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Role of P53-Senescence Induction in Suppression of Lncap Prostate Cancer Growth by Cardiotonic Compound Bufalin Yong Zhang1, Yinhui Dong1,2, Michael W

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Role of P53-Senescence Induction in Suppression of Lncap Prostate Cancer Growth by Cardiotonic Compound Bufalin Yong Zhang1, Yinhui Dong1,2, Michael W Published OnlineFirst August 30, 2018; DOI: 10.1158/1535-7163.MCT-17-1296 Small Molecule Therapeutics Molecular Cancer Therapeutics Role of P53-Senescence Induction in Suppression of LNCaP Prostate Cancer Growth by Cardiotonic Compound Bufalin Yong Zhang1, Yinhui Dong1,2, Michael W. Melkus1, Shutao Yin1,2, Su-Ni Tang1, Peixin Jiang1, Kartick Pramanik1,3,WeiWu1,3, Sangyub Kim3,MinYe4, Hongbo Hu2, Junxuan Lu1,3,5, and Cheng Jiang1,3 Abstract Bufalin is a major cardiotonic compound in the tradi- Bufalin exposure induced protein abundance of P53 (not tional Chinese medicine, Chansu, prepared from toad skin mRNA) and P21CIP1 (CDKN1A), G2 arrest, and increased secretions. Cell culture studies have suggested an anticancer senescence-like phenotype (SA-galactosidase). Small RNAi potential involving multiple cellular processes, including knocking down of P53 attenuated bufalin-induced senes- differentiation, apoptosis, senescence, and angiogenesis. In cence, whereas knocking down of P21CIP1 exacerbated prostate cancer cell models, P53-dependent and indepen- bufalin-induced caspase-mediated apoptosis. In vivo, daily dent caspase-mediated apoptosis and androgen receptor intraperitoneal injection of bufalin (1.5 mg/kg body weight) (AR) antagonism have been described for bufalin at micro- for 9 weeks delayed LNCaP subcutaneous xenograft tumor molar concentrations. Because a human pharmacokinetic growth in NSG SCID mice with a 67% decrease of final study indicated that single nanomolar bufalin was safely weight without affecting body weight. Tumors from bufalin- achievable in the peripheral circulation, we evaluated its treated mice exhibited increased phospho-P53 and SA- cellular activity within range with the AR-positive and P53 galactosidase without detectable caspase-mediated apopto- wild-type human LNCaP prostate cancer cells in vitro.Our sis or suppression of AR and PSA. Our data suggest potential data show that bufalin induced caspase-mediated apoptosis applications of bufalin in therapy of prostate cancer in at 20 nmol/L or higher concentration with concomitant patients or chemo-interception of prostate precancerous suppression of AR protein and its best-known target, PSA lesions, engaging a selective activation of P53 senescence. and steroid receptor coactivator 1 and 3 (SRC-1, SRC-3). Mol Cancer Ther; 17(11); 2341–52. Ó2018 AACR. Introduction ities that have improved patient survival measured by months, there is currently no cure for castration-resistant prostate cancer Prostate cancer is the second leading cause of cancer-related (CRPC), following standard-of-care androgen deprivation thera- death in American men. In spite of significant advances in hor- py (ADT). Unfortunately, the heavily treated CRPC does not monal- (1, 2), and taxane-based chemotherapeutic (3, 4) modal- respond to checkpoint inhibitor immunotherapies that exert durable remission in some "high mutation load" malignancies such as melanoma and non–small cell lung cancer. New drugs 1Department of Biomedical Sciences, Texas Tech University Health Sciences with mechanisms distinct from current modalities will likely, 2 Center School of Pharmacy, Amarillo, Texas. Department of Nutrition and therefore, help to improve patient survival to prevent or delay Health, College of Food Science and Nutritional Engineering, China Agricultural recurrence at different stages of a patient's therapeutic course. University, Beijing, China. 3Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania. 4The State Key Laboratory of Natural and Chansu, a traditional Chinese medicine prepared from the skin Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beij- and auricular gland secretions of giant toads, has been used to ing, China. 5Penn State Cancer Institute, Pennsylvania State University, Hershey, treat various cardiac diseases, infections, and pains for millen- Pennsylvania. niums (5). Chansu-containing prescriptions have been used for Note: Supplementary data for this article are available at Molecular Cancer cancer treatment in China and are being evaluated clinically Therapeutics Online (http://mct.aacrjournals.org/). elsewhere (6). Bufalin (Fig. 1A) is a major cardiotonic steroid- Current address for K. Pramanik: Kentucky College of Osteopathic Medicine like compound isolated from Chansu (5, 7). In cell culture (KYCOM), University of Pikeville, Pikeville, Kentucky; and current address for W. models, bufalin exerts growth inhibitory and/or cytotoxic activity Wu, Department of Chemistry and Pharmacology, Zhuhai