GSK-3β抑制剂增强前列腺癌22RV1细胞化疗敏感性的体外研究
发布时间：2019-01-07 20:15
【摘要】：[目的]在体外对GSK-3β抑制剂调控DTX诱导的前列腺癌22RV1细胞化疗敏感性的作用机制进行初步研究。 [方法] 1.DTX联合GSK-3β抑制剂对前列腺癌细胞的毒性作用 22RV1细胞经DTX单独处理或联合GSK-3β抑制剂LiCl处理后,经结晶紫染色分析细胞毒性作用。 2. GSK-3β抑制剂对DTX诱导的细胞自噬活性的调控 22RV1细胞经DTX单独处理或联合GSK-3β抑制剂处理后,细胞经NP40液裂解,Western Blot检测自噬相关蛋白表达(LC-3、Beclin-1, ATG14L, ATG5及Bif-1蛋白等)及GSK-3β活性(总GSK-3β蛋白、pGSK-3β-S9磷酸化蛋白)。 3.GSK-3p抑制剂对自噬复合体与下游分子(ATG14L或Rubicon)结合的调控 22RV1细胞经DTX单独或联合GSK-3β抑制剂处理,收集细胞经NP40液裂解后,细胞裂解液分别通过Beclin-1抗体免疫沉淀,蛋白A/G琼脂糖孵育、洗脱后,4×SDS上样缓冲液重悬,通过ATG14L及Rubicon抗体Western Blot检测,明确各处理组VPS34/Beclin-1自噬复合体与上述蛋白分子的结合情况。 [结果]在DTX基础上联合应用LiCl,可以下调GSK-3β活性；上调自噬促进蛋白ATG14L, Beclin-1水平,下调自噬抑制蛋白Rubicon水平；上调自噬促进复合物beclin-1/ATG14的形成,下调自噬抑制复合物beclin-1/Rubicon的形成。 [结论]GSK-3β抑制剂Lic1可以通过上调DTX诱导的前列腺癌细胞自噬水平,促进自噬性死亡,提高化疗敏感性。
[Abstract]:[objective] to study the mechanism of GSK-3 尾 inhibitor regulating chemosensitivity of prostate cancer 22RV1 cells induced by DTX in vitro. [methods] the toxic effects of 1.DTX combined with GSK-3 尾 inhibitor on prostate cancer cells were analyzed by crystal violet staining after 22RV1 cells were treated with DTX alone or GSK-3 尾 inhibitor LiCl. 2. Regulation of GSK-3 尾 inhibitor on DTX induced autophagy activity of 22RV1 cells treated with DTX alone or in combination with GSK-3 尾 inhibitor, the expression of autophagy associated protein (LC-3,Beclin-1, ATG14L,) was detected by NP40 cleavage, Western Blot. ATG5 and Bif-1 protein) and GSK-3 尾 activity (total GSK-3 尾 protein, pGSK-3 尾 -S9 phosphorylated protein). Regulation of autophagy complex combined with downstream molecules (ATG14L or Rubicon) by 3.GSK-3p inhibitor, 22RV1 cells were treated with DTX alone or in combination with GSK-3 尾 inhibitor, and the collected cells were lysed in NP40 solution. The cell lysate was immunoprecipitated by Beclin-1 antibody and incubated with protein A / G agarose. After elution, the buffer on 4 脳 SDS was resuspended, and detected by ATG14L and Rubicon antibody Western Blot. To determine the binding of VPS34/Beclin-1 autophagy to the above protein molecules in each treatment group. [results] combined application of LiCl, on the basis of DTX could down-regulate the activity of GSK-3 尾, up-regulate the ATG14L, Beclin-1 level of autophagy promoter protein and down-regulate the Rubicon level of autophagy inhibitor protein. Upregulated autophagy promoted the formation of beclin-1/ATG14 and down-regulated the formation of autophagy inhibitory complex beclin-1/Rubicon. [conclusion] GSK-3 尾 inhibitor Lic1 can promote autophagic death and chemosensitivity by up-regulating DTX induced autophagy of prostate cancer cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
本文编号：2404107
[Abstract]:[objective] to study the mechanism of GSK-3 尾 inhibitor regulating chemosensitivity of prostate cancer 22RV1 cells induced by DTX in vitro. [methods] the toxic effects of 1.DTX combined with GSK-3 尾 inhibitor on prostate cancer cells were analyzed by crystal violet staining after 22RV1 cells were treated with DTX alone or GSK-3 尾 inhibitor LiCl. 2. Regulation of GSK-3 尾 inhibitor on DTX induced autophagy activity of 22RV1 cells treated with DTX alone or in combination with GSK-3 尾 inhibitor, the expression of autophagy associated protein (LC-3,Beclin-1, ATG14L,) was detected by NP40 cleavage, Western Blot. ATG5 and Bif-1 protein) and GSK-3 尾 activity (total GSK-3 尾 protein, pGSK-3 尾 -S9 phosphorylated protein). Regulation of autophagy complex combined with downstream molecules (ATG14L or Rubicon) by 3.GSK-3p inhibitor, 22RV1 cells were treated with DTX alone or in combination with GSK-3 尾 inhibitor, and the collected cells were lysed in NP40 solution. The cell lysate was immunoprecipitated by Beclin-1 antibody and incubated with protein A / G agarose. After elution, the buffer on 4 脳 SDS was resuspended, and detected by ATG14L and Rubicon antibody Western Blot. To determine the binding of VPS34/Beclin-1 autophagy to the above protein molecules in each treatment group. [results] combined application of LiCl, on the basis of DTX could down-regulate the activity of GSK-3 尾, up-regulate the ATG14L, Beclin-1 level of autophagy promoter protein and down-regulate the Rubicon level of autophagy inhibitor protein. Upregulated autophagy promoted the formation of beclin-1/ATG14 and down-regulated the formation of autophagy inhibitory complex beclin-1/Rubicon. [conclusion] GSK-3 尾 inhibitor Lic1 can promote autophagic death and chemosensitivity by up-regulating DTX induced autophagy of prostate cancer cells.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25
【参考文献】
相关期刊论文 前1条
1 潘半舟;封冰;宋海珠;;自噬在调控抗肿瘤药物耐药中的研究进展[J];医学研究生学报;2012年12期
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