Growth and Molecular Characteristics of Temozolomide-Resistant Human A172 and R1 Glioblastoma Cells

Abstract

Glioblastoma recurrence is caused by initial and acquired as a result of therapy resistance of tumor cells. Studies searching the markers that would allow predicting the level of glioblastoma cell resistance to therapy are in progress. The complexity of the problem is related to the high heterogeneity of individual tumors and the cellular content of each tumor. In present work, a comparative study of the influence of single temozolomide (in Temodal® form) ex-posure on the well-known glioblastoma cell line A172 and a new one R1 was performed. In A172 (highly tem-ozolomide-sensitive cell line) after treatment with 0.1 mM of this drug only individual cells persisted and resumed proliferation. In R1 glioblastoma cell line single cells survived and resumed proliferation after treatment with 1 mM temozolomide. The populations resulting from the proliferation of these cells were designated as resistant. The expression of MGMT, as well as genes responsible for resistance to chemotherapy and tumor progression (MGMT, ABCB1, ABCC1, ABCG2), growth factor genes (VEGF, HGF), cytokines IL-6 and IL-8, and their encoding genes was examined in resistant A172 and R1 cells. In A172 cells, the methylated status of MGMT gene promoter was confirmed, as well as the absence of the corresponding gene expression. It was shown for the first time that glioblastoma R1 is heterogeneous by the methylation status of MGMT gene promoter and expression of the relevant enzyme. In A172 and R1 resistant cell populations, the level of MGMT gene promoter methylation was lower than in the intact cells, and MGMT gene expression was enhanced. We suspect that this may be the reason for greater resistance of such cells to chemotherapy. The expression of most genes associated with resistance to chemotherapy and a more aggressive course of the disease, genes of growth factors, and interleukins in resistant A172 cells was higher than in intact cells. In contrast, in resistant R1 cells, the expression of most of the same genes (with the exception of ABCC1 and VEGF, for which the expression level changed insignificantly) was lower than in the intact cells. These results confirm the significance of MGMT in the formation of glioblastoma cell resistance to temozolomide. The prognostic value of the other studied parameters is still ambiguous.