Chk1与Chk2高表达对DADS阻滞人胃癌BGC823细胞G2/M期的影响
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摘要
目的:在建立Chk1/2基因高表达人胃癌BGC823细胞的基础上,探讨Chk1/2基因在二烯丙基二硫(DADS)诱导人胃癌BGC823细胞G2/M期阻滞中的作用。方法:采用流式细胞术检测DADS作用于Chk1/2高表达BGC823细胞周期分布情况。Western blot检测Chk1、p-Chk1、Chk2、p-Chk2、Cdc25C与Cyclin B1表达变化。结果:流式细胞术显示,Chk1高表达组G2/M期细胞较对照组与空载体组增加(P<0.05)。而15 mg·L~(-1) DADS处理后,各组G2/M期细胞较处理前增加,Chk1高表达组较空载体组差异有统计学意义(P<0.05)。Chk2高表达组G2/M期细胞较对照组与空载体组差异无统计学意义(P>0.05)。而DADS处理Chk2高表达组后,G2/M期细胞较对照组与空载体组差异有统计学意义(P<0.05)。Western blot显示,Chk1/2蛋白及p-Chk2表达水平不受DADS的影响,但p-Chk1呈时间依赖性上调(P<0.05)。DADS处理Chk1高表达BGC823细胞12、24、36、48 h后,Cdc25C磷酸酶与Cyclin B1表达较对照组呈时间依赖性下降(P<0.05)。结论:DADS阻滞人胃癌BGC823细胞G2/M期与激活Chk1/Cdc25C/Cyclin B1通路有关。Chk1高表达可增强DADS阻滞G2/M期细胞的作用,而Chk2高表达对DADS无影响。
Objective: To investigate the role of Chk1/2 on the G2/M phase arrest in gastric cancer BGC823 cells induced by diallyl disulfide(DADS) on the basis of the establishment of Chk1/2 gene overexpression BGC823 cells. Methods: Flow cytometry was used to detect the cell cycle distribution in Chk1/2 overexpression BGC823 cells treated by DADS.The expressions of Chk1, p-Chk1,Chk2,p-Chk2, Cdc25 C and Cyclin B1 were detected by Western blot. Results:Flow cytometry showed that the G2/M phase cells in Chk1 overexpression group were increased compared with the control group and the vector group(P<0.05).After 15 mg·L~(-1) DADS treatment,G2/M phase cells in each group were increased compared with those before treatment,and there was statistically significant of the difference between the Chk1 overexpression group and the vector group(P<0.05). The G2/M phase cells in the Chk2 overexpression group exhibited no statistically significant of the difference with the control group and the vector group(P>0.05). The G2/M phase cells were different from those in the control group and the vector group after DADS treated Chk2 overexpression group(P<0.05). Western blot showed that the expression level of Chk1/2 protein and p-Chk2 was not affected by DADS, but the p-Chk1 was time-dependent increased(P<0.05). The expression of Cdc25 C and Cyclin B1 showed time-dependent decrease after DADS treated Chk1 overexpression BGC823 cells for 12,24,36 and 48 h(P<0.05). Conclusion:DADS arrest the G2/M phase in BGC823 cells is related to the activation of Chk1/Cdc25 C/Cyclin B1 pathway.Chk1 overexpression can enhance the role of DADS arrest G2/M phase, while Chk2 overexpression has no effect on DADS.
Objective: To investigate the role of Chk1/2 on the G2/M phase arrest in gastric cancer BGC823 cells induced by diallyl disulfide(DADS) on the basis of the establishment of Chk1/2 gene overexpression BGC823 cells. Methods: Flow cytometry was used to detect the cell cycle distribution in Chk1/2 overexpression BGC823 cells treated by DADS.The expressions of Chk1, p-Chk1,Chk2,p-Chk2, Cdc25 C and Cyclin B1 were detected by Western blot. Results:Flow cytometry showed that the G2/M phase cells in Chk1 overexpression group were increased compared with the control group and the vector group(P<0.05).After 15 mg·L~(-1) DADS treatment,G2/M phase cells in each group were increased compared with those before treatment,and there was statistically significant of the difference between the Chk1 overexpression group and the vector group(P<0.05). The G2/M phase cells in the Chk2 overexpression group exhibited no statistically significant of the difference with the control group and the vector group(P>0.05). The G2/M phase cells were different from those in the control group and the vector group after DADS treated Chk2 overexpression group(P<0.05). Western blot showed that the expression level of Chk1/2 protein and p-Chk2 was not affected by DADS, but the p-Chk1 was time-dependent increased(P<0.05). The expression of Cdc25 C and Cyclin B1 showed time-dependent decrease after DADS treated Chk1 overexpression BGC823 cells for 12,24,36 and 48 h(P<0.05). Conclusion:DADS arrest the G2/M phase in BGC823 cells is related to the activation of Chk1/Cdc25 C/Cyclin B1 pathway.Chk1 overexpression can enhance the role of DADS arrest G2/M phase, while Chk2 overexpression has no effect on DADS.
