二甲双胍下调谷胱甘肽S转移酶和拓扑异构酶Ⅱα对SiHa/cDDP增殖的抑制作用
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摘要
目的探讨二甲双胍下调谷胱甘肽S转移酶(GSTpi)和拓扑异构酶Ⅱα(TopIIα)对顺铂(cDDP)耐药的宫颈癌细胞系SiHa/cDDP增殖的抑制作用。方法构建SiHa/cDDP耐药细胞株并进行鉴定。CCK8法测定cDDP对Si Ha和Si Ha/c DDP细胞的抑制率;绘制SiHa、SiHa/cDDP细胞的生长曲线,按Patterson公式计算Si Ha和Si Ha/c DDP细胞的群体倍增时间;观察c DDP对SiHa、SiHa/cDDP细胞形态的影响;CCK8法测定不同浓度二甲双胍对SiHa和SiHa/cDDP细胞的抑制率;细胞迁移实验、Transwell细胞侵袭实验分别检测二甲双胍对SiHa、SiHa/cDDP细胞侧向迁移能力及纵向迁移能力的影响;RT-qPCR法检测SiHa/cDDP细胞较SiHa细胞GSTpi、TopIIαmRNA的表达变化,以及二甲双胍对SiHa/cDDP细胞作用后GSTpi、TopIIαm RNA的表达变化。结果 cDDP对SiHa、SiHa/cDDP细胞的抑制作用与cDDP的浓度正相关,相同浓度下对SiHa/cDDP细胞的抑制率明显低于对SiHa细胞的抑制率(P<0.01);SiHa、SiHa/cDDP细胞的群体倍增时间分别为(30.69±0.58)、(38.39±1.05)h,比较差异具有统计学意义(P<0.001);顺铂对SiHa细胞形态的影响较大,而对SiHa/cDDP细胞基本没有影响;二甲双胍对SiHa、SiHa/cDDP细胞生长均有抑制作用,抑制率与二甲双胍浓度正相关,相同浓度下对SiHa/cDDP细胞的抑制率明显高于对SiHa细胞的抑制率(P<0.01);与SiHa细胞相比,二甲双胍能更明显抑制SiHa/cDDP细胞迁移和侵袭(P<0.01);RT-qPCR结果显示,与SiHa细胞相比,SiHa/cDDP中GSTpi、TopIIαm RNA的表达明显升高(P<0.01、0.05),且二甲双胍能抑制SiHa/cDDP细胞中GSTpi、Top IIαm RNA的表达(P<0.01)。结论二甲双胍能通过下调GSTpi、Top IIα的表达抑制Si Ha/c DDP的增殖、侵袭和迁移。
Objective To investigate the effects of metformin on SiHa/cDDP cells proliferation by down-regulating GSTpi and TopIIα.Methods SiHa/cDDP cells were constructed and authenticated. The inhibition rates of cDDP on SiHa and SiHa/cDDP cells were estimated by Cell Counting Kit-8(CCK8) assay. Cell growth curve of SiHa and SiHa/cDDP cells was drawed, and population doubling time of SiHa and SiHa/cDDP cells was calculated by the manner of Patterson formula. The effects of cDDP on the morphology of SiHa and SiHa/cDDP cells were observed. The inhibition rates of metformin in different concentration on SiHa and SiHa/cDDP cells were estimated by CCK8 assay. The effects of metformin on SiHa and SiHa/cDDP cells were detected by wound healing assay to evaluate lateral migration, and by transwell assay to evaluate longitudinal migration, respectively. Changes of expression of GSTpi and TopIIαm RNA in SiHa/cDDP cells compared with those in SiHa cells were detected by RT-qPCR, and changes of expression of GSTpi and TopIIα mRNA in SiHa/cDDP cells treated with metformin were detected. Results The inhibition of cDDP on SiHa and SiHa/cDDP cells was positively correlated with the concentration of cDDP. The inhibition rate in SiHa/cDDP cells was significantly lower than that in SiHa cells(P < 0.01) under the same concentration of cDDP. The population doubling time of SiHa and SiHa/cDDP cells was(30.69 ± 0.58) h and(38.39 ± 1.05) h respectively,(P < 0.001). Cisplatin had a significant effect on the morphology of SiHa cells, but had few effects on SiHa/cDDP cells. Metformin inhibited the growth of both SiHa and SiHa/cDDP cells, with positively correlated with the concentration of merformin. The inhibition rate in SiHa/cDDP cells was significantly higher than that in SiHa cells(P < 0.01) under the same concentration of merformin. Compared with SiHa cells, metformin inhibited the migration and invasion of SiHa/cDDP cells more significantly(P < 0.01). RT-qPCR results showed that the expression of GSTpi, and TopIIα mRNA in SiHa/cDDP cells was significantly higher than that in SiHa cells(P < 0.01, 0.05), and metformin could inhibit m RNA expression of GSTpi, TopIIα in SiHa/cDDP cells(P < 0.01). Concousion Metformin inhibits SiHa/cDDP cells proliferation, motility, and invasiveness by down-regulating expression levels of genes GSTpi and TopIIα.
