protection against the Caspase 3/7 activity induced by CuB in the cells transiently transfected

with the pCMV6-ACLY (Fig. 5E and F). These data confirmed that the ACLY actively contributed to the CuB-induced growth inhibition of human prostate cancer. The anticancer effect of CuB was involved in mitochondrial ROS production in prostate cancer cells It has been reported that that the inhibition of tumor growth based on ACLY inhibition is regulated by ROS generation and mitochondrial dysfunction [14,15]. Our present results have revealed that the ACLY plays an important role in CuB-induced apoptosis in human prostate cancer cells. More recently, CuB has been shown to cause human breast [26] and colon [34] cancer cell death via increased ROS generation. We therefore used a cell-permeable and mitochondria-targeting chemical probe (MitoSOX Red) to measure ROS production by CuB treatment in LNCaP and PC-3 cells. Flow

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Fig. 3. Average tumor volume (A), tumor weight (B) and body weight (C) in vehicle-treated control mice and mice treated with 0.1 lmol CuB (Monday–Friday). The CuB administration commenced two weeks before the tumor cell injection. Points, mean (tumor; n = 8; four mice per group with tumor cells injected on both left and right flank of each mouse); bars, SE. ⁄, P < 0.05, significantly different compared with control by two-way ANOVA. The body weights of the control and CuB-treated mice did not differ significantly throughout the experimental protocol. Points, mean (n = 4); bars, SE. D, Immunoblotting for cleavage of PARP, Caspase 3, ACLY and p-ACLY using tumor supernatants from control and CuB-treated mice. The blots were stripped and reprobed with anti-b-tubulin antibody to correct for differences in protein loading. Densitometric scanning data for these protein levels in tumors from control and CuB-treated mice were shown on top of the immunoreactive bands. Tumor tissues from three mice of each group were used for immunoblotting. Columns, mean (n = 3); bars, SE. ⁄, P < 0.05, significantly different compared with control by paired t test.

cytometry revealed statistically significant enrichment of MitoSOX Red fluorescence over DMSO-treated controls in PC-3 (Fig. 6A) and LNCaP (data not shown) cells following treatment 1 h with 0.2 or 0.4 lM CuB. These results indicated that CuB treatment caused mitochondrial superoxide generation. Next, we designed experiments to test the contribution of ROS to CuB-induced apoptosis and ACLY inhibition. As shown in Fig. 6, the CuB-mediated apoptotic cell death evidenced by the increase in Caspase 3/7 activity and cleaved Caspase 3 in PC-3 (Fig. 6B and C) and LNCaP (Fig. 6E and F) cells was markedly suppressed in the presence of NAC (2 h pretreatment with NAC and then a 24-h treatment with CuB in the presence of NAC). The NAC also conferred protection against the CuB-mediated increase in MitoSOX Red fluorescence (data not shown) as well as inhibition of ACLY activity (Fig. 6D, PC-3) and phospho-ACLY in both PC-3 (Fig. 6C) and LNCaP cells (Fig. 6F). Taken together, these results indicated that the CuB-inhibited tumor growth is mediated by ROS-dependent ACLY inhibition. Effect of CuB treatment on levels of Bcl-2 family proteins The Bcl-2 family proteins have emerged as critical regulators of mitochondria-mediated apoptosis by functioning as either promoter (e.g., Bax) or inhibitors (e.g., Bcl-2 and Bcl-xL) of the cell death process [38]. We proceeded to test whether CuB-induced apoptosis was regulated by Bcl-2 family proteins. The effect of CuB treatment on the levels of Bcl-2 family proteins in PC-3 and LNCaP cells was determined by immunoblotting, and representative blots are shown in Fig. S2. The level of antiapoptotic proteins Bcl-2 was significantly decreased on treatment of both cells with CuB (Fig. S2). The CuB treatment did not alter the protein

expression of Bcl-xL in both cells (Fig. S2). However, the present results showed that a remarkably down-regulation of Survivin protein expression and a Mcl-1 cleavage were observed in both cells treated with CuB (Fig. S2). The increased levels of multidomain proapoptotic proteins Bax and Bim-S were found in LNCaP cells treated with CuB but not in PC-3 cells with the same doses of CuB treatment (Fig. S2). These results indicated that CuB treatment altered the Bcl-2 family proteins in prostate cancer cells. Discussion CUs, which have been used as traditional medicine for thousands of years in East Asian countries, have the potential to be used for cancer chemoprevention and chemotherapy [17–20]. CuB is one of the most promising agents as it is reported to have anticancer effects on a variety of tumors [17–34]. The efficacy of CuB against human prostate cancer, however, has not previously been examined. In our present study, we, for the first time, showed that CuB treatment significantly inhibited human prostate cancer cell growth and caused apoptotic cell death both in vitro and in vivo. Our results revealed a novel anti-cancer mechanism of CuB, in which the ACLY signaling pathway is involved in CuB-induced apoptosis in human prostate cancer. Our data indicates that CuB has anti-cancer potential in human prostate cancer cells as evidenced by the inhibition of cell growth and the induction of apoptotic cell death. The inhibition of cell survival by 24-h treatment with CuB was statistically significant at IC50 0.3 lmol/L concentrations, as determined by CellTiter-GloÒ luminescent cell viability assays (Fig. 1A). The results of both the clonogenic survival assay and trypan blue dye exclusion assays

