去甲斑蝥素诱导人皮肤鳞状细胞癌A431细胞凋亡作用
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摘要
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-dose NCTD group and high-dose 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 cells(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.
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-dose NCTD group and high-dose 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 cells(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.
引文
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[2]周钧,王琴,王群伟,等.去甲斑蝥素对LPS诱导的体外培养的肝细胞损伤的保护作用[J].中南大学学报(医学版),2012,37(3):285-289.
[3]REN J,LI G,ZHAO W,et al.Norcantharidin combined with ABT-737 for hepatocellular carcinoma:Therapeutic effects and molecular mechanisms[J].World Journal of Gastroenterology,2016,22(15):3962-3968.
[4]张双伟,李润美,徐进文,等.毛冬青皂苷E对H9C2心肌细胞缺氧/复氧损伤的影响[J].中药新药与临床药理,2015,26(5):591-595.
[5]张军,谷翔,黄问银,等.GLP-1对AGEs诱导H9C2心肌细胞凋亡的保护作用研究[J].中国药理学通报,2017,33(1):120-126.
[6]邹翔,曲中原,季宇彬.胡桃醌经由活性氧介导JNK,p38通路诱导SGC-7901细胞凋亡[J].中国药学杂志,2016,51(7):544-549.
[7]张志强,高体云,李暐,等.雷公藤红素通过活性氧诱导结肠癌SW620细胞凋亡[J].中国医院药学杂志,2015,35(17):1558-1563.
[8]ZAMAN S,WANG R,GANDHI V.Targeting the apoptosis pathway in hematologic malignancies[J].Leukemia&Lymphoma,2014,55(9):1980-1992.
[9]FICKOVA M,MACHO L,BRTKO J.A comparison of the effects of tributyltin chloride and triphenyltin chloride on cell proliferation,proapoptotic p53,Bax,and antiapoptotic Bcl-2 protein levels in human breast cancer MCF-7 cell line[J].Toxicology in Vitro,2015,29(4):727-731.
[10]宋金莲,马中良,迟晓伟,等.天然活性分子isatin经p53介导的线粒体途径诱导乳腺癌细胞MCF-7凋亡[J].中国药理学通报,2016,32(6):773-778.
[11]DEL PRINCIPE M I,DAL BO M,BITTOLO T,et al.Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia[J].Haematologica,2016,101(1):77-85.