Article pubs.acs.org/JAFC
Physapubescin B Exhibits Potent Activity against Human Prostate Cancer In Vitro and In Vivo Wanjing Ding,† Zhijuan Hu,† Zhewen Zhang,‡ Qiaoqiao Ma,† Huifang Tang,*,‡ and Zhongjun Ma*,† †
Institute of Marine Biology, Ocean College of Zhejiang University, Hangzhou, Zhejiang 310058, China Department of Pharmacology, School of Basic Medical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
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S Supporting Information *
ABSTRACT: The present data showed that a natural compound isolated from the plant Physalis pubescens L. (Solanaceae), physapubescin B, exhibited antitumor activity against prostate cancer in vitro and in vivo. Treating prostate cancer cells with physapubescin B resulted in the accumulation of cells in the G2/M phase, which was associated with reduced Cdc25C levels and increased levels of CyclinB1, P21 as well as p-Cdk1 (Tyr15). Additionally, reactive oxygen species (ROS) generation was increased in physapubescin B-treated PC-3 cells. Furthermore, the physapubescin B-induced decrease of Cdc25C protein expression together with the G2/M phase cell cycle arrest were significantly abrogated by antioxidant NAC and GSH. Our data also demonstrated that physapubescin B exhibited strong in vivo antitumor efficacy in human prostate cancer PC3 xenograft. In conclusion, our results provide clear evidence that physapubescin B exhibits antitumor activity both in vitro and in vivo and deserves further development as an anticancer agent. KEYWORDS: physapubescin B, antitumor activity, prostate cancer, G2/M phase arrest, ROS, Cdc25C
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xenograft models.13 Hence, it is important to develop a bioactive inhibitor of Cdc25 phosphatases. In the past 30 years, natural products played a significant role in the process of drug discovery and development.14,15 Many compounds used in cancer chemotherapy are derived from natural products, such as paclitaxel, camptothecin, and etoposide. Evidence is mounting to indicate that some naturally occurring withanolides, a group of C28 steroids, exert an antiproliferative effect by inducing apoptosis or cell cycle arrest.16,17 Physapubescin B (Figure 1A) is a natural withanolide isolated from the plant Physalis pubescens L. (Solanaceae). The plant is cultivated for its nutritious fruits in China. Our previous results showed that physapubescin B possessed potent quinone reductase induction activities and inhibited the proliferation of mouse hepatoma Hepa1c1c7 cells.18 In the present study, we further evaluated its effects against prostate cancer in vitro and in vivo. We also provide evidence to indicate that physapubescin B treatment causes G2/M cell cycle arrest in PC3 cells via a mechanism involving the generation of ROS. In summary, the results of our study suggest that physapubescin B could be a potential drug candidate for the treatment of prostate cancer.
INTRODUCTION Prostate cancer is the second most common cancer in men throughout the world, accounting for 250 000 deaths per year. Although androgen ablation therapy has transient effects in the majority of patients, many patients develop more aggressive androgen-independent tumors, which are resistant to the chemotherapeutic agents, and eventually die of their disease.1,2 Therefore, there is a critical need to develop new therapies against prostate cancer. Dysregulation of cell proliferation is one of the hallmarks of tumorigenesis which is thus believed to be connected with disorders of the cell cycle.3 Sequential activation of cyclindependent kinases (CDKs) controls cell cycle progression. Cyclins, cyclin-dependent kinase inhibitors, and a variety of other proteins are involved in regulating CDK activities. The cell division cycle 25 (Cdc25) family of proteins are important regulators of G2-M transition, which are responsible for the removal of inhibitory phosphate groups from Thr14 and Tyr15, thus activating CyclinB/Cdk.4,5 Overexpression of Cdc25C and Cdc25B are related to malignant features and aggressive cancer phenotypes in vulvar squamous cell carcinomas.6 Frequent mutation of Cdc25C promoted malignant transformation in FPD/AML patients patients.7 Furthermore, abnormal expression of Cdc25C has been reported in prostate carcinomas.8 Androgen treatment in prostate cancer cells resulted in increased Cdc25C protein expression partly through reducing its degradation, suggesting that up-regulating the degradation pathways of Cdc25C protein could be a promising therapy for prostate cancer.9 A group of agents has been reported to exert anticancer effects through inducing Cdc25C degradation.10−12 Numerous potent quinoid Cdc25 inhibitors have been shown to inhibit the proliferation of tumor cell lines, but very few of these have exhibited antitumor activity in human tumor cell © XXXX American Chemical Society
