The Effect And Its Underlying Mechanism Of Vemurafenib Sensitizing Temozolomide(TMZ)-induced Cytotoxicity On Malignant Melanoma Cells

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.