引文
[1]Bray F,Ferlay J,Soerjomataram I,et al.Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in185 countries[J].CA Cancer J Clin,2018,68(6):394-424.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]Visconti R,Della Monica R,Grieco D.Cell cycle checkpoint in cancer:a therapeutically targetable double-edged sword[J].J Exp Clin Cancer Res,2016,35(1):153.
[4]Yi L,Su Q.Molecular mechanisms for the anti-cancer effects of diallyl disulfide[J].Food Chem Toxicol,2013,57(7):362-370.
[5]Yuan JP,Wang GH,Ling H,et al.Diallyl disulfide-induced G2/M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways[J].World J Gastroenterol,2004,10(18):2731-2734.
[6]Ling H,Zhang LY,Su Q,et al.Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803[J].Cell Mol Biol Lett,2006,11(3):408-423.
[7]Su B,Xiang SL,Su J,et al.Diallyl disulfide increased histone acetylation and p21WAF1 expression in human gastric cancer cells in vivo and in vitro[J].Biochem&Pharmacol,2012,1(7):1-10.
[8]Ling H,Lu LF,He J,et al.Diallyl disulfide selectively causes Checkpoint kinase-1 mediated G2/M arrest in human MGC803 gastric cancer cell line[J].Oncol Rep,2014,32(5):2274-2282.
[9]Tang H,Kong Y,Guo J,et al.Diallyl disulfide suppresses proliferation and induces apoptosis in human gastric cancer through Wnt-1 signaling pathway by up-regulation of miR-200b and miR-22[J].Cancer Lett,2013,340(1):72-81.
[10]谭亚丽,夏红,曾颖,等.沉默Chk1基因对DADS阻滞人胃癌BGC823细胞G2/M期的影响[J].中国医药导刊,2018,20(6):346-350.
[11]王莉,曾颖,夏红,等.Chk1和Chk2高表达人胃癌BGC823细胞的建立与鉴定[J].中南医学科学杂志,2018,46(5):468-472.
[12]Zhang Y,Hunter T.Roles of Chk1 in cell biology and cancer therapy[J].Int J Cancer,2014,134(5):1013-1023.
[13]Li QQ,Hsu I,Sanford T,et al.Protein kinase D inhibitor CRT0066101suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M[J].Cell Mol Life Sci,2018,75(5):939-963.
[14]Wang J,Zhang Z,Che Y,et al.Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma[J].Cancer Chemother Pharmacol,2018,81(3):469-481.
[15]Jayasooriya RGPT,Molagoda IMN,Park C,et al.Molecular chemotherapeutic potential of butein:a concise review[J].Food Chem Toxicol,2018,112:1-10.
[16]Nikolos F,Thomas C,Bado I,et al.ERβsensitizes NSCLC to chemotherapy by regulating DNA damage response[J].Mol Cancer Res,2018,16(2):233-242.
[17]Yu CY,Jerry Teng CL,Hung PS,et al.Ovatodiolide isolated from anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways[J].Eur J Pharmacol,2018,819:16-29.
[18]Ma YC,Su N,Shi XJ,et al.Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM-Chk1/2-Cdc25C pathway[J].Toxicol Appl Pharmacol,2015,282(2):227-236.
[19]Huang H,Hu M,Zhao R,et al.Dihydromyricetin suppresses the proliferation of hepatocellular carcinoma cells by inducing G2/M arrest through the Chk1/Chk2/Cdc25C pathway[J].Oncol Rep,2013,30(5):2467-2475.
[20]Shin SS,Song JH,Hwang B,et al.Angiopoietin-like protein 4 potentiates DATS-induced inhibition of proliferation,migration,and invasion of bladder cancer EJ cells;involvement of G2/M-phase cell cycle arrest,signaling pathways,and transcription factors-mediated MMP-9 expression[J].Food Nutr Res,2017,61(1):1338918.
[21]Liu H,Luo Q,Cui H,et al.Sodium fluoride causes hepatocellular S-phase arrest by activating ATM-p53-p21 and ATR-Chk1-Cdc25A pathways in mice[J].Oncotarget,2017,9(4):4318-4337.
