T-钙黏蛋白联合顺铂对黑色素瘤顺铂耐药株的作用研究
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摘要
目的探讨T-钙黏蛋白联合顺铂对黑色素瘤顺铂耐药株的作用。方法采用大剂量冲击和逐步增加剂量相结合的方法诱导建立小鼠黑色素瘤B16F10顺铂耐药细胞株(CDDP-R B16F10)。MTT法检测CDDP-R B16F10的增殖能力。将T-钙黏蛋白转染肿瘤细胞。分别采用反转录聚合酶链式反应(RT-PCR)和免疫组化SP法检测转染后T-钙黏蛋白mRNA和蛋白的表达。实验将CDDP-R B16F10细胞分为空白对照组、pEGFP-N1组、pEGFP-N1-Tcadherin组、顺铂组、pEGFP-N1联合顺铂组、pEGFP-N1-T-cadherin联合顺铂组。采用Wound-healing划痕实验和transwell侵袭实验检测T-钙黏蛋白联合顺铂对CDDP-R B16F10细胞迁移和侵袭力的影响。结果成功建立黑色素瘤顺铂耐药细胞株。MTT法结果显示,CDDP-R B16F10细胞增殖能力与B16F10细胞相比差异无统计学意义(P>0.05)。RT-PCR和免疫组化SP法检测表明,转染后细胞可稳定转录和表达T-钙黏蛋白。pEGFP-N1-T-cadherin联合顺铂组细胞迁移率和穿膜细胞数低于pEGFP-N1-T-cadherin组(P<0.05),而pEGFP-N1-T-cadherin组低于空白对照组、pEGFP-N1组、顺铂组和pEGFP-N1联合顺铂组(P<0.05)。析因分析显示,T-钙黏蛋白与顺铂联合对CDDP-R B16F10细胞迁移率及侵袭力的抑制有交互作用(P<0.05)。结论 T-钙黏蛋白可恢复顺铂对黑色素瘤顺铂耐药细胞株迁移及侵袭力的抑制作用。
Objective To investigate the effect of T-cadherin combined with cisplatin on cisplatin resistant malignant melanoma cell line.Methods CDDP resistance B16F10(CDDP-R B16F10) was induced by using high and gradually increased dose of CDDP.MTT assay was used to test the proliferation of CDDP-R B16F10. The T-cadherin was transfected into CDDP-R B16F10 cells. The expressions of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction(RT-PCR) and SP immunohistochemistry method. There were six groups in this study including control group, pEGFP-N1 group, pEGFP-N1-T-cadherin group, cisplatin group, pEGFP-N1 combined with cisplatin group and pEGFP-N1-T-cadherin combined with cisplatin group. The effects of T-cadherin combined with cisplatin on migration and invasion of CDDP-R B16F10 were determined by Wound-healing assay and Transwell invasion assay. Factor analysis method was used to evaluate the interactions of T-cadherin and CDDP on migration and invasion of CDDP-R B16F10.Results The CDDP-R B16F10 cell line was successfully established. There was no statistical difference in proliferation between CDDP-R B16F10 cells and B16F10 cells(P>0.05). RT-PCR and SP immunohistochemistry assay showed that Tcadherin could be transcribed and expressed in cells. The cell migration rate and transmembrane number were significantly lower in pEGFP-N1-T-cadherin combined with cisplatin group than those of pEGFP-N1-T-cadherin group(P<0.05). The cell migration rate and transmembrane number were significantly lower in pEGFP-N1-T-cadherin group than those of control group, pEGFP-N1 group, cisplatin group and pEGFP-N1 combined with cisplatin group(P<0.05). There were interaction between T-cadherin and cisplatin in inhibiting the migration and invasiveness of CDDP-R B16F10(P<0.05).Conclusion T-cadherin gene can restore the inhibitory effect of cisplatin on the migration and invasiveness of cisplatin resistant melanoma cell line.
