顺铂诱导胃腺癌细胞凋亡及非凋亡性死亡

Cisplatin induces apoptotic and non apoptotic cell death of gastric adenocarcinoma cells

目的:阐明顺铂诱导胃腺癌细胞死亡的方式并进一步探讨其分子机制。方法:MTT法检测细胞生存率;AnnexinⅤ-FITC流式细胞术检测细胞凋亡;PI流式细胞术检测细胞膜的完整性;吖啶橙染色后荧光显微镜下观察细胞酸性自噬泡(AVO)的形成;半定量RT-PCR法检测p53、Noxa、Beclin1的mRNA水平。结果:顺铂诱导胃腺癌细胞死亡具有剂量和时间依赖性,48 h时IC50为3μg/mL;顺铂诱导后SGC-7901细胞主要以凋亡方式死亡,而BGC-823细胞主要以非凋亡方式死亡;顺铂作用早期BGC-823细胞膜的完整性就被破坏;顺铂诱导前后两种细胞AVO没有明显变化,并且两种细胞中自噬基因Beclin1的表达水平都无显著增高;在对顺铂诱导的凋亡相对敏感的SGC-7901细胞系中,顺铂诱导后p53、Noxa的表达水平显著上调,而在不敏感的BGC-823细胞系中p53、Noxa的表达水平变化不显著。结论:顺铂不但诱导胃腺癌细胞凋亡,而且诱导凋亡耐受的胃腺癌细胞非凋亡性死亡,后者可能是坏死;p53及其靶基因Noxa的活化可能是顺铂诱导SGC-7901细胞凋亡的机制之一。

Objective： To clarify the modes of cisplatin（CDDP）-induced cell death of gastric adenocarcinoma and to study the underlying mechanisms. Methods：Cell viability was measured with MTT assay. Apoptosis was assessed by flow cytometry using FITC-conjugated Annexin V. Uptake of PI measured by flow cytometry was used to monitor plasma membrane perturbations. Formation of acidic vesicle organelles （AVO） was observed by staining with acridine orange under fluorescence microscope. The expression levels of mRNA for p53, Noxa and Beclin 1 were measured by semi quantitative reverse transcription polymerase chain reaction （RT PCR）. Results：CDDP induced cell death of gastric adenocarcinoma in a dose and time-dependent manner. The IC50 was 3 mg/ml, after cells were treated with CDDP for 48 h. After exposure to CDDP, SGC 7901 cells died primarily by apop tosis, while BGC 823 cells died mainly by non apoptosis. The integrity of plasma membrane of BGC 823 cells was destroyed at the early stage of CDDP treatment. CDDP did not induce any increase in formation of AVO and did not cause any change in the expression levels of the autophagy gene Beclin 1 in both BGC 823 and SGC 7901 cell lines. The expression levels of p53 and Noxa were markedly increased in the apoptosis sensitive cell line SGC 7901 but remained unchanged in BGC 823 cells after treatment with CDDP. Conclusion：CDDP induces both apoptotic and non apoptotic, presumably necrotic, cell death in gastric adenocarcinoma cells. Activation of p53 and its target gene Noxa may play a role in CDDP induced apoptosis of SGC-7901 cells.