Overexpression of miR-126 sensitizes osteosarcoma cells to apoptosis induced by epigallocatechin-3-gallate

Jiang et al. World Journal of Surgical Oncology 2014, 12:383 http://www.wjso.com/content/12/1/383 WORLD JOURNAL OF SURGICAL ONCOLOGY RESEARCH Open Access Overexpression of miR-126 sensitizes osteosarcoma cells to apoptosis induced by epigallocatechin-3-gallate Liangdong Jiang1, Cheng Tao1, Aiyong He1 and Xiaojie He2* Abstract Background: miR-126 plays an important role in the proliferation, invasion, migration, and chemotherapeutics resistance in cancer. Epigallocatechin-3-gallate (EGCG), as the major polyphenolic constituent present in green tea, is a promising anticancer agent. However, the role of miR-126 in EGCG anticancer remains unclear. Here, we investigated the effects of miR-126 and EGCG on cell viability, apoptosis, cell cycle distribution of osteosarcoma cells and the sensitization of miR-126 on osteosarcoma cells to EGCG. Methods: The cell viability, apoptosis and cycle distribution were analyzed using MTT assay and flow cytometry. Results: Our results showed that EGCG (0.025, 0.05, 0.1, 0.2 g/L) suppresses proliferation of osteosarcoma MG63 and U2OS cells in a concentration-dependent and time-dependent manner and the inhibitory effects of 0.05 g/L EGCG on U2OS cells were roughly equivalent to 20 μM cisplatin (DDP); miR-126 could promote apoptosis and inhibit proliferation in U2OS cells but without significant effects on cell cycle G1 phase arrest; EGCG suppressed proliferation of U2OS cells through induction of cell cycle G1 arrest and apoptotic death; overexpression of miR-126 enhanced the inhibitory effects of EGCG on proliferation in U2OS cells via promotion of apoptosis. Conclusions: Our results demonstrate that enhanced expression of miR-126 increased the sensitivity of osteosarcoma cells to EGCG through induction of apoptosis. Keywords: Osteosarcoma, miR-126, Epigallocatechin-3-gallate, Sensitization, Apoptosis Background Osteosarcoma is a primary malignant bone tumor with high morbidity that occurs mainly in children and adolescents. Multiple options for the treatment of osteosarcoma have been described, including chemotherapy, radiation, and so on, however, therapeutic efficacy is typically transient and mostly absent with advanced disease [1]. Therefore, the need for more rational approaches to osteosarcoma therapy is essential. Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, showing anti-inflammatory, antioxidant, anticancer and immunomodulatory activities [2-5]. It has been shown that EGCG has extensive anticancer activities, including in breast cancer [6], cervical cancer [7], * Correspondence: 2 Children’s Medical Center, The Second Xiangya Hospital, Central South University, No. 139 Middle Renmin Road, Changsha, Hunan 410011, P.R. China Full list of author information is available at the end of the article lung cancer [8], prostate cancer [9], colon cancer [10], head and neck cancer [11], gastric cancer [12], ovarian cancer [13], even cancer stem cells [14], and so on. It was reported that combined administration of EGCG and interleukin 1 (IL-1) receptor antagonist could efficiently decrease IL-1-induced tumorigenic factors, leading to reduction in angiogenesis and invasiveness in human osteosarcoma cells [15]. It suggests that a combination of EGCG and interfering approaches against factors that play a crucial role in tumor progression is a promising strategy for improving therapeutic efficacy, including osteosarcoma. MicroRNAs (miRs), a class of 22-nucleotide noncoding RNAs, have emerged as critical components of generegulatory networks controlling numerous important pathophysiological processes, including the initiation and progression of tumors. Studies on miRs have opened new avenues for both the diagnosis and treatment of © 2014 Jiang et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jiang et al. World Journal of Surgical Oncology 2014, 12:383 http://www.wjso.com/content/12/1/383 cancer. Many miRs, [16-18] including miR-126 [19], are found to be involved in the proliferation, invasion, migration, and drug resistance [20] in osteosarcoma cells. It was found that miR-126 was consistently underexpressed in osteosarcoma tissues and cell lines and functioned as a tumor suppressor in osteosarcoma [19]. Studies showed that miR-126 could enhance the sensitivity of lung cancer [21] and cervical cancer [22] cells to anticancer agents. However, whether miR-126 can sensitize osteosarcoma cells to EGCG remains to be elucidated. In this study, the effects of miR-126 and EGCG on the proliferation, apoptosis and cell cycle in osteosarcoma cells were investigated. Our results showed that miR-126 could enhance the sensitivity of osteosarcoma U2OS cells to EGCG, providing novel approaches or targets for reducing drug resistance in cancer. Methods Cell culture and stimulation MG63 and U2OS cell lines (American Type Culture Collection (ATCC), USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) culture medium at 5% CO2. Cells were treated with EGCG (0.025, 0.05, 0.1, 0.2 g/L) for 24, 48 and 72 hours, or EGCG (0.05 g/L), cisplatin (DDP, 20 μM) or rapamycin (RAPA, 100 nm) for 48 hours as indicated. To investigate the roles of miR-126 in U2OS cells, the lentiviral vectors comprising pre-miR-126 or anti-miR-126 were constructed and used to infect U2OS cells, establishing stable U2OS cell lines overexpressing or silencing miR-126. Cell proliferation assay Cells treated with indicated reagents or samples in exponential growth were plated at a final concentration of 2 × 103 cells per well in 96-well plates. The viability of cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay after 24, 48 and 72 hours of seeding. The optical density at 570 nm (OD570) of each well was measured with an enzymelinked immunosorbent assay (ELISA) reader (ELX-800 type, BioTek Winooski, VT, USA). Cell apoptosis assay After being incubated in the presence of EGCG or RAPA for 48 hours, cells were harvested and treated with fluorescein isothiocyanate (FITC, BioVision, Mountain View, CA, USA) according to the manufacturer’s instructions. Cell apoptosis was detected by analyzing Annexin V-FITC binding by flow cytometry (Ex = 488 nm; Em = 530 nm) using a FITC signal detector and a propidium iodide (PI) signal detector. Page 2 of 7 Cell cycle analysis The cells were digested with trypsin (Auragene Bioscience Corporation, Changsha, China) and collected after treatment for 48 hours, and washed with phosphate-buffered saline (PBS) twice. The cells were resuspende (...truncated)