Objective To investigate the effect of T-cadherin combined with cisplatin on cisplatin resistant malignant melanoma cell line.Methods CDDP resistance B16F10(CDDP-R B16F10) was induced by using high and gradually increased dose of CDDP.MTT assay was used to test the proliferation of CDDP-R B16F10. The T-cadherin was transfected into CDDP-R B16F10 cells. The expressions of T-cadherin mRNA and protein were measured by reverse transcription polymerase chain reaction(RT-PCR) and SP immunohistochemistry method. There were six groups in this study including control group, pEGFP-N1 group, pEGFP-N1-T-cadherin group, cisplatin group, pEGFP-N1 combined with cisplatin group and pEGFP-N1-T-cadherin combined with cisplatin group. The effects of T-cadherin combined with cisplatin on migration and invasion of CDDP-R B16F10 were determined by Wound-healing assay and Transwell invasion assay. Factor analysis method was used to evaluate the interactions of T-cadherin and CDDP on migration and invasion of CDDP-R B16F10.Results The CDDP-R B16F10 cell line was successfully established. There was no statistical difference in proliferation between CDDP-R B16F10 cells and B16F10 cells(P>0.05). RT-PCR and SP immunohistochemistry assay showed that Tcadherin could be transcribed and expressed in cells. The cell migration rate and transmembrane number were significantly lower in pEGFP-N1-T-cadherin combined with cisplatin group than those of pEGFP-N1-T-cadherin group(P<0.05). The cell migration rate and transmembrane number were significantly lower in pEGFP-N1-T-cadherin group than those of control group, pEGFP-N1 group, cisplatin group and pEGFP-N1 combined with cisplatin group(P<0.05). There were interaction between T-cadherin and cisplatin in inhibiting the migration and invasiveness of CDDP-R B16F10(P<0.05).Conclusion T-cadherin gene can restore the inhibitory effect of cisplatin on the migration and invasiveness of cisplatin resistant melanoma cell line.
引文
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[13]Bosserhoff AK,Ellmann L,Quast AS,et al.Loss of T-cadherin(CDH-13)regulates AKT signaling and desensitizes cells to apoptosis in melanoma[J].Mol Carcinog,2014,53(8):635-647.
[14]Adachi Y,Takeuchi T,Sonobe H,et a1.An adiponectin receptor,T-cadherin,was selectively expressed in intratumoral capillary endothelial cells in hepatocellular carcinoma:possible cross talk between T-cadherin and FGF-2 pathways[J].Virchows Arch,2006,448(3):311-3l8.doi:10.1002/mc.22018.
[15]Kalayda GV,Wagner CH,Jaehde U.Relevance of copper transporter 1 for cisplatin resistance in human ovarian carcinoma cells[J].J Inorg Biochem,2012,116:1-10.doi:10.1016/j.jinorgbio.2012.07.010.
[16]唐春兰,杨和平,周向东.DNA损伤修复与肺癌顺铂耐药机制的研究进展[J].中国肺癌杂志,2011,14(12):960-964.Tang CL,Yang HP,Zhou XD.Advances of DNA damage repair and cisplatin resistance mechanisms in lung cancer[J].Chin J Lung Cancer,2011,14(12):960-964.doi:10.3779/j.issn.1009-3419.2011.12.11.
[17]Citri A,Yarden Y.EGF-ERBB signalling:towards the systems level[J].Nat Rev Mol Cell Biol,2006,7(7):505-516.
[2]赵庆芳,霍焱,姜战胜,等.肉叶芸香碱对黑色素瘤A375细胞上皮间充质转化的影响[J].天津医药,2017,45(7):691-695.Zhao QF,Huo Y,Jiang ZH,et al.Effects of harmine on the epithelial mesenchymal transition of melanoma A375 cells[J].Tianjin Med J,2017,45(7):691-695.doi:10.11958/20170329.
[3]Alrwas A,Papadopoulos NE,Cain S,et al.Phase I trial of biochemotherapy with cisplatin,temozolomide,And dose escalation of nab-paclitaxel combined with interleukin-2 and interferon-αin patients with metastatic melanoma[J].Melanoma Res,2014,24(4):342-348.doi:10.1097/CMR.0000000000000062.
[4]Duan XS,Lu J,Ge ZH,et al.Effects of T-cadherin expression on B16F10 melanoma cells[J].Oncology Letters,2013,5(4):1205-1210.doi:10.3892/o1.2013.1164.
[5]陆海涛,孙立新,陆洁,等.T-钙黏蛋白对黑素瘤侵袭力的影响[J].中国皮肤性病学杂志,2011,25(5):329-332.Lu HT,Sun LX,Lu J,et al.The Effect of T-cadherin on the Invasion of Malignant Melanoma[J].Chin J Derm Venereol,2011,25(5):329-332.doi:1001-7089(2011)05-0329-04.