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Fig. 4. Immunoblotting analysis of cleavage of ACLY and p-ACLY was performed using lysates from CuB-treated or DMSO-treated PC-3 (A) and LNCaP (B) cells. The blot was stripped and reprobed with-anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in levels relative to DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates. Effects of HT on survival and apoptosis of PC-3 and LNCaP cells determined by CellTiter-GloÒ luminescent cell viability assays (C) and by analysis of Caspase 3/7 activity using a Caspase-GloÒ3/7 activity assay kit (D). Cells were treated with different concentrations of HT for 24 h. Columns, mean of three determinations; bars, SE. ⁄Significantly different (P < 0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown. E, Immunoblotting analysis of cleavage of PARP, Caspase 3 and ACLY was performed using lysates from HT-treated or DMSO-treated PC-3 cells. The blot was stripped and reprobed with-anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in levels relative to DMSOtreated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates.

show that the IC50 concentrations for CuB on cell viability were 0.1 lmol/L (Fig. 1B and C). These results indicate that the clonogenic survival assay and trypan blue dye exclusion assays are more sensitive than the CellTiter-GloÒ luminescent cell viability assays. Previous studies [19–34] have reported that CuB inhibited cell growth in other types of cancer cells with IC50 at 0.1–1.0 lmol/L concentrations. Our present results indicate that prostate cancer cells are sensitive to cell death induced by CuB because the IC50 0.1–0.3 lmol/L concentrations of CuB to prostate cancer cells are within the IC50 0.1–1.0 lmol/L range of CuB for other types of cancer cells. Next, we investigated whether CuB treatment has a selective activity on human prostate cancer cells. To address this question, we conducted an experiment to show the effects of CuB on the proliferation of PrEC, a normal human prostate epithelial cell line. Our data clearly showed that CuB-treated PrEC cells displayed significantly less cell growth inhibition than the CuBtreated human prostate cancer cells (Fig. 1). Consistent with our data, Dakeng et al. [20] reported that CuB exerts a strong anticancer activity in human breast cancer cells but only has a slight effect on the proliferation of non-malignant HBL-100 cells. Collectively,

these results indicate that the human prostate cancer cells are more sensitive to growth inhibition by CuB than is the normal human prostate epithelial cell line PrEC. Although the prostate cancer cell lines PC3 is androgen-independent whereas LNCaP is androgen-dependent, the present data showed that the LNCaP and PC-3 cells exhibited comparable sensitivity to CuB. We, therefore, concluded that androgen-responsiveness is not a critical factor in CuB-mediated growth inhibition in prostate cancer cells. The present results indicate that the anticancer activity of CuB against human prostate cancer cells is associated with apoptosis induction. This conclusion is based on the following: (a) CuB treatment resulted in a significant increase of Caspase 3/7 activity in human prostate cancer cells, but not in the normal human prostate epithelial cell line (Fig. 2C and E), (b) an increase in the Sub-G0/G1 phase was observed in CuB-treated prostate cancer cells (Fig. 2D), (c) CuB treatment increased an immunoblotting band of cleaved PARP and cleaved Caspase 3 in the prostate cancer cells (Fig. 2A and B) and (d) CuB-induced apoptotic cell death was also determined by flow cytometric analysis of apoptosis using the Alexa FluorÒ488-annexin V binding assay (Fig. S1).

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Fig. 5. A, Immunoblotting for ACLY and p-ACLY using lysates from PC-3 cells transiently transfected with a control nonspecific siRNA or ACLY-targeted SiRNA and treated for 24 h with DMSO or 0.2 or 0.4 lmol/L CuB. The blots were stripped and reprobed with anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated nonspecific control siRNA-transfected cells. Caspase 3/7 activity in PC-3 (B) or LNCaP (C) cells transiently transfected with a control nonspecific siRNA or ACLY-targeted SiRNA and treated for 24 h with DMSO or 0.2 or 0.4 lmol/L CuB. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean (n = 3); bars, SE. ⁄Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by by Dunnett’s test. (D) Immunoblotting for ACLY and p-ACLY using lysates from LNCaP cells transiently transfected with pCMV6 vector or pCMV6-AC-GFP and treated for 24 h with DMSO or 0.2 or 0.4 lmol/L CuB. The blots were stripped and reprobed with anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent changes in protein levels relative to DMSO-treated pCMV6 vector-transfected cells. Caspase 3/7 activity in PC-3 (E) or LNCaP (F) cells transiently transfected with pCMV6 vector or pCMV6-AC-GFP and treated for 24 h with DMSO or 0.2 or 0.4 lmol/L CuB. The results are expressed as enrichment factor relative to DMSO-treated control cells transiently transfected with the control nonspecific siRNA. Each experiment was done twice, and representative data from a single experiment are shown. Columns, mean (n = 3); bars, SE. ⁄Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Dunnett’s test.