College of Jilin on leukemia (8–10) and solid cancers of many organ sites, University, Zhuhai, Guangdong, China. including prostate (7, 11, 12) and breast (13–15). Reported Corresponding Authors: Cheng Jiang, Penn State College of Medicine, Hershey, cellular responses include differentiation, apoptosis, cell-cycle PA 17033. Phone: 717-531-8964; Fax 717- 531- 5013; E-mail: arrests, autophagy, antiangiogenesis, and antimetastasis (5). [email protected]; and Junxuan Lu, Some of these reports suggest a preferential growth inhibition [email protected] of malignant cells over "normal" noncancer cells. Most studies doi: 10.1158/1535-7163.MCT-17-1296 employed bufalin exposure concentrations in sub- to micromolar Ó2018 American Association for Cancer Research. range, except the few early studies with leukemia differentiation www.aacrjournals.org 2341 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 30, 2018; DOI: 10.1158/1535-7163.MCT-17-1296 Zhang et al. Figure 1. Effects of bufalin on apoptosis in LNCaP, DU145, and PC-3 prostate cancer cells and on AR, P53, and DDR signaling in relationship to apoptosis in LNCaP cells. A, Chemical structure of bufalin. B, Concentration-dependent reduction of number of adherent LNCaP cells exposed to bufalin for 48 hours. The data were shown as Mean Æ SEM of crystal violet stain optical density, n ¼ 3 wells. C, Western blot analysis detection of caspase-driven apoptotic marker (c-PARP) and P53 protein in LNCaP (wild-type P53), DU145 (mutant P53), and PC-3 (P53 null) cells after bufalin treatment for 48 hours. D, Western blot analysis detection of c-PARP, AR, PSA, SRC-1, and SRC-3 in LNCaP cells exposed to bufalin for 48 hours. E, Growth suppression by bufalin on PC-3 cells estimated by crystal violet stain optical density, n ¼ 3 wells, Mean Æ SEM. F, Western blot analysis detection of P53, p-P53 (ser15), P21CIP1, and key DDR proteins in LNCaP cells exposed to bufalin for 48 hours. G, Western blot analysis detection of LC3 lipid modification as a marker of autophagy in LNCaP cells exposed to bufalin for 24 hours and 72 hours. 2342 Mol Cancer Ther; 17(11) November 2018 Molecular Cancer Therapeutics Downloaded from mct.aacrjournals.org on September 27, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst August 30, 2018; DOI: 10.1158/1535-7163.MCT-17-1296 Bufalin and Prostate Cancer Cell Senescence induction (5, 9, 16). For example, prostate cancer cell culture The DU145 and PC-3 prostate cancer cells were also obtained studies reported concentrations up to 15 mmol/L (7, 11, 12). from the ATCC and used within 25 passages of thawing from Within such ranges, bufalin was shown to induce caspase- cryopreservation. In terms of P53 functional status, DU145 cells mediated apoptosis in association with changes of BAX/BCL2 contain nonfunctional mutant protein and PC3 cells are null (23, þþ ratio, cytochrome release, and cellular Ca level. It is note- 24). DU145 cells were cultured in EMEM essential medium worthy that one study reported the mediator role of P53 (TP53) (ATCC) supplemented with 10% FBS without antibiotics. PC-3 tumor suppressor protein in micromolar bufalin–induced cells were cultured in F-12K medium (ATCC) supplemented with caspase activation and apoptosis of LNCaP cells (11). 10% FBS without antibiotics. Mycoplasma testing was performed In light of the potent cardiotonic indication of bufalin, which on LNCaP cells and was found negative. also represents a major adverse effect of overdosing, the thera- peutic window is likely tight (5). A human pharmacokinetic study Flow cytometry analyses with Huachansu (a commercial Chansu preparation) at up to The LNCaP cells were harvested with trypsinization for flow 8-fold higher doses than typical use showed that bufalin infusion cytometry as previously described (25). Phospho-histone H3 could reach a plasma concentration of approximately 9 nmol/L, (Ser10) was used as a marker of mitotic cells to distinguish the without apparent side effects (6). Therefore, the relevance of cell cycle phase between G2 and M. Cells were isolated and fixed mechanisms studied in cell culture models should be validated (4% formaldehyde þ 0.1% Triton X-100 in PBS) at room tem- with in vivo efficacy and safety data to facilitate meaningful perature for 10 minutes. The cells were washed with PBS and translation into clinical benefits. Given the crucial role of andro- resuspened in 75 mL of 2% FBS in PBS. The cells were then gen receptor (AR) signaling for prostate cancer genesis and pro- incubated with anti-phospho-histone H3 (Ser10; Cell Signaling gression (17), the steroid-like backbone of bufalin and its Technology) antibody together with propidium iodide/RNAse A reported AR antagonism (18, 19) and inhibition of select steroid solution at room temperature in the dark for 60 minutes. The cells receptor coactivator SRCs (SRC1 and SRC3, but
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