二甲双胍与顺铂联合诱导人结肠癌HCT-8细胞凋亡的研究
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摘要
目的研究二甲双胍联合顺铂对HCT-8人类结肠癌细胞凋亡的影响及其可能机制。方法将HCT-8人类结肠癌细胞分成二甲双胍组(MET)、顺铂组(DDP)、二甲双胍联合顺铂组(MET+DDP)、空白对照组(CK) 4组,MTT实验观察4组细胞于24 h、48 h、72 h时的增殖能力;流式细胞术观察各组细胞48 h的凋亡比例;Western blotting观察各组细胞48 h时凋亡相关蛋白Bcl-2、Bax、caspase-3(激活型)以及AKT/GSK-3通路蛋白的表达水平。结果该实验中MET+DDP组的细胞增殖能力弱于其它三组(P<0. 05);细胞凋亡比例高于其它三组(P<0. 05); Bax、caspase-3(激活型)表达量高于其它组群,Bcl-2、P-AKT、P-GSK-3表达量低于其它组群,AKT、GSK-3在4组中表达量相对恒定(P>0. 05)。结论二甲双胍、顺铂可能通过下调AKT/GSK-3信号通路,改变Bcl-2家族蛋白的表达活性从而促进HCT-8人类结肠癌细胞的凋亡,并促进p-caspase-3剪切转化为caspase-3(激活型);二甲双胍与顺铂可协同发挥促HCT-8人类结肠癌细胞凋亡的作用。
Objective To investigate the effects and mechanisms of action of metformin combined with cisplatin on the apoptosis in human colon cancer HCT-8 cells. Methods Human colon cancer HCT-8 cells were divided into four groups: metformin group( MET),cisplatin group( DDP),metformin combined with cisplatin group( MET + DDP),and blank control group( CK). The proliferative ability of HCT-8 cells was determined by an MTT experiment at 24,48 and 72 h. The apoptosis rate was determined by flow cytometry at 48 h. Western blotting was used to reveal the expression of apoptosis-related proteins such as bcl-2,Bax and caspase-3( activated),as well as proteins of the AKT/GSK-3β pathwayat 48 h. Results The cell proliferative ability in the MET+DDP group was weaker than that of the other three groups( P<0. 05). Moreover,its apoptosis rate was higher than those of the other three groups( P<0. 05). The expressions of Bax and caspase-3( activated) was higher than that of the other groups,the expression of Bcl-2,p-AKT and p-GSK-3β was lower than that of the other groups,and the expression of AKT and Gsk-3 was relatively consistent among the four groups.Conclusions Metformin and cisplatin in combination can reduce the activity of the AKT/GSK-3β signaling pathway,and change the expression of Bcl-2 family proteins,to promote the apoptosis of human colon cancer HCT-8 cells. They also promote the p-caspase-3 conversion to caspase-3 p-caspase-3 conversion to caspase-3( activated),metformin and cisplatin can thus play a role in promoting the apoptosis of HCT-8 human colon cancer cells.
Objective To investigate the effects and mechanisms of action of metformin combined with cisplatin on the apoptosis in human colon cancer HCT-8 cells. Methods Human colon cancer HCT-8 cells were divided into four groups: metformin group( MET),cisplatin group( DDP),metformin combined with cisplatin group( MET + DDP),and blank control group( CK). The proliferative ability of HCT-8 cells was determined by an MTT experiment at 24,48 and 72 h. The apoptosis rate was determined by flow cytometry at 48 h. Western blotting was used to reveal the expression of apoptosis-related proteins such as bcl-2,Bax and caspase-3( activated),as well as proteins of the AKT/GSK-3β pathwayat 48 h. Results The cell proliferative ability in the MET+DDP group was weaker than that of the other three groups( P<0. 05). Moreover,its apoptosis rate was higher than those of the other three groups( P<0. 05). The expressions of Bax and caspase-3( activated) was higher than that of the other groups,the expression of Bcl-2,p-AKT and p-GSK-3β was lower than that of the other groups,and the expression of AKT and Gsk-3 was relatively consistent among the four groups.Conclusions Metformin and cisplatin in combination can reduce the activity of the AKT/GSK-3β signaling pathway,and change the expression of Bcl-2 family proteins,to promote the apoptosis of human colon cancer HCT-8 cells. They also promote the p-caspase-3 conversion to caspase-3 p-caspase-3 conversion to caspase-3( activated),metformin and cisplatin can thus play a role in promoting the apoptosis of HCT-8 human colon cancer cells.
引文
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[26]赵瑞杰,李引乾,王会,等. Caspase家族与细胞凋亡的关系[J].中国畜牧杂志,2010,46(17):73-78.
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[29]石雪萍,李静,冉建华,等.人参皂苷Rh2调控PI3K/AKT/GSK-3β信号通路诱导人结肠癌细胞凋亡[J].中国药理学通报,2017,33(01):114-119.