Demonstration of in vivo efficacy of potential chemopreventive agents in animal models is necessary for their clinical development. Our present study provides experimental evidence that CuB 0.1 lmol by oral administration (for 2 weeks before the cancer cell injection, 5 days per week) significantly inhibits the growth of PC-3 xenografts in athymic mice without causing weight loss or any other side effects. Similarly, CuB has been reported to inhibit the growth of several types of human cancer cells in xenograft mouse models, including non-small-cell lung cancer H1299 cell [25], pancreatic cancer Panc-1 cell [21,22], hepatocellular carcinoma BEL-7402 cell [24] and HepG2 [23], and breast cancer MDA-MB-231 cell [30]. These studies [21–25,30] showed the chemotherapeutic effect of CuB in vivo. Our results are the first, however, to show the potential chemoprevention of CuB in prostate cancer. We also found that the CuB-mediated inhibition of PC-3 xenograft growth in vivo is associated with an increase in an immunoblotting band of cleaved PARP and cleaved Caspase 3 in

tumors from CuB-treated mice as compared to those of the control mice (Fig. 3D). These observations are consistent with cellular studies in which treatment of PC-3 cells with CuB at 0.05– 0.4 lmol/L results in a concentration-dependent induction of apoptosis (Fig. 2). Thus, it is reasonable to conclude that the induction of apoptosis is a critical event in CuB-mediated growth inhibition of PC-3 cells in vivo. ACLY signaling is involved in the progress of many diseases including cancer [4–16]. Inactivation of ACLY signaling has been implicated in the pathogenesis of many kinds of human cancers including prostate cancer [4–16]. Our present results indeed suggest that ACLY signaling is also involved in CuB-induced prostate cancer cell growth inhibition and apoptosis induction. First, the phospho-ACLY levels were significantly reduced in PC-3 xenograft tumors in mice treated with CuB compared to those in the control mice (Fig. 3D). Next, down-regulation of the phospho-ACLY and ACLY proteins was observed in both prostate cancer PC-3 and

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Fig. 6. Anticancer effect of CuB is mediated by mitochondrial ROS generation. (A) Flow cytometric measurement of MitoSOX Red fluorescence in PC-3 cells treated with DMSO (control) or 0.2 and 0.4 lM CuB for 1 h. Results shown are mean ± S.D. total sample size is n = 4 per group. ⁄Significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Dunnett’s test. Effect of NAC on CuB (0.2 lM for 24 h)-mediated Caspase 3/7 activity (B), cleaved Caspase 3 and phosphor-ACLY (C) and ACLY activity (D) in PC-3 cells treated with or without 2 h pretreatment of 4 mM NAC. The detailed treatment information was described in the section of Methods. For B and D, Columns, mean of three determinations; bars, SE. ⁄Significantly different (aP < 0.05) compared with DMSO-treated control and (bP < 0.05) compared with CuB-treated group by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown. For C, immunoblotting analysis of cleavage of Caspase 3 and phospho-ACLY was performed using lysates from the PC-3 cells. The blot was stripped and reprobed with-anti-btubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in levels relative to DMSO-treated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates. Effect of NAC on CuB (0.2 lM for 24 h)-mediated Caspase 3/7 activity (E) and cleaved Caspase 3 and phosphor-ACLY (F) in LNCaP cells treated with or without 2 h pretreatment of 4 mM NAC. The detailed treatment information was described in the section of Methods. For E, Columns, mean of three determinations; bars, SE. ⁄Significantly different (aP < 0.05) compared with DMSO-treated control and (bP < 0.05) compared with CuB-treated group by one-way ANOVA followed by Dunnett’s test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown. For F, immunoblotting analysis of cleavage of Caspase 3 and phospho-ACLY was performed using lysates from the LNCaP cells. The blot was stripped and reprobed with-anti-b-tubulin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent change in levels relative to DMSOtreated control. Immunoblotting for each protein was performed at least twice using independently prepared lysates.

