Zheng Li-Duan, Tong Qiang-Song, Tao Kai-Xiong, Wang Liang, Zhang Bo
Department of Pathology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, PR China.
Ai Zheng. 2004 Apr;23(4):361-6.
BACKGROUND & OBJECTIVE: Abnormal apoptosis is one of the key factors for drug resistance of neoplasms. Smac, a novel gene involved in regulation of apoptosis,plays an important role in tumor cell apoptosis induced by chemotherapeutic drugs. This study was designed to explore the effects of overexpressed Smac gene on chemotherapeutic sensitivity of gastric cancer cell line.
By liposome GeneSHUTTLE-40, Smac gene was transfected into gastric cancer cell line MKN-45. Cellular Smac gene expression were determined by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot analysis. Cisplatin (1, 5, 10 microg/ml), mitomycin C (0.1,1,10 microg/ml), and curcumin (10, 20, 40 micromol/L) were selected to treat untransfected and transfected MKN-45 cells. Cellular proliferation activities were assayed by tetrazolium bromide (MTT) colorimetry. The morphological changes of cancer cells were observed under inverted microscope. Cellular apoptosis was determined by Annexin V-FITC and propidium iodide staining flow cytometry.
Compared with untransfected control group,Smac mRNA and protein levels in transfected MKN-45 cells were significantly increased (P< 0.01). The growth inhibition rates of MKN-45 cells treated with various concentration of cisplatin, mitomycin C, and curcumin on MKN-45 cells were increased by 10.10%-23.80% (P< 0.01), 10.01%-15.86% (P< 0.01), 11.28%-22.12% (P< 0.01), respectively, with the cells becoming round, obviously refracted, and fragmented. The cellular apoptosis rates were increased by 6.7%-20.2% (P< 0.01), 5.4%-13.2% (P< 0.01), and 10.6%-20.1% (P< 0.01), respectively.
Transfection of extrinsic Smac gene results in its over-expression in MKN-45 cells,which could increase the chemotherapeutic sensitivity of MKN-45 cells.
异常凋亡是肿瘤耐药的关键因素之一。Smac是一种参与凋亡调控的新基因，在化疗药物诱导的肿瘤细胞凋亡中起重要作用。本研究旨在探讨Smac基因过表达对胃癌细胞系化疗敏感性的影响。
采用脂质体GeneSHUTTLE-40将Smac基因转染至胃癌细胞系MKN-45。通过逆转录聚合酶链反应（RT-PCR）和蛋白质印迹分析检测细胞Smac基因表达。选用顺铂（1、5、10μg/ml）、丝裂霉素C（0.1、1、10μg/ml）和姜黄素（10、20、40μmol/L）处理未转染和转染的MKN-45细胞。采用四氮唑蓝（MTT）比色法检测细胞增殖活性。在倒置显微镜下观察癌细胞的形态变化。采用膜联蛋白V-FITC和碘化丙啶染色流式细胞术检测细胞凋亡。
与未转染对照组相比，转染的MKN-45细胞中Smac mRNA和蛋白水平显著升高（P<0.01）。不同浓度顺铂、丝裂霉素C和姜黄素处理MKN-45细胞的生长抑制率分别提高了10.10%-23.80%（P<0.01）、10.01%-15.86%（P<0.01）、11.28%-22.12%（P<0.01），细胞变为圆形，折光明显，出现破碎。细胞凋亡率分别提高了6.7%-20.2%（P<0.01）、5.4%-13.2%（P<0.01）、10.6%-20.1%（P<0.01）。
外源性Smac基因转染导致其在MKN-45细胞中过表达，可提高MKN-45细胞的化疗敏感性。
