去甲斑蝥素诱导人皮肤鳞状细胞癌A431细胞凋亡作用

Effect of Norcantharidin on Inducing Apoptosis of Human Cutaneous Squamous Cell Carcinoma A431 Cells

目的探讨去甲斑蝥素(Norcantharidin,NCTD)诱导人皮肤鳞状细胞癌A431细胞凋亡作用及其相关分子机制。方法体外培养A431细胞,分为对照组和NCTD低、高剂量组(20,40μmol·L^(-1))。采用MTT法检测对细胞增殖的抑制作用;Annexin V/PI染色法检测细胞凋亡;试剂盒检测细胞活性氧(ROS)水平、丙二醛(MDA)含量、线粒体膜电位(MMP)变化、Caspase-3及Caspase-9酶活性变化;Western Blot法检测对细胞Bcl-2及Bax蛋白表达水平的影响。结果采用NTCD处理A431细胞48 h,与对照组相比,NCTD低、高剂量组均能抑制细胞增殖能力(P<0.05),并明显诱导细胞凋亡(P<0.05),提高细胞内的ROS水平(P<0.05,P<0.01)和MDA含量(P<0.05,P<0.01),降低细胞MMP(P<0.05,P<0.01)。同时,NTCD可以抑制Bcl-2蛋白表达及上调Bax蛋白表达,并能够明显升高细胞的Caspase-3及Caspase-9酶活性。结论 NCTD可能通过提高细胞内ROS水平的途径诱导A431细胞发生凋亡。

Objective To investigate the effects of norcantharidin （NCTD） on the proliferation and apoptosis of human skin squamous cell carcinoma A431 cells and to explore its related molecular mechanisms. Methods A431 cells were cultured in vitro. The cells were divided into control group, low-close NCTD group and high-close NCTD group. MTT assay was used to detect the effect on cell proliferation. Apoptosis was detected after Annexin V/PI staining. The level of reactive oxygen species （ROS）, malondialdehyde （MDA） content, the change of mitochondrial membrane potential （MMP） and the activities of Caspase-3 and Caspase-9 were measured by the kits. Western Blot method was used to detect the protein expression of Bcl-2 and Bax. Results Compared with the control group, NTCD could significantly inhibit the proliferation and induce the apoptosis of A431 ceils （P 〈 0.05 ） after treatment for 48 h, increase ROS level and MDA content, and decrease the cell MMP （P 〈 0.05, P 〈 0.01 ）. At the same time, NTCD could inhibit the protein expression of Bcl-2 and up-regulate the protein expression of Bax, and significantly increase the activities of Caspase-3 and Caspase-9. Conclusion NCTD might induce the apoptosis of A431 cells by increasing the intracellular ROS level.