LNCaP cells treated with CuB compared with the DMSO-treated control cells (Fig. 4A and B). In addition, CuB treatment caused a significant reduction of ACLY activity in both PC-3 (Fig. 6D) and LNCaP (data not shown) cells, as determined by a quantitative ACLY ELISA kit. These results suggest that CuB-induced prostate cancer growth inhibition was mediated by the downregulation of ACLY. Recently, it has been reported that the genetic or pharmacologic down-regulation of ACLY activity in cancer cells results in the inhibition of cell proliferation and induction of apoptosis in vitro and in vivo [7–15]. A more recent publication showed that ACLY inhibition may affect cancer stem cells in a broad range of genetic backgrounds and thus has widespread applicability [8]. Beckner et al. [16] reported that inhibition of ACLY with HT in a 12–24 mmol/L

treatment resulted in the suppression of in vitro glioblastoma cell migration, clonogenicity and brain invasion under glycolytic conditions. We therefore tested the effect of HT on cell growth and apoptosis in human prostate cancer cells. As shown in Fig. 4C–E, treatment with 20 mmol/L HT significantly inhibited the proliferation and induced apoptosis, as well as downregulating the protein expression of ACLY. To determine the real role of ACLY in the CuBinduced apoptotic cell death in our models, we knocked down ACLY in the cells with ACLY-siRNA and then measured the ACLY protein expression and the induction of apoptosis. The present data showed that the inhibition of ACLY resulted in a reduction of cell viability and an increase of apoptosis in human prostate cancer cells. The CuB-induced downregulation of ACLY and induction of apoptosis were significantly enhanced by the siRNA knockdown

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in both cancer cell lines (Fig. 5A–C). To further support the regulatory role of ACLY in CuB-induced apoptosis, we overexpressed ACLY via pCMV-ACLY in the cancer cell lines. Interestingly, the overexpression of ACLY significantly protected against the induction of apoptosis induction by CuB in these cells (Fig. 5D–F). Taken together, these results indicated that ACLY is the real target for the CuB-induced apoptosis in human prostate cancer cells. The inhibition of ACLY induces an anticancer effect that has been reported to be involved in mitochondrial reactive oxygen species (ROS) generation [14,16], dual blockade of mitogen-activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways [11], and the glycolytic phenotype of tumor [14]. More recently, Lei et al. reported that ACLY acetylation stabilizes ACLY and promotes the development of lung cancer [45]. CuB has been revealed as a multitargeted cancer chemopreventive and chemotherapeutic agent. The mechanisms underlying CuB-induced apoptosis are associated with the signal transducer and activator of transcription, cyclooxygenase-2, BRCA1, p34CDC2/Cyclin B1 complex, ROS regulation [19–34], and the disruption of microtubule polymerization and nucleophosmin/B23 translocation [46]. Our present data showed that CuB treatment induced mitochondrial ROS generation in human prostate cancer cells. However, the CuB-induced mitochondrial ROS production and apoptosis as well as ACLY inhibition were almost completely blocked by NAC treatment. These results suggest that mitochondrial ROS-dependent ACLY signaling is involved in CuB-induced apoptotic cell death in our models. The proapoptotic Bcl-2 family proteins, which can be subdivided into the Bax subfamily of multidomain proteins (e.g., Bax and Bak) or BH3-only subfamily (e.g., Bid and Bim), induce mitochondrial membrane permeabilization and release of apoptogenic molecules from mitochondria to the cytosol [38]. A down-regulation of Bcl-2 and Survivin protein expressions (PC-3 and LNCaP) and an increase in protein levels of Bax (LNCaP) and Bim-S (LNCaP) were observed in our present study (Fig. S2). Furthermore, CuB treatment resulted in a remarkable cleavage of Mcl-1 protein in both PC-3 and LNCaP cells (Fig. S2). The present study indicates that the multidomain proapoptotic Bcl-2 family members Bax/ Bim and/or antiapoptic Bcl-2 family members Bcl-2 and Mcl-1 play a critical role in regulation of CuB-induced apoptosis. Therefore, further studies are needed to determine the role of antiapoptotic Bcl-2 family members, such as Bcl-2 and Mcl-1 in regulation of CuB-mediated cell death in prostate cancer cells. Conclusion We are the first to report that CuB is a potent inhibitor of prostate cancer cell growth both in vitro and in vivo. Our present study reveals a novel mechanism of CuB-anticancer activity namely, that ACLY plays an important role in CuB-induced apoptotic cell death in human prostate cancer. To our knowledge, this is the first study to report that a natural compound inhibited cancer growth by inactivation of ACLY signaling. Therefore, CuB could potentially be useful as a leading compound for future anti-prostate cancer research, as well as for clinical studies that aim to investigate prostate cancer prevention and therapy. Conflict of Interest No potential conflicts of interest were disclosed. Acknowledgments The authors thank Drs. Joel Nelson and Zhou Wang of Department of Urology and Department of Urology Research for their support. The research related to this article is partially supported

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