13-Methyl-Palmatrubine Induces Apoptosis and Cell Cycle Arrest in A549 Cells in Vitro and in Vivo

2526 ONCOLOGY REPORTS 36: 2526-2534, 2016 13-Methyl-palmatrubine induces apoptosis and cell cycle arrest in A549 cells in vitro and in vivo JINGXIAN CHEN1*, XINGANG LU2*, CHENGHUA LU3, CHUNYING WANG4, HAIZHU XU2, XIAOLI XU2, HAIXIN GOU2, BING ZHU2 and WANGCHUN DU5 1Department of Traditional Chinese Medicine, RuiJin Hospital, JiaoTong University School of Medicine, Shanghai 200025; 2Department of Traditional Chinese Medicine, HuaDong Hospital, FuDan University School of Medicine, Shanghai 200040; Departments of 3Respiration and 4Oncology, LongHua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032; 5Clinical Medicine College, Shanghai University of Medicine and Health Sciences, Shanghai 201318, P.R. China Received April 5, 2016; Accepted June 24, 2016 DOI: 10.3892/or.2016.5093 Abstract. Corydalis yanhusuo, a well-known herbaceous plant, Despite modern advancements in diagnosis, prevention and is commonly used in the treatment of inflammation, injury and therapy, cancer still affects millions of patients worldwide, pain. One natural agent isolated from Corydalis yanhusuo, reduces their quality of life and is one of the leading causes 13-methyl-palmatrubine, was found to have a cytotoxic effect of mortality worldwide (1,2). Natural products including on cancer cells as reported in published studies. In the present plants, microorganisms and marines provide rich resources for study, we synthesized a potential anti-lung tumor agent, anticancer drug discovery (3,4). Based on ancient and modern 13-methyl-palmatrubine and analyzed its activity. 13-Methyl- Chinese herbal medicine books and Pharmacopoeia of China, palmatrubine exhibited a cytotoxic effect on a panel of cancer there are many natural anticancer plants or herbal formula- cell lines in a time- and concentration-dependent manner. tions which provide a guide, along with clinical evidence, for Among all the tested cancer cell lines, lung cancer A549 cells the identification of new anticancer agents or a natural source were most sensitive to 13-methyl-palmatrubine treatment. of alternative cancer therapy, and these have recently received Meanwhile 13-methyl-palmatrubine showed less cytotox- increasing scientific attention (5,6). icity in human normal cells. Our investigation revealed that Corydalis yanhusuo, also named Rhizoma Corydalis, is a 13-methyl-palmatrubine induced apoptosis and cell cycle well-known herbaceous plant commonly used in the treatment arrest in A549 cells in a dose-dependent manner. Furthermore, of pain, injuries and coronary diseases in Traditional Chinese 13-methyl-palmatrubine treatment caused activation of P38 Medicine (7,8). Alkaloids are important biological active constit- and JNK pathways and blocked the EGFR pathway. In conclu- uents of Corydalis yanhusuo (9). 13-Methyl-palmatrubine was sion, our findings demonstrated that 13-methyl-palmatrubine isolated as a natural protoberberine alkaloid from the methanol inhibited the growth of A549 cells mediated by blocking of extract of the tubes of Corydalis yanhusuo (10). The chemical the EGFR signaling pathway and activation of the MAPK structure of 13-methyl-palmatrubine is shown in Fig. 1A. One signaling pathway and provides a better understanding of the study found that 13-methyl-palmatrubine exhibited antitumor molecular mechanisms of 13-methyl-palmatrubine. activity in 3 types of human cancer cell lines (11). In our large scale screening for suitable anticancer agents from herbal Introduction plants, 13-methyl-palmatrubine exhibited an antiproliferative effect on various cell lines. Further experiments indicated that Cancer is still a serious clinical issue with significant social 13-methyl-palmatrubine induced apoptosis and arrested the and economic impact on the human health care system (1). cell cycle in lung cancer A549 cells, suggesting that 13-methyl- palmatrubine may be an antitumor compound for lung cancer treatment. Materials and methods Correspondence to: Dr Wangchun Du, Clinical Medicine College, Shanghai University of Medicine and Health Sciences, 279 ZhouZhu Road, Shanghai 201318, P.R. China Materials. RPMI-1640 medium, fetal bovine serum (FBS), E-mail: [email protected] pancreatin, penicillin and streptomycin were obtained from Gibco (Carlsbad, CA, USA). Terminal deoxynucleo- *Contributed equally tidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) Apo-Green Detection kit was supplied by Roche Key words: A549 cells, apoptosis, cell cycle arrest, 13-methyl- (Roche, Basel, Switzerland). The Annexin V and PI kit was palmatrubine, EGFR-MAPK purchased from Biotool (Selleck Chemicals, Houston, TX, USA). Protein and RNA extraction kits, BCA protein assay chen et al: 13-METHYL-PALMATRUBINE EXHIBITS ANTIPROLIFERATIve EFFECTS ON A549 CELLS 