Li et al Tropical Journal of Pharmaceutical Research April 2015; 14 (4): 611-617 ISSN: 1596-5996 (print); 1596-9827 (electronic) © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. All rights reserved. Available online at http://www.tjpr.org http://dx.doi.org/10.4314/tjpr.v14i4.8 Original Research Article Effect of Scopoletin on Apoptosis and Cell Cycle Arrest in Human Prostate Cancer Cells In vitro Chun-Long Li*, Xian-Cheng Han, Hong Zhang, Jin-Sheng Wu and Bao Li Department of Urology, Affiliated Hospital of Weifang Medical University, Weifang 261031, China *For correspondence: Email: [email protected]; Tel/Fax: 0086-536-8068850 Received: 5 November 2014 Revised accepted: 14 March 2015 Abstract Purpose: To investigate the anticancer activity of scopoletin against human prostate cancer. Methods: The anticancer activity of scopoletin was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MMT) assay. Flow cytometry using propidium iodide and annexin V-FITC was employed to study apoptosis and cell cycle analysis. Hoechst 33258 staining was used to assess the effect of scopoletin on cell morphology and apoptotic body formation in human prostate carcinoma (LNCaP) cells via Florescence microscopy and finally Western blotting was used to evaluate the effect of scopoletin on cyclin D1 and cyclin B1 expressions. Results: Scopoletin induced a dose-dependent growth inhibition in LNCaP prostate cancer cells. It induced G2/M phase growth arrest and led to an increase in the sub-G0/G1 cell population after treatment with increasing doses compared to control cells, scopoletin treatment resulted in cell shrinkage along with membrane blebbing which are characteristic features of cell apoptosis. Approximately 15.45, 32.6 and 21.71 % of the cells underwent early apoptosis after treatment with 40, 80 and 100 µM of scopoletin respectively. Cyclin D expression diminished in a concentration-dependent manner when LNCaP cells were treated with different concentrations of scopoletin. Conclusion: These results reveal that scopoletin may be used as a natural chemotherapeutic agent against prostate cancer. Keywords: Prostate cancer, Apoptosis, Cell cycle analysis, Scopoletin, Flow cytometry, Fluorescence microscopy Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts INTRODUCTION prostate cancer accounts for less than 1 % of all cancers in men. Prostate cancer is the most frequently diagnosed malignancy in males and the second leading However, recent data suggest that prostate cause of cancer-related death in men after lung cancer rates are increasing [1]. In spite of cancer in United States of America [1]. Despite a substantial progress in prostate cancer similar prevalence of latent prostate tumors treatment, the survival rate for advanced stages around the globe, incidence rates for clinical of this particular cancer has not considerably prostate cancer in Western men are 30-50 times improved during the past decade [2,3]. higher than those for Asian men and the reason Furthermore, standard treatment preferences for for this difference is unclear. In the United States, localized prostate cancer (surgical, radiation, and prostate cancer accounts for 32 % of all cancer hormonal therapy) are accompanied with cases in men whereas in Shanghai (China), complications that often deteriorate patients’ Trop J Pharm Res, April 2015; 14(4): 611 Li et al quality of life, such as urinary incontinence and Cell viability assay sexual dysfunction. Therefore, prostate cancer patients seek complementary and alternative The in vitro cytotoxic effect of scopoletin toward medicine (CAM) treatments in the hope of human prostate cancer cells (LNCaP) was suppressing or slowing down disease determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. progression to improve the general quality of life. 3 Recent surveys demonstrated that approximately Cells (5 × 10 cells per well) were seeded onto a 40 % of prostate cancer patients utilize various 96-well plate for 24 h, treated with various CAM modalities as a component of therapy [4-6]. concentrations (20, 40, 60, 80 and 100 µM) of Scopoletin, and incubated for an additional 2 days at 37 oC. Thereafter, 10 μL of MTT at a Scopoletin (6-methoxy-7-hydroxycomarin) is a concentration of 5 mg/mL was added to each phenolic coumarin and an important member of well, and cells were incubated for an additional the group of phytoalexins isolated from many 4–6 h. The supernatant was aspirated, and 100 plants, and also a prominent coumarin derivative μL of DMSO was added to the wells to dissolve occurring