【作者】 李强；

【导师】 陶敏；

【作者基本信息】 苏州大学 ， 肿瘤学（专业学位）， 2021， 博士

【摘要】 目的:本研究旨在探讨维莫非尼(Vemurafenib,vMF)与替莫唑胺(Temozolomide,TMZ)联用对恶性黑色素瘤细胞杀伤效果的影响及其作用机制。方法:(1)选择相应的恶性黑色素瘤组织芯片,免疫组化染色法检测芯片中MGMT和p-ERK1/2的表达情况,分析MGMT和p-ERK1/2的表达与恶性黑色素瘤患者临床病理因素的相关性,统计分析MGMT和p-ERK1/2在恶性黑色素瘤组织中表达的相关性。(2)MTT法用于检测不同浓度的TMZ单用或联合vMF作用后对恶性黑色素瘤细胞活力的影响,并计算相应处理方法对恶性黑色素瘤细胞的抑制率。(3)建立BALB/c裸鼠皮下移植恶性黑色素瘤模型,绘制成瘤曲线,对比TMZ单用和TMZ联合vMF对体内模型移植瘤生长的影响。(4)RT-qPCR检测不同浓度ERK抑制剂U0126与TMZ联用对恶性黑色素瘤A375细胞株中MGMT mRNA水平的影响;对比BRAF抑制剂vMF与TMZ联用对A375细胞株中MGMT mRNA水平的影响。(5)Western Blot检测vMF对ERK通路相关蛋白(ERK1/2、pERK1/2、MEK1、pMEK1)表达水平的影响;检测vMF与TMZ联用后A375亲本细胞以及MGMT过表达(Over expression,OE)细胞内MGMT蛋白水平的变化;检测vMF与TMZ联用对A375细胞内DNA损伤相关蛋白(pATR、γ-H2AX、pATM、pchk1、pchk2)表达水平的影响。(6)细胞免疫荧光检测vMF与TMZ联用对A375细胞核中DNA损伤相关蛋白γ-H2AX表达水平的影响。(7)流式细胞计数法(Annexin V-FITC/PI双染)检测vMF与TMZ联用对恶性黑色素瘤细胞A375凋亡的影响。结果:(1)恶性黑色素瘤组织芯片中可检测到MGMT和p-ERK1/2的表达。相关性结果显示在恶性黑色素瘤组织中MGMT与p-ERK1/2的表达水平呈正相关(相关系数r=0.567)。MGMT和p-ERK1/2的表达与恶性黑色素瘤患者年龄、病理分期及标本来源等无显著相关性。(2)TMZ可有效抑制恶性黑色素瘤细胞株A375和SK-MEL-28的活性,诱导恶性黑色素瘤细胞凋亡,且TMZ抑制恶性黑色素瘤细胞生长的作用呈剂量-反应关系。联用vMF可以增强TMZ对A375和SK-MEL-28细胞活力的抑制作用,并增强TMZ介导的肿瘤细胞凋亡。裸鼠实验结果显示vMF在移植瘤模型中同样可以增加TMZ对恶性黑色素肿瘤的抑制作用。DNA损伤相关数据显示TMZ可引起A375细胞中的DNA损伤,vMF可以增强TMZ所致DNA损伤的效果,但单用vMF对A375细胞内的DNA损伤水平无明显影响。(3)在体内外,vMF可以加快TMZ对A375细胞中MGMT蛋白的耗竭。ERK通路抑制剂U0126可抑制A375细胞中MGMT mRNA的转录水平。BRAF抑制剂vMF抑制ERK通路相关蛋白p-ERK1/2、p-MEK1的表达,并下调A375细胞中MGMT mRNA的转录水平。在A375-MGMT OE细胞中,TMZ对MGMT蛋白表达无抑制作用,vMF与TMZ联用对MGMT蛋白表达仍无明显抑制作用,且vMF对TMZ杀伤恶性黑色素瘤细胞的增敏作用减弱。结论:在恶性黑色素瘤组织中ERK通路活性与MGMT的表达水平正相关。vMF在体外、体内均可增加恶性黑色素瘤对TMZ的敏感性,其机制可能是VMF通过抑制ERK通路从而下调MGMT,进而增强TMZ对恶性黑色素瘤细胞的DNA损伤。vMF联合TMZ有协同增敏作用,该联合方案有望成为ERK高表达晚期恶性黑色素瘤患者新的治疗选择。更多还原

