芹菜素诱导乳腺癌MDA-MB-231细胞系非P53依赖性凋亡的研究

  目的  探讨芹菜素(Apigenin)对乳腺癌MDA-MB-231细胞系凋亡的影响。  方法  常规培养MDA-MB-231细胞，应用四甲基偶氮唑盐(MTT)法评价芹菜素对乳腺癌MDA-MB-231细胞增殖的影响。荧光染色法观察细胞形态学变化。流式细胞术(FCM)Annexin V/PI双染法检测细胞凋亡率。Western blot检测不同浓度芹菜素对Caspase-3，PARP，突变型P53，P73，PIG3，Bax，Bcl-2等凋亡相关蛋白表达的影响。RNA干扰技术检测P73对凋亡及下游靶基因PIG3表达的影响。  结果  MTT结果显示，芹菜素10、20及40 μM分别处理细胞24 h，发现芹菜素可有效抑制MDA-MB-231细胞增殖，且具有浓度依赖性和时间依赖性，实验组与对照组比较差异具有统计学意义(P < 0.05)，两组组间比较差异具有统计学意义(P < 0.05)；荧光染色结果显示，对照组未观察到凋亡细胞，芹菜素10 μM、20 μM及40 μM组细胞中均可见凋亡小体，其数目随芹菜素浓度的增加而逐渐增加，具有浓度依赖性；流式细胞术结果显示，与对照组比较，芹菜素10 μM，20 μM及40 μM组早期凋亡率逐渐增加，呈明显的量效关系，实验组与对照组比较差异具有统计学意义(P < 0.05)；Western blot结果显示，与对照组相比，芹菜素10μM、20μM及40μM组Caspase-3、PARP剪切带含量明显增加，P73、PIG3及Bax表达增加，而突变型P53与Bcl-2表达减少，且具有浓度依赖性。RNA干扰结果显示，与阴性对照组相比，p73-siRNA可有效抑制芹菜素诱导的PIG3表达，Caspase-3和PARP剪切含量也相应降低，实验组与对照组比较差异具有统计学意义(P < 0.05)。  结论  芹菜素可通过非P53依赖性凋亡有效抑制乳腺癌MDA-MB-231细胞增殖，其机制可能与下调突变型p53表达，上调P73介导的PIG3表达，以及Bax/Bcl-2比值增加有关。 

Apigenin induction of p53-independent apoptosis in MDA-MB-231 breast cancers

  Objective  This work aims to investigate the anticancer effect of apigenin in the MDA-MB-231 breast cancer cell line.  Methods  The MDA-MB-231 cells were cultured in vitro. Cell proliferation was measured using the methyl thiazolyl tetrazolium (MTT) assay. Morphological changes in the apoptotic cells were observed by fluorescent microscopy. Flow cytometry was used to detect the rate of apoptotic cells with longer treatments of apigenin. Apoptosis-related proteins were detected by Western blot. The ribonucleic acid interference (RNAi) experiment was applied using chemically synthesized siRNA.  Results  The MTT assay revealed that cell proliferation significantly decreased in a dose- and time-dependent manner. Fluorescent staining and flow cytometry showed that the number of apoptotic cells increased with a longer treatment of apigenin. Caspase 3 activation and the poly ADP ribose polymerase (PARP) cleavage were observed after treatment with apigenin, indicating the death of apoptotic cells. The upregulation of p73, PIG3, and Bax expressions, as well as the downregulation of mutant p53 and Bcl-2 expressions, was also observed in the apigenin-treated cells. The RNAi assay showed that the silencing of p73 significantly inhibited PIG3 expression, as well as the cleavage of Caspase 3 and PARP induced by the apigenin treatment.  Conclusion  Apigenin exhibits an inhibitory effect against cell proliferation by inducing the p53-independent apoptosis of the MDA-MB-231 cells. The underlying mechanism may be associated with the downregulation of mutant p53, the upregulation of p73-mediated PIG3, and the increase of the Bax/Bcl-2 ratio.