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MATERIALS AND METHODS
Drugs and Chemicals. Physapubescin B was isolated from dried fruits of P. pubescens in our laboratory. Its purity was determined to be 98% by HPLC measurement. It was dissolved in DMSO with a stock solution at 40 mM, which was stored in aliquots at −20 °C. 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Received: June 19, 2015 Revised: September 28, 2015 Accepted: September 29, 2015
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DOI: 10.1021/acs.jafc.5b03045 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
tumor tissues were homogenated with lysis buffer on ice, then centrifuged at 10 000g for 25 min at 4 °C. Standard Bradford assay (Bio-Rad, San Diego, CA) was used to determine protein concentration. 40−80 μg of protein was electrophoresed by SDS− PAGE. Primary antibodies to p-Cdc25C (ser216), P21, p-Cdk1 were from Cell Signaling Technology (Beverly, MA) and other antibodies we used were from Santa Cruz Biotechnology (Santa Cruz, CA). IRDye 800 antirabbit and IRDye 680 antimouse secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE). Proteins were visualized using LI-COR Odyssey Infrared Imaging System. Normalization was ensured by β-actin or GAPDH and densitometry of the Western blot protein bands was analyzed using ImageJ software. Real-Time Reverse Transcription-PCR. Total RNA was isolated with the Trizol rea-gent, precipitated by isopropyl alcohol and rinsed with 70% ethanol. Thermoscript RT ki-t (Invitrogen, Carlsbad, CA) was used to prepare single-strand cDNA from the purified RNA. The cDNA was subjected to the quantitative real-time PCR (Biorad, Hercules, CA). The primers are as follows: Cdc25c, 5′-TTTTTCCAAGGTATGTGCGCTG-3′, 5′-TGGAACTTCCCCGACAGTAAGG-3′; GAPDH, 5′-GAGTCAACGGATTTGGTCGT-3′, 5′TTGATTTTGGAGGGATCTCG-3′. Intracellular ROS Detection. Cells were exposed to physapubescin B for 1−3 h and stained with 10 μM DCFDA for 30 min at 37 °C. Then cells were harvested, washed with PBS, and measured immediately by Beckman FC500 (excitation at 488 nm, emission at 525 nm). In Vivo Antitumor Activity. Human prostate cancer PC3 xenograft was established by injecting 5 × 106 cells subcutaneously into nude mice. The mice were divided into control and physapubescin B treatment groups randomly. Physapubescin B (50 mg/kg) was dissolved in Cremophor EL/ethanol/PBS solution (1:1:8) and administrated intraperitoneally twice a week for 30 days. Mice in the control group received vehicle. Mice tumor volume was recorded every 4 days until animals were sacrificed at 30 days. The size of tumors (mm3) was measured with calipers. Tumor volume was calculated as (W2 × L)/2 (W: width; L: length). The relative tumor volume was calculated based on the formula: RTV = Vn /V0 (Vn: the tumor volume at day n; V0: the tumor volume at day 0). The T/C % was determined using the calculation formula T/C % = mean RTV of treated group/mean RTV of control group ×100. The tumor growth inhibition rate was calculated according to the formula IR (%) = (1 − Wt/Wc) × 100 (Wt: the mean tumor weight of treated group; Wc: the mean tumor weight of control group). Animal care was in accordance with institutional guidelines. Histology, Immunohistochemistry, and TUNEL Assay. The tumors were fixed in 10% neutral formalin and embedded in paraffin. Histopathological changes in the tumor were assessed in at least 5 randomly selected tissue sections from each group studied. The tissues were cut into sections (4 μm) and stained with hematoxylin and eosin (H&E) to examine tissue and cell structure. Ki 67 protein in tumor tissues was detected by immunohistochemistry. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was applied with the TUNEL apoptosis assay kit (Roche Diagnostics GmbH, Mannheim, Germany) to paraffin sections of formalin-fixed tumor samples in according to the manufacturer’s instructures. Statistical Analyses. All values were presented as means ± SD and significance between the values of the groups was analyzed using Student’s t-test. If p values
