双硫仑通过上调GADD45A抑制胰腺癌细胞增殖的机制研究

Disulfiram inhibits the growth of pancreatic cancer cell via upregulation of GADD45A expression

目的:通过体外实验评估双硫仑(disulfiram,DSF)对人胰腺癌细胞增殖、细胞周期的影响并初步探讨可能作用机制。方法:不同浓度梯度的DSF联合Cu(DSF/Cu)处理人胰腺癌PANC-1和PATU8988T细胞,CCK-8法检测细胞增殖活性及药物对细胞的增殖抑制率;选取IC50浓度的DSF/Cu处理细胞,流式细胞术检测细胞周期分布情况,并采用荧光定量PCR和Western blot法检测各组细胞中生长阻滞和DNA损伤诱生基因45A(growth arrest and DNA damage inducible 45A,GADD45A),G2/M周期特异性蛋白CCNB1、CDC25C、CDK1的转录和翻译水平,同时检测MAPK通路相关蛋白P38和JNK蛋白磷酸化水平;siRNA干扰试验检测抑制GADD45A基因后细胞相对活力、细胞周期及MAPK通路相关蛋白磷酸化水平的变化。结果:DSF联合Cu对人胰腺癌细胞增殖具有明显抑制作用,且抑制率随DSF浓度增加而增加。DSF/Cu可明显上调GADD45A基因的表达,诱导细胞周期G2/M期阻滞,抑制G2/M周期特异性基因及蛋白的表达;Real-time PCR结果表明DSF/Cu处理PANC-1细胞和PATU8988T细胞24 h后GADD45A表达量升高,分别为对照组的11.4和7.99倍(P均<0.001);相应地,PANC-1细胞和PATU8988T细胞CCNB1、CDC25C、CDK1表达量下降,分别为对照组的31%、35%和37%(P均<0.05)及48%、24%和29%(P均<0.05),差异有统计学意义。Western blot结果与RNA表达结果相对应,且呈浓度和时间依赖趋势。同时,MAPK信号通路相关蛋白磷酸化水平增高。siRNA-GADD45A干扰后细胞活力增加,G2/M细胞比例降低,MAPK信号通路磷酸化水平降低。结论:DSF/Cu可通过上调GADD45A并激活JNK/P38-MAPK信号通路诱导细胞周期G2/M阻滞,进而引起细胞增殖抑制,发挥抗肿瘤作用。

Objective:This study aims to observe the effects of disulfiram(DSF)on the proliferation and cell cycle arrest in human pancreatic cancer cells,and to explore its mechanism.Methods:CCK-8 was used to detect the effects of DSF on the proliferation activity and the cycle distribution was detected by flow cytometry.Real-time PCR and Western blot were used to detect mRNA and protein expression of GADD45 A,G2/M related CCNB1,CDC25 C,and CDK1 genes and proteins.The phosphorylation of P38 and JNK in MAPK pathway were also detected.SiRNA assay was used to detect the changes of cell cycle and phosphorylation of proteins in MAPK pathway after growth arrest and DNA damage inducible 45 A(GADD45 A)genes were decreased.Results:DSF combined with Cu(DSF/Cu)inhibited the proliferation of pancreatic cancer cells in concentration-dependent trend.GADD45 A was significantly increased in DSF treated groups(11.4 times of control group in PANC-1 cells and 7.99 times of control group in PATU8988 T cells,P<0.001,respectively).The cells were mainly blocked in G2/M phase and CCNB1,CDC25 C and CDK1 genes were significantly decreased(31%,35%,37%of control in PANC-1 cells and 48%,24%,29%of control in PATU8988 T cells,P<0.05,respectively)after treated 24 hours.The proteins showed the same results with the mRNA expression while the p-P38 and p-JNK were increased in dosage and time-dependent trend.Compared with the DSF treated groups,the groups of cells pretreated with siRNA-GADD45 A showed an increasing relative viability and the percentage of G2/M phase cells was decreased.Meanwhile,the p-P38 and p-JNK were also decreased.Conclusion:DSF/Cu can significantly inhibit the proliferation and induce G2/M arrest in pancreatic cancer cells.Its antitumor effect may attribute to upregulating GADD45 A and activating JNK/P38 MAPK signaling pathway.