Effect of lenalidomide combined with temozolomide on drug resistance and O6-methylguanine-DNA methyltransferase gene epigenetic modification of U251/TR cells
10.3760/cma.j.cn115354-20210423-00261
- VernacularTitle:来那度胺联合替莫唑胺对U251/TR细胞耐药性及 MGMT基因表观遗传修饰的影响
- Author:
Qiang PAN
1
;
Lin ZHU
;
Xiao YUE
;
Yong GAO
;
Hua HUANG
;
Zongfei JIANG
;
Yunfeng MA
;
Chunyu SONG
Author Information
1. 山东第一医科大学附属济南人民医院神经外科，济南市神经肿瘤分子生物学重点实验室 271199
- Keywords:
Glioblastoma;
Lenalidomide;
Temozolomide;
Drug-resistance;
O6-methylguanine-DNA methyltransferase
- From:
Chinese Journal of Neuromedicine
2021;20(9):865-872
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of lenalidomide (LEN) combined with temozolomide (TMZ) on proliferation, invasion, drug resistance and O6-methylguanine-DNA methyltransferase ( MGMT) gene epigenetic modification of TMZ-resistant human glioblastoma cell line U251/TR. Methods:A TMZ-resistant human glioma cell line, U251/TR, was successfully established by stepwise exposure of U251 parental cells to TMZ. U251/TR cells were divided into dimethyl sulfoxide (DMSO) group, LEN group, TMZ group and LEN+TMZ group (DMSO group: without any drug intervention; LEN group, TMZ group, and LEN+TMZ group were pretreated with 100 μmol/L LEN, 200 μmol/L TMZ, 100 μmol/L LEN+200 μmol/L TMZ, respectively; the drugs were administered once every 24 h). The proliferation rate of these cells in each group was detected by sulfonylrhodamine B colorimetric assay at different time points (24, 48, 72, and 96 h after treatment). At 96 h after treatment, the invasion and migration abilities of cells in each group were detected by Transwell assay; the proliferation cycle of cells in each group was detected by flow cytometry; Western blotting, immunohistochemical staining and immunofluorescence staining were used to detect the MGMT protein expression, and the MGMT mRNA expression in cells of each group was detected by reverse transcription-PCR; methylation specific PCR was used to detect the MGMT gene promoter methylation in each group of cells. Results:The cell proliferation rate of LEN+TMZ group was significantly decreased as compared with TMZ, LEN, and DMSO groups at 24, 48, 72 and 96 h after treatment ( P<0.05). At 96 h after treatment, LEN+TMZ group had significantly decreased number of transmembrane cells, and significantly increased ratio of cells at G0/G1 phase as compared with the other 3 groups ( P<0.05); the MGMT protein and mRNA expression levels in TMZ group and LEN+TMZ group were significantly lower than those in LEN group and DMSO group ( P<0.05); and the number of cells with strong or moderate MGMT expression in TMZ group and LEN+TMZ group was obviously less than that in LEN group and DMSO group, and the MGMT fluorescence intensity in TMZ group and LEN+TMZ group (+) was obviously lower than that in LEN group (+++) and DMSO group (+++). The MGMT gene promoter was unmethylated in all groups. Conclusion:LEN alone does not obviously inhibit the proliferation and invasion of U251/TR cells; but LEN combined with TMZ could inhