Relationship between mismatch repair gene hMLH1, hMSH2 and temozolomide-resistance of glioma stem cells

Abstract

Objective To analyze the status of hMLH1 and hMSH2 of the glioma stem cells (GSC) during TMZ chemotherapy and the relationship between them and temozolomide-resistance of GSC.Methods GSC line U251g and A172g were isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium.CD133,Nestin and GFAP of GSC were examined by Immunofluorescence.The half inhibition concentration (IC50) of TMZ for U251g and A172g were calculated through Cytotoxic Test with CCK-8,then the corresponding two kinds of concentration of TMZ,IC50 and 150％ IC50 as the condition culture mediums,were used to induce U251 g and A172g.At the end,the IC50 of TMZ for U251g and A172g after induction were calculated.Methylation -specific PCR (MSP) and Western blot were used to detect gene promoter methylation and the corresponding protein expressions of hMLH1,hMSH2 of the GSC before and after induction.Results (1) The GSC line U251g and A172g were successfully isolated from the glioma cell line U251 and A172 respectively by suspension culture in the neural stem cell culture medium,which met the definition of cancer stem cells through the identification.(2) The IC50 of TMZ was 1 695.41 μmol / L for U251g,724.63 μmol for A172g,1 913 μmol / L for U251g-1,2 555 μmol / L for U251g-2,754 μmol / L for A172g -1,1 549 μmol / L for A172g-2.(3) After TMZ induction,hMLH1 gene promoter abnormal methylation and the corresponding protein deletion were detected in U251g and A172g,and methylation level promoted by the increase of drug concentration in A172g.hMSH2 gene promoter was found no methylation and protein expression deletion in U251g and A172g.Conclusions There were GSC in the glioma cell line U251 and A172,which had different levels of temozolomide-resistance.During the process of treatment with TMZ,the function of mismatch repair system was abnormalized in U251g and A172g,mainly hMLH1 gene promoter abnormal methylation and the corresponding protein deletion. Key words: Glioma stem cells; Mismatch repair gene; Temozolomide