Objective To investigate the effects of metformin on SiHa/cDDP cells proliferation by down-regulating GSTpi and TopIIα.Methods SiHa/cDDP cells were constructed and authenticated. The inhibition rates of cDDP on SiHa and SiHa/cDDP cells were estimated by Cell Counting Kit-8(CCK8) assay. Cell growth curve of SiHa and SiHa/cDDP cells was drawed, and population doubling time of SiHa and SiHa/cDDP cells was calculated by the manner of Patterson formula. The effects of cDDP on the morphology of SiHa and SiHa/cDDP cells were observed. The inhibition rates of metformin in different concentration on SiHa and SiHa/cDDP cells were estimated by CCK8 assay. The effects of metformin on SiHa and SiHa/cDDP cells were detected by wound healing assay to evaluate lateral migration, and by transwell assay to evaluate longitudinal migration, respectively. Changes of expression of GSTpi and TopIIαm RNA in SiHa/cDDP cells compared with those in SiHa cells were detected by RT-qPCR, and changes of expression of GSTpi and TopIIα mRNA in SiHa/cDDP cells treated with metformin were detected. Results The inhibition of cDDP on SiHa and SiHa/cDDP cells was positively correlated with the concentration of cDDP. The inhibition rate in SiHa/cDDP cells was significantly lower than that in SiHa cells(P < 0.01) under the same concentration of cDDP. The population doubling time of SiHa and SiHa/cDDP cells was(30.69 ± 0.58) h and(38.39 ± 1.05) h respectively,(P < 0.001). Cisplatin had a significant effect on the morphology of SiHa cells, but had few effects on SiHa/cDDP cells. Metformin inhibited the growth of both SiHa and SiHa/cDDP cells, with positively correlated with the concentration of merformin. The inhibition rate in SiHa/cDDP cells was significantly higher than that in SiHa cells(P < 0.01) under the same concentration of merformin. Compared with SiHa cells, metformin inhibited the migration and invasion of SiHa/cDDP cells more significantly(P < 0.01). RT-qPCR results showed that the expression of GSTpi, and TopIIα mRNA in SiHa/cDDP cells was significantly higher than that in SiHa cells(P < 0.01, 0.05), and metformin could inhibit m RNA expression of GSTpi, TopIIα in SiHa/cDDP cells(P < 0.01). Concousion Metformin inhibits SiHa/cDDP cells proliferation, motility, and invasiveness by down-regulating expression levels of genes GSTpi and TopIIα.
引文
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[24]Kaplan E,Gunduz U.Expression analysis of TOP2A,MSH2 and MLH1 genes in MCF7 cells at different levels of etoposide resistance[J].Biomed Pharmacother,2012,66(1):29-35.
[2]Chen W,Zheng R,Baade P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[3]Siddik Z H.Cisplatin:mode of cytotoxic action and molecular basis of resistance[J].Oncogene,2003,22(47):7265-7279.
[4]Brabec V,Kasparkova J.Modifications of DNA by platinum complexes.Relation to resistance of tumors to platinum antitumor drugs[J].Drug Resist Updat,2005,8(3):131-146.