in various plants [7-10]. It has also any precipitate present. The absorbance was been reported to be an active component in the measured in an ELISA reader (Thermo Molecular plants such as Erycibe obtusifolia, Aster tataricus Devices Co, Union City, USA) at 570 nm. and Foeniculum vulgare. These plants have been used in traditional Chinese medicines for Cell inhibition ratio (C) was calculated as in Eq 1. the treatment of various diseases with a long history. A variety of biological activities like anti- C (%) = {(Ac At)/Ac}100 ……………………. (1) inflammatory, anti- allergy and anti-angiogenesis have been reported for scopoletin [11-14]. The Cytotoxicity was expressed as the concentration aim of the current research work was to evaluate of scopoletin inhibiting cell growth by 50 % (IC50). the anticancer activity of scopoletin against prostate cancer cells in vitro by studying its effect Cell cycle analysis by flow cytometry on apoptosis and cell cycle phase distribution. 6 LNCaP cells (1 × 10 ) were seeded in 60-mm EXPERIMENTAL dishes and subjected to various concentrations (0, 40, 80, 100 μM) of scopoletin for 48 h. Chemicals and reagents Floating and adherent cells were trypsinized and washed three times with PBS. Cells were Scopoletin was purchased from Sigma Chemical incubated in 70 % ethanol at -20 °C overnight, Company (St Louis, MO, USA).Growth medium treated with 20 µg/mL RNase A, and stained with RPMI-1640 (Hangzhou Sijiqing Biological 5.0 µg/mL of propidium iodide. Finally the stained Products Co, Ltd, China), Minimum Essential cells were analyzed and studied by Flow Medium (MEM) (Invitrogen Corp, Carlsbad, CA, cytometry at wavelength of 488 nm (FACS USA), Fetal Calf Serum (Gibco Corp, Carlsbad, Calibur instrument, BD Biosciences, San Jose, CA, USA), trypsin, penicillin, MTT, streptomycin, CA, USA) equipped with Cell Quest 3.3 software. DMSO and phosphate buffer saline (PBS) (Sigma) were used in this study. MTT kit was Apoptosis detection using Hoechst 33258 obtained from Roche (USA). Annexin V-FITC- staining Propidium Iodide Apoptosis Detection Kit was purchased from Sigma (USA). All other LNCaP cells were fixed with 4 % formaldehyde in chemicals and solvents used were of the highest phosphate buffered saline (PBS) for 30 min before staining with 10 μg/mL of Hoechst 33258 purity grade. Cell culture plastic ware was o purchased from BD Falcon (USA). at 37 C for 20 min. The cells were treated with different concentrations (0, 40, 80, 100 μM) of Cell line scopoletin for 48 h. They were then washed once with PBS, and observed under a fluorescence Human prostate carcinoma (LNCaP) cells were microscope (Nikon). A minimum of 600 cells was procured from the Shanghai Institute of Cell counted, and each experiment was performed in Resource Center of Life Science (Shanghai, triplicate. China). The cells were grown in a humidified 5 % o Detection of early and late apoptosis, and CO2 atmosphere at 37 C in an incubator, and cultured in RPMI-1640 medium supplemented necrosis by annexin V binding assay with 10 % heat-inactivated newborn calf serum, 100 IU/mL penicillin and 100 μg/mL Apoptosis was demonstrated by annexin V streptomycin. binding assay using flow cytometry. Briefly, Trop J Pharm Res, April 2015; 14(4): 612 Li et al LNCaP cells were treated with different rabbit IgG antibodies at a 1:200 dilution (Santa concentrations (0, 40, 80 and 100 μM) of Cruz Biotechnology, Santa Cruz, CA). scopoletin for 48 h. Subsequently, treated and untreated cells were harvested by trypsinization. Statistical analysis Harvested cells were then incubated in annexin V-FITC (100ng/mL) and propidium iodide (40 All statistical analyses were carried out with µg/mL), at room temperature for 30 min in dark, SPSS® software (version 19.0) based on one- and analysed using a FACS Calibur flow way analysis of variance (ANOVA) and Tukey cytometer (BD Bioscience). test. Data are expressed as mean ± SEM, and p < 0.05 was considered significant. Western blot analysis RESULTS To assess the dose-dependent effect of scopoletin, LNCaP cells were treated for 48 h Anti-proliferative activity of scopoletin with various concentrations of scopoletin, (20, 40, 60 and 80 µM to determine the expression The effect of scopoletin treatment on the prostate levels of cyclin D1 and cyclin B1). Cellular cancer cell viability is shown in Fig 1. Scopoletin proteins were extracted using a cell lysis buffer exhibited potent and dose-dependent cytotoxic containing 75 mM Tris-HCl (pH 7.6), 125 mM NaCl, 0.1 % sodium dodecyl sulfate (SDS), 1 % activity