文章摘要
李明俊,赵冬.异丙安替比林通过抑制蛋白激酶B通路诱导人肾癌细胞凋亡和自噬[J].安徽医药,2019,23(8):1501-1504.
异丙安替比林通过抑制蛋白激酶B通路诱导人肾癌细胞凋亡和自噬
Propyphenazone induces apoptosis and autophagy through inhibiting AKT signalling pathway in human renal carcinoma cells
投稿时间:2018-02-12  
DOI:
中文关键词: 肾肿瘤  异丙安替比林  Caki-1细胞  细胞凋亡  自噬  蛋白质丝氨酸苏氨酸激酶  流式细胞术
英文关键词: Kidney neoplasms  Propyphenazone  Caki-1 cells  Apoptosis  Flow cytometry  Protein-serine-threonine kinases  Autophagy
基金项目:
作者单位
李明俊 广东省第二中医院外一科,广东 广州 510095 
赵冬 广东省第二中医院外一科,广东 广州 510095 
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中文摘要:
      目的 探究异丙安替比林(Propyphenazone)对人肾癌细胞Caki-1增殖抑制和凋亡的影响及其分子机制。方法 使用MTT(四甲基偶氮唑盐)法和克隆形成抑制实验检测异丙安替比林在不同时间和浓度下对Caki-1细胞增殖能力的影响;Annexin V-FITC/PI双染色检测对Caki-1细胞凋亡的影响;Hoechst 33342染色法检测Caki-1细胞染色质固缩状态;蛋白质印迹法(Western Blot)检测Caki-1细胞的丝氨酸/苏氨酸蛋白激酶AKT(蛋白激酶B)、磷酸化的丝氨酸/苏氨酸蛋白激酶p-AKT以及DNA修复酶PARP蛋白表达变化;激光共聚焦显微镜检测LC3蛋白在细胞中的点状聚集。结果 MTT实验结果表明异丙安替比林可抑制人肾癌细胞Caki-1的增殖(P<0.05),同时,异丙安替比林分别处理24 h、48 h、72 h后,所对应的半抑制浓度(IC50)分别为105 μM、83 μM、56 μM;同时,克隆形成抑制实验表明20 μM和40 μM的异丙安替比林处理Caki-1细胞后,克隆形成率分别降为38%(P<0.05)和20%(P<0.01);流式结果表明,当使用0 μM、40 μM、80 μM和100 μM 的异丙安替林处理Caki-1细胞后,细胞凋亡率分别为7.4%、13.5%、34.5%和50.9%;Western Blot结果表明,随着异丙安替比林浓度的增加,p-AKT蛋白表达降低,并伴随DNA修复酶PARP失活。免疫荧光实验结果表明异丙安替比林可能诱导细胞发生自噬。结论异丙安替比林显著抑制人肾癌Caki-1细胞增殖,并通过抑制AKT通路诱导肾癌细胞发生凋亡和自噬。
英文摘要:
      Objective To investigate the effects of propyphenazone on growth and apoptosis,and the antitumor mechanism in Caki-1 cells (human renal carcinoma cells).Methods The effects of propyphenazone on proliferation of Caki-1 cells were detected by MTT assay and colony formation assay.Cell apoptosis was analyzed using Annexin V-FITC/PI staining by flow cytometry.The cellular morphology changes of Caki-1 cells were observed by fluorescence microscopy and Hoechst 33342 staining was used to observe chromatin state of Caki-1 cells treated with propyphenazone.The expression of AKT,p-AKT and cleaved-PARP in Caki-1 cells was measured using Western Blot.The punctate dots of LC3 were evaluated using confocal-immunofluorescence.Results MTT assay showed that the proliferation of Caki-1 cells was inhibited by propyphenazone in a dose- and time-dependent manner (P<0.05).IC50 in different time (24 h,48 h,72 h) was 105 μM,83 μM,56 μM,respectively.Colony formation assay showed that propyphenazone inhibited colony formation,and colony forming efficiency was decreased to 38% (P<0.05) and 20% (P<0.01) by the treatment of 20μM and 40μM propyphenazone.Flow cytometry analysis indicated when Caki-1 cells were treated with propyphenazone (0 μM,40 μM,80 μM,100 μM),apoptosis ratio was 7.4%,13.5%,34.5%,50.9%,respectively.Propyphenazone downregulated the expression of p-AKT with different concentrations.In addition,cleaved-PARP could be detected by western blot,which demonstrated that propyphenazone induced apoptosis.Propyphenazone induced autophagy by increasing LC3 punctate dots.Conclusion Propyphenazone significantly inhibited growth of Caki-1,and induced cell apoptosis and autophagy through inhibition of AKT pathway.
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