[5]Bailey C J,Turner R C.Metformin[J].N Engl J Med,1996,334(9):574-579.
[6]Muszynska-Oglaza A,Zarzycka-Lindner G,Olejniczak H,et al.Use of metformin is associated with lower incidence of cancer in patients with type 2 diabetes[J].Endokrynol Pol,2017,68(6):652-658.
[7]Kim H J,Lee S,Chun K H,et al.Metformin reduces the risk of cancer in patients with type 2 diabetes:an analysis based on the Korean National Diabetes Program Cohort[J].Medicine(Baltimore),2018,97(8):e0036.
[8]Stambolic V,Woodgett J R,Fantus I G,et al.Utility of metformin in breast cancer treatment,is neoangiogenesis a risk factor?[J].Breast Cancer Res Treat,2009,114(2):387-389.
[9]Hadad S M,Appleyard V,Thompson A M.Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERalpha negative MDA-MB-435 breast cancer model[J].Breast Cancer Res Treat,2009,114(2):391.
[10]Cai X,Hu X,Cai B,et al.Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIPexpression and downregulation of cyclin D1 in vitro and in vivo[J].Oncol Rep,2013,30(5):2449-2457.
[11]Jeong Y K,Kim M S,Lee J Y,et al.Metformin radiosensitizes p53-deficient colorectal cancer cells through induction of G2/M arrest and inhibition of DNArepair proteins[J].PLoS One,2015,10(11):e0143596.
[12]Song C W,Lee H,Dings R P,et al.Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells[J].Sci Rep,2012,2:362.
[13]Xia C,Chen R,Chen J,et al.Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice[J].Sci Rep,2017,7:43373.
[14]Xia C,Liang S,He Z,et al.Metformin,a first-line drug for type 2 diabetes mellitus,disrupts the MALAT1/miR-142-3p sponge to decrease invasion and migration in cervical cancer cells[J].Eur J Pharmacol,2018,830:59-67.
[15]严泉剑,郭金龙,刘恩靖,等.绘制细胞生长曲线及细胞群体倍增时间的简化计算[J].前卫医药杂志,2000,17(4):228-229.
[16]陈新莲,王和,张雪梅,等.人宫颈癌顺铂耐药细胞系SiHa/cDDP的建立[J].四川大学学报:医学版,2012,43(2):151-155.
[17]林雯,李德锐,王鸿彪,等.人鼻咽癌顺铂耐药细胞系建立及其与凋亡关系的研究[J].中华肿瘤防治杂志,2008,15(8):572-575.
[18]Bai T,Tanaka T,Yukawa K,et al.A novel mechanism for acquired cisplatin-resistance:suppressed translation of death-associated protein kinase m RNA is insensitive to5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells[J].Int J Oncol,2006,28(2):497-508.
[19]Ellerhorst J A,Frost P,Abbruzzese J L,et al.2'-deoxy-5-azacytidine increases binding of cisplatin to DNA by a mechanism independent of DNA hypomethylation[J].Br J Cancer,1993,67(2):209-215.
[20]潘洪明,费洪新,杜静平,等.人胃癌顺铂耐药细胞系的建立过程[J].世界华人消化杂志,2007,15(18):2009-2013.
[21]Tew K D,Manevich Y,Grek C,et al.The role of glutathione S-transferase P in signaling pathways and S-glutathionylation in cancer[J].Free Radic Biol Med,2011,51(2):299-313.
[22]Checa-Rojas A,Delgadillo-Silva L F,Velasco-Herrera MD C,et al.GSTM3 and GSTP1:novel players driving tumor progression in cervical cancer[J].Oncotarget,2018,9(31):21696-21714.
[23]Wang T L,Song Y Q,Ren Y W,et al.Dual specificity phosphatase 6(DUSP6)polymorphism predicts prognosis of inoperable non-small cell lung cancer after chemoradiotherapy[J].Clin Lab,2016,62(3):301-310.
[24]Kaplan E,Gunduz U.Expression analysis of TOP2A,MSH2 and MLH1 genes in MCF7 cells at different levels of etoposide resistance[J].Biomed Pharmacother,2012,66(1):29-35.