2527 kit, propidium iodide (PI), caspase-3 and -9 activity kit, treated with increasing doses of 13-methyl-palmatrubine. The dimethyl sulfoxide (DMSO), 4',6-diamidino-2-phenylindole cells were fixed with 4% paraformaldehyde and incubated (DAPI) and Hoechst 33243 were purchased from Beyotime with Hoechst 33342 (5 µg/ml) for 10 min. After washing with Institute of Biotechnology (Beyotime, Haimeng, China). cold PBS, the A549 cells after treatment and the control cells 13-Methyl-palmatrubine standard preparation (purity >98%) were observed by inverted fluorescence microscopy (D5100; was purchased from the National Institute for the Control Nikon, Tokyo, Japan). of Pharmaceutical and Biological Products (Beijing, China). The JC-10 Mitochondrion Membrane Potential Assay Kit TUNEL staining. The NCSLC A549 cells were cultured and was obtained from AAT Bioquest (Sunnyvale, CA, USA). treated on a specific glass cover in 6-well plates and treated ECL Advanced Detection kit was provided by Thermo Fisher as mentioned above. TUNEL kit was used to determine DNA (Waltham, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5- fragmentation in apoptotic cells according to the manufac- diphenyl-2H tetrazolium bromide (MTT) and bovine serum turer's instructions. The cells were stained by TUNEL and albumin (BSA) was purchased from Sigma-Aldrich (St. Louis, DAPI for image analysis. The samples after treatment and MO, USA). Primary antibodies and HRP-labeled secondary the control cells were viewed by Apo-Green fluorescence at anti-mouse/anti-rabbit antibodies were provided by Cell 520 nm and blue DAPI at 460 nm using a fluorescence micro- Signaling Technology (CST; Beverly, MA, USA). All other scope (D5100). chemicals needed were of analytic grade. Apoptosis analysis with Annexin V/PI. The A549 cells were Cell lines and cell culture. The human cell lines used in the cultured and treated as mentioned above. A FITC-Annexin V/ present study included A549, a human lung cancer cell line; PI cell apoptosis assay was conducted to evaluate the apop- HCT116, a human colon carcinoma cell line; MCF-7, a human tosis ratio in the cells according to the kit manufacturer's breast cancer cell line; MKN-45, a human cancer cell line; instructions. In brief, the untreated and treated cells were HepG2, a human hepatocellular carcinoma cell line, L02, a washed and harvested in 100 µl 1X binding buffer, and 5 µl human normal liver cell line; and HEK293 a human kidney Annexin V-FITC solution and 5 µl PI staining solution were normal cell line obtained from the Cell Bank of the Chinese added to each 100 µl of cell suspension. Then, the cells were Academy of Sciences (Shanghai, China). Cells were grown in incubated for 15 min. Binding buffer (400 µl) was added to the a humidified atmosphere of 5% CO2 at 37˚C with RPMI-1640 cell suspensions before determination by FACSCalibur flow medium. Cells were supplemented with 10% fetal calf serum cytometry (BD Biosciences). containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin). Mitochondrial membrane potential (MMP) assay. The AAT JC-10 assay kit was used to evaluate the changes in MMP. Cytotoxicity assay in vitro. Cells were seeded and treated with The kit was used according to the manufacturer's instruc- 13-methyl-palmatrubine at increasing concentrations. The tions. Briefly, A549 cells were seeded in a 96-well plate, and 13-methyl-palmatrubine standard was dissolved in DMSO 50 µl/well JC-10 solution was added into the cell suspensions. at a concentration of 300 µg/ml as the initial dose. Then, the Cell suspensions were incubate in a 37˚C incubator with stock solution was maintained at -80˚C. The solutions were 5% CO2 for 30 min. The cells were washed twice with pre- diluted to the desired concentrations when used. 13-Methyl- cold PBS, re-suspended in assay buffer B and immediately palmatrubine was dissolved in DMSO with a final DMSO examined on a microplate reader under excitation filter of concentration <0.1%. Cytotoxicity of the control and treated 490 nm while emission filter of 525 and 590 nm, separately cells was measured using the MTT assay. Cells (5x103) were (PerkinElmer, Inc.). cultured in 96-well plates treated with the indicated concentra- tins for the indicated times. The absorbance of the treated and Animals. The present study was strictly conducted according control cells at 570 nm was assessed using a microplate reader to the Declaration of Helsinki and the Guide for the Care and (PerkinElmer, Inc., Waltham, MA, USA). the Use of Laboratory Animals as adopted and promulgated by the United States National Institutes of Health. All animal Cell cycle distribution assay by flow cytometry. Cell cycle experiments were approved by the Institutional Animal analysis was determined by PI staining. Briefly, A549

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