[2]Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]Visconti R,Della Monica R,Grieco D.Cell cycle checkpoint in cancer:a therapeutically targetable double-edged sword[J].J Exp Clin Cancer Res,2016,35(1):153.
[4]Yi L,Su Q.Molecular mechanisms for the anti-cancer effects of diallyl disulfide[J].Food Chem Toxicol,2013,57(7):362-370.
[5]Yuan JP,Wang GH,Ling H,et al.Diallyl disulfide-induced G2/M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways[J].World J Gastroenterol,2004,10(18):2731-2734.
[6]Ling H,Zhang LY,Su Q,et al.Erk is involved in the differentiation induced by diallyl disulfide in the human gastric cancer cell line MGC803[J].Cell Mol Biol Lett,2006,11(3):408-423.
[7]Su B,Xiang SL,Su J,et al.Diallyl disulfide increased histone acetylation and p21WAF1 expression in human gastric cancer cells in vivo and in vitro[J].Biochem&Pharmacol,2012,1(7):1-10.
[8]Ling H,Lu LF,He J,et al.Diallyl disulfide selectively causes Checkpoint kinase-1 mediated G2/M arrest in human MGC803 gastric cancer cell line[J].Oncol Rep,2014,32(5):2274-2282.
[9]Tang H,Kong Y,Guo J,et al.Diallyl disulfide suppresses proliferation and induces apoptosis in human gastric cancer through Wnt-1 signaling pathway by up-regulation of miR-200b and miR-22[J].Cancer Lett,2013,340(1):72-81.
[10]谭亚丽,夏红,曾颖,等.沉默Chk1基因对DADS阻滞人胃癌BGC823细胞G2/M期的影响[J].中国医药导刊,2018,20(6):346-350.
[11]王莉,曾颖,夏红,等.Chk1和Chk2高表达人胃癌BGC823细胞的建立与鉴定[J].中南医学科学杂志,2018,46(5):468-472.
[12]Zhang Y,Hunter T.Roles of Chk1 in cell biology and cancer therapy[J].Int J Cancer,2014,134(5):1013-1023.
[13]Li QQ,Hsu I,Sanford T,et al.Protein kinase D inhibitor CRT0066101suppresses bladder cancer growth in vitro and xenografts via blockade of the cell cycle at G2/M[J].Cell Mol Life Sci,2018,75(5):939-963.
[14]Wang J,Zhang Z,Che Y,et al.Rabdocoestin B exhibits antitumor activity by inducing G2/M phase arrest and apoptosis in esophageal squamous cell carcinoma[J].Cancer Chemother Pharmacol,2018,81(3):469-481.
[15]Jayasooriya RGPT,Molagoda IMN,Park C,et al.Molecular chemotherapeutic potential of butein:a concise review[J].Food Chem Toxicol,2018,112:1-10.
[16]Nikolos F,Thomas C,Bado I,et al.ERβsensitizes NSCLC to chemotherapy by regulating DNA damage response[J].Mol Cancer Res,2018,16(2):233-242.
[17]Yu CY,Jerry Teng CL,Hung PS,et al.Ovatodiolide isolated from anisomeles indica induces cell cycle G2/M arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways[J].Eur J Pharmacol,2018,819:16-29.
[18]Ma YC,Su N,Shi XJ,et al.Jaridonin-induced G2/M phase arrest in human esophageal cancer cells is caused by reactive oxygen species-dependent Cdc2-tyr15 phosphorylation via ATM-Chk1/2-Cdc25C pathway[J].Toxicol Appl Pharmacol,2015,282(2):227-236.
[19]Huang H,Hu M,Zhao R,et al.Dihydromyricetin suppresses the proliferation of hepatocellular carcinoma cells by inducing G2/M arrest through the Chk1/Chk2/Cdc25C pathway[J].Oncol Rep,2013,30(5):2467-2475.
[20]Shin SS,Song JH,Hwang B,et al.Angiopoietin-like protein 4 potentiates DATS-induced inhibition of proliferation,migration,and invasion of bladder cancer EJ cells;involvement of G2/M-phase cell cycle arrest,signaling pathways,and transcription factors-mediated MMP-9 expression[J].Food Nutr Res,2017,61(1):1338918.
[21]Liu H,Luo Q,Cui H,et al.Sodium fluoride causes hepatocellular S-phase arrest by activating ATM-p53-p21 and ATR-Chk1-Cdc25A pathways in mice[J].Oncotarget,2017,9(4):4318-4337.