【Abstract】 Objective:This study is to investigate the effect and mechanism of the combination of vemurafenib(vMF)and temozolomide(TMZ)-induced cytotoxicity on malignant melanoma cells.Methods:(1)Immunohistochemical assay was used to detect the expression of MGMT and p-ERK1/2 in malignant melanoma tissue chips,and to analyze the expression levels and correlation of MGMT and p-ERK1/2.Its correlation with clinicopathological parameters of malignant melanoma patients was analyzed.(2)MTT assay was used to examine the toxic effect of TMZ or the combination of vMF and TMZ on malignant melanoma cells.MTT assay was used for detecting the combination of TMZ and vMF-induced cytotoxicity on the overexpression of MGMT malignant melanoma cells.The melanoma cell inhibition rate of the corresponding treatment methods on malignant melanoma cells was calculated.(3)The effect of vMF on TMZ-inducing tumor growth inhibition of malignant melanoma in vivo was examined by using nude mice with xenografts.(4)RT-qPCR assay was used to detect the MGMT mRNA expression effect of the combination of TMZ and U0126 or TMZ and vMF in A375 cells.(5)Western Blot assay was used to detect the expression effect of vMF on ERK pathway-related proteins(ERK1/2、pERK1/2、MEK1、pMEK1).Western Blot assay was used to detect the MGMT protein expression effect of the combination of vMF and TMZ on malignant melanoma cells.Western Blot assay was used to examine the MGMT protein expression effect of the combination of vMF and TMZ on the overexpression(OE)of MGMT A375 cells.Western Blot assay was used to examine the DNA damage-related proteins(pATR,y-H2AX,pATM,pchkl,pchk2)expression effect of the combination of vMF and TMZ in A375 cells.(6)Immunofluorescence assay was used to detect of the expression effect of the combination of vMF and TMZ on γ-H2AX.(7)Flow cytometry(Annexin V-FITC and PI double staining)assay was used to detect the apoptosis effect of the combination of vMF and TMZ on A3 75 cells.Results:(1)The expression of MGMT and p-ERK1/2 can be detected in the malignant melanoma tissue chip.The correlation result shows that the expression level of MGMT and p-ERK1/2 in malignant melanoma tissue is positively correlated(correlation coefficient r=0.567).The expressions of MGMT and p-ERK1/2 are not related to the age,pathological stage and specimen source of patients in malignant melanoma tissue(p>0.05).(2)The proliferation activity of A375 and SK-MEL-28 is decreased significantly and the apoptosis of malignant melanoma cells is induced by TMZ.The effect of TMZ on the growth of malignant melanoma cells is in a dose-response relationship.The inhibitory effect of TMZ on the viability of A375 and SK-MEL-28 cells and TMZ-mediated tumor cell apoptosis can be enhanced by the combined use of vMF.Experimental results in nude mice show that the inhibitory effect of TMZ on malignant melanoma in the transplanted tumor model can also be increased by vMF.DNA damage related data show that TMZ can cause DNA damage in A375 cells,which can be increased by vMF.But the DNA damage can not be caused by vMF.(3)In vitro or vivo,the depletion of MGMT protein in A375 cells by TMZ can be accelerated by vMF.The transcription level of MGMT mRNA in A3 75 cells can be inhibited by ERK pathway inhibitor U0126 or vMF.The expression of p-ERK1/2 and p-MEK1 protein-related ERK pathway can be inhibited by BRAF inhibitor vMF.In A375-MGMT OE cells,TMZ can not inhibit the expression of MGMT protein.The combined use of vMF and TMZ still has no obvious inhibitory effect on the expression of MGMT protein,and the sensitizing effect of vMF on the killing of malignant melanoma cells by TMZ is weakened.Conclusions:In malignant melanoma tissue,the activity of ERK pathway is positively correlated with the expression level of MGMT.The sensitivity of malignant melanoma to TMZ can be increased by vMF in vitro and in vivo.The mechanism may be that VMF inhibits the ERK pathway to down-regulate the expression of MGMT,thereby increasing the DNA damage of TMZ to malignant melanoma cells.vMF combined with TMZ has a synergistic sensitization effect,and this combination is expected to become a new treatment option for patients with advanced malignant melanoma with high ERK expression.更多还原