高氧刺激下ＰＡＣ１诱导乳腺癌细胞凋亡的机制探讨

目的　初步探讨高氧刺激下ＰＡＣ１对人乳腺癌细胞株ＭＤＡ-ＭＢ４３５的凋亡诱导作用及其机制。方法　通过脂质体法转染目的质粒ｐｃＤＮＡ３.１（＋）／ＰＡＣ１进入人乳腺癌细胞株ＭＤＡ ＭＢ４３５，筛选稳定表达ＰＡＣ１的细胞克隆，并使用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ法鉴定高表达ＰＡＣ１的ＭＢ４３５细胞克隆。应用台盼蓝染色法检测不同细胞在Ｈ２Ｏ２处理后的存活率变化，同时进一步使用Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ法检测ＭＢ４３５／ＰＡＣ１细胞克隆经Ｈ２Ｏ２处理后其ＥＲＫ１／２磷酸化的水平。结果　在细胞克隆ＭＢ４３５／ＰＡＣ１-Ｃ２和ＭＢ４３５／ＰＡＣ１-Ｃ６中均有高水平的ＰＡＣ１表达，这些高表达ＰＡＣ１的肿瘤细胞经高氧化合物Ｈ２Ｏ２刺激后细胞存活率相对于对照组均明显降低（Ｐ＜０.０１），而且ＰＡＣ１的高表达可以引起细胞内ＭＡＰＫ激酶ＥＲＫ１／２的活性受到抑制，使磷酸化水平降低。结论　ＰＡＣ１可以通过抑制ＥＲＫ１／２的磷酸化水平而介导细胞对高氧刺激的反应，从而诱导细胞发生凋亡。

Mechanism of PAC1 in signaling oxidative stress-induced apoptosis of MDA-MB435

Objective To study the mechanism of PAC1 in signaling apoptosis of MDA-MB435 cell line under oxidative stress.Methods Gene transfection of pcDNA3.1（＋）/PAC1 on MDA-MB435 cells was conducted by lipofectamine and stable cell clones expressing high level PAC1 were selected,and the expression level of PAC1 were tested by Western blotting.PAC1-mediated induction of apoptotic cell death and dephosphorylation of ERK in response to H2O2 were examined by trypan blue exclusion and Western blotting.Results There were high levels of PAC1 protein in the two stable clones expressing ectopic PAC1,and PAC1 could induce cell apoptosis and suppress the activity of ERK1/2 in response to H2O2 treatment（P0.01）.Conclusion PAC1 acts in the apoptotic pathway to elicit cancer cell killing under oxidative stress through suppressing the MAP kinase survival pathway.