三氧化二砷诱导人卵巢癌耐药细胞系3AO/cDDP细胞凋亡及机制的研究

目的：探讨三氧化二砷（As2O3）对人卵巢癌耐药细胞系3AO／cDDP细胞生长的影响及其机制。方法：采用四甲基偶氮唑蓝（MTT）法,检测不同浓度As2O3作用后,3AO／cDDP细胞的生长抑制率;采用流式细胞技术检测细胞凋亡率,细胞周期变化,以及Fas、FasL基因表达的变化。所有结果均与人卵巢癌细胞系3AO细胞相比较;采用细胞骨架染色法观察3AO／cDDP细胞凋亡细胞形态变化,通过吖啶橙染色,荧光显微镜观察As2O3作用后3AO细胞的形态变化。结果：As2O3能明显抑制3AO／cDDP细胞的增殖,抑制作用呈时间和剂量依赖性（P＜0．05）;在定一浓度范围内,3AO／cDDP细胞凋亡率与As2O3的浓度和作用时间呈依赖关系,诱导凋亡的最适浓度是3μmol/L;As2O3低浓度时,3AO／cDDP细胞周期S期通过受阻,高浓度时诱导S期细胞凋亡;As2O3作用后,两种细胞系Fas基因的表达均呈升调节,FasL基因的表达无变化;与3AO细胞相比,差异无显著意义（P＞0．05）;As2O3作用后3AO及3AO／cDDP形成典型的凋亡小体。结论：As2O3通过Fas/FasL系统,诱导S期细胞凋亡,有效地抑制人卵巢癌耐药细胞系细胞的生长。

Apoptosis of drug-resistant human ovarian carcinoma cell line 3AO/cDDP induced by arsenic trioxide and its mechanism

OBJECTIVE: To explore the effects of arsenic trioxide (As(2)O(3)) on the growth of drug-resistant human epithelial ovarian carcinoma cell line 3AO/cDDP and its possible mechanism. METHODS: Human epithelial ovarian carcinoma cell line 3AO and drug- resistant human epithelial ovarian carcinoma cell line 3AO/cDDP were cultured As(2)O(3) of different concentrations was added into the media. Cell culture without addition of As(2)O(3) was used as control. The growth inhibiting rates of 3AO/cDDP cells with various concentrations of As(2)O(3) in different time course (24, 48, 72, and 96 hours after) were studied by methyl thiazolyl tetrazolium (MTT) method. The apoptosis percentage, cell cycle phase distribution and expression of Fas/FasL gene were estimated by flow cytometry (FCM). The apoptosis phenotype of 3AO/cDDP cells was observed by cytoskeleton dying, and the apoptosis phenotype of 3AO cells was observed by acridine dying under fluorescent microscopy. RESULTS: 3AO/cDDP cell growing inhibiting rates by As(2)O(3) were different significantly in dose-dependent and time-dependent manners (P < 0.05). Within a certain concentration range, 3AO/cDDP apoptosis inducing rates by As(2)O(3) were dose -and time- dependent, and the most appropriate concentration was 3.0 micromol/L; lower concentrations of As(2)O(3) perturbed the cells to progress through S/G(2) phase, while higher concentrations of As(2)O(3) selectively induced apoptosis of S phase cells. As(2)O(3) up-regulated Fas gene expression, but did not affect FasL gene expression in both cell lines without significant difference (P > 0.05). Morphological observation indicated that As(2)O(3) induced typical apoptotic bodies in 3AO/cDDP and 3AO cells. CONCLUSION: As(2)O(3) effectively inhibits the proliferation of drug-resistant human ovarian carcinoma cell line through up-regulating Fas gene expression and inducing apoptosis of cells in S phase.