[6]Selga E,Morales C,NoéV,et al.Role of Caveolin 1,E-Cadherin,Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells[J].BMC Medical Genomics,2008,1:35.doi:10.1186/1755-8794-1-35.
[7]郭科,莫乃新.下调丙酮酸激酶2表达对膀胱癌T24细胞株生物学特性的影响[J].江苏大学学报(医学版),2015,25(1):22-26.Guo K,Mo NX.Expression of PKM2 in bladder carcinoma and its effect of down-regulation on biological characteristics of bladder carcinoma cell line T24[J].Journal of Jiangsu University(Medicine Edition),2015,25(1):22-26.
[8]李素,周芳,张庆瑜,等.ZEB2基因3'UTR区转染对人胃黏膜上皮细胞GES-1增殖、侵袭、迁移的影响[J].天津医药,2014,42(5):401-405.Li S,Zhou F,Zhang QY,et al.Effects of ZEB2 3'UTR transfection on proliferation,invasion and migration in human gastric epithelial cell GES-1[J].Tianjin Med J,2014,42(5):401-405.doi:10.3969/j.issn.0253-9896.2014.05.001.
[9]Chekhun VF,Lukyanova NY,Urchenko OV,et al.The role of the components of proteome in the formation of molecular profile of human ovarian carcinoma A2780 cells sensitive and resistant to cisplatin[J].Exp Oncol,2005,27(3):191-195.
[10]Fricke E,Hermannst?dter C,Keller G,et a1.Effect of wild-type and mutant E-cadherin on cell proliferation and responsiveness to the chemotherapeutic agents cisplatin,etoposide,and 5-fluorouracil[J].Oncology,2004,66(2):150-159.doi:10.1159/000077442.
[11]王梅红,孔德娣,周艳贞,等.T-钙黏蛋白在乳腺癌中表达及其与预后的关系[J].中华普外科手术学杂志(电子版),2014,8(2):157-160.Wang MH,Kong DD,Zhou YZ,et al.Expression and predictive value of prognosis of T-cadherin in breast cancer[J].Chin J Oper Proc Gen Surg(Electronic Edition),2014,8(2):157-160.doi:10.3877/cma.j.issn.1674-3946.2014.02.046.
[12]李鲲,冯旭,石俊杰,等.T-钙黏蛋白基因甲基化在肺癌发生中的作用[J].中华实验外科杂志,2016,33(8):1897-1899.Li K,Feng X,Shi JJ,et al.Role of T-cadherin genomic methylation in the development of lung cancer[J].Chin J Exp Surg,2016,33(8):1897-1899.doi:10.3760/cma.j.issn.1001-9030.2016.08.003.
[13]Bosserhoff AK,Ellmann L,Quast AS,et al.Loss of T-cadherin(CDH-13)regulates AKT signaling and desensitizes cells to apoptosis in melanoma[J].Mol Carcinog,2014,53(8):635-647.
[14]Adachi Y,Takeuchi T,Sonobe H,et a1.An adiponectin receptor,T-cadherin,was selectively expressed in intratumoral capillary endothelial cells in hepatocellular carcinoma:possible cross talk between T-cadherin and FGF-2 pathways[J].Virchows Arch,2006,448(3):311-3l8.doi:10.1002/mc.22018.
[15]Kalayda GV,Wagner CH,Jaehde U.Relevance of copper transporter 1 for cisplatin resistance in human ovarian carcinoma cells[J].J Inorg Biochem,2012,116:1-10.doi:10.1016/j.jinorgbio.2012.07.010.
[16]唐春兰,杨和平,周向东.DNA损伤修复与肺癌顺铂耐药机制的研究进展[J].中国肺癌杂志,2011,14(12):960-964.Tang CL,Yang HP,Zhou XD.Advances of DNA damage repair and cisplatin resistance mechanisms in lung cancer[J].Chin J Lung Cancer,2011,14(12):960-964.doi:10.3779/j.issn.1009-3419.2011.12.11.
[17]Citri A,Yarden Y.EGF-ERBB signalling:towards the systems level[J].Nat Rev Mol Cell Biol,2006,7(7):505-516.