[30]朱敬丽,路赵硕,吴越,等.铜、铁、锌、铝通过Akt/GSK-3β信号通路诱导SH-SY5Y细胞的凋亡[J].实用老年医学,2017,31(08):723-727.
[2] Evans JM,Donnelly LA,Emslie-Smith AM,et al. Metformin and reduced risk of cancer in diabetic patients[J]. BMJ,2005,330(7503):1304-1305.
[3]母义明,纪立农,宁光,等.二甲双胍临床应用专家共识(2016年版)[J].中国糖尿病杂志,2016,24(10):871-884.
[4] Chen W,Zheng R,Zhang S,et al. Report of cancer incidence and mortality in China,2010[J]. Ann Transl Med,2014,2(7):61.
[5] Torre LA,Bray F,Siegel RL,et al. Global cancer statistics,2012[J].CA Cancer J Clin,2015,65(2):87-108.
[6]姜艳芳,魏志,孙自勤.中国青年大肠癌发病趋势分析[J].胃肠病学和肝病学杂志,2016,25(09):982-987.
[7] Matsuo K,Mizoue T,Tanaka K,et al. Association between body mass index and the colorectal cancer risk in Japan:pooled analysis of population-based cohort studies in Japan[J]. Ann Oncol,2012,23(2):479-490.
[8] Ben Q,An W,Jiang Y,et al. Body mass index increases risk for colorectal adenomas based on meta-analysis[J]. Gastroenterol,2012,142(4):762-772.
[9] De Bruijn KM,Arends LR,Hansen BE,et al. Systematic review and meta-analysis of the association between diabetes mellitus and incidence and mortality in breast and colorectal cancer[J].Br J Surg,2013,100(11):1421-1429.
[10] Chen X,Wu Y,Dong H,et al. Platinum-based agents for individualized cancer treatment[J]. Curr Mol Med,2013,13(10):1603-1612.
[11] Hato SV,Khong A,De Vries IJ,et al. Molecular pathways:the immunogenic effects of platinum-based chemotherapeutics[J].Clin Cancer Res,2014,20(11):2831-2837.
[12] Kelland L. The resurgence of platinum-based cancer chemotherapy[J].Nat Rev Cancer,2007,7(8):573-584.
[13] Monneret C. Platinum anticancer drugs. From serendipity to rational design[J].Ann Pharm Fr,2011,69(6):286-295.
[14]高传柱,王天帅,陈佳,等.铂类抗肿瘤药物作用机制研究进展[J].昆明理工大学学报(自然科学版),2014,39(04):83-92.
[15]李华梅.静脉输注顺铂副作用的预防措施及效果观察[J].医疗装备,2014,27(10):42-43.
[16]郗照勇,刘扬中.顺铂耐药的分子机制[J].中国科学:化学,2014,44(04):410-422.
[17] DILMAN VM. Age-associated elevation of hypothalamic,threshold to feedback control, and its role in development,ageine,and disease[J]. Lancet,1971,297(7711):1211-1219.
[18]周侠,缪珩.二甲双胍抗癌作用的研究进展[J].天津医药,2017,45(07):767-771.
[19]许关煜.二甲双胍或延缓肿瘤生长[N].医药经济报,2013-04-05(003).
[20]姜宁,路平.二甲双胍抗肿瘤作用机制[J].新乡医学院学报,2016,33(2):150-152.
[21]陈津,张如松.细胞凋亡机制概述[J].中华中医药学刊,2011,29(04):886-889.
[22]张浩赟,夏艳,王鹏飞,等.胞质空泡化的研究进展[J].世界最新医学信息文摘,2017,17(34):66-68.
[23] Liu L,Zhang Z,Xing D. Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage[J].Free Radic Biol Med,2011; 51(1):53-68.
[24] Ola MS,Nawaz M,Ahsan H. Role of Bcl-2 family proteins and caspases in the regulation of apoptosis[J]. Mol Cell Biochem,2011; 351:41-58.
[25]许茸茸,李应东.Bcl-2家族与线粒体凋亡通路相互作用研究进展[J].中国老年学杂志,2013,33(12):2977-2979.
[26]赵瑞杰,李引乾,王会,等. Caspase家族与细胞凋亡的关系[J].中国畜牧杂志,2010,46(17):73-78.
[27] Shang Y,Myers M,Brown M. Formation of the androgen receptor transcription complex[J]. Mol Cell 2002,9:601-610.
[28]龚青,陈洁.PTEN/PI3K/Akt信号转导通路与结直肠癌关系的研究现状[J].胃肠病学和肝病学杂志,2016,25(06):706-710.
[29]石雪萍,李静,冉建华,等.人参皂苷Rh2调控PI3K/AKT/GSK-3β信号通路诱导人结肠癌细胞凋亡[J].中国药理学通报,2017,33(01):114-119.
[30]朱敬丽,路赵硕,吴越,等.铜、铁、锌、铝通过Akt/GSK-3β信号通路诱导SH-SY5Y细胞的凋亡[J].实用老年医学,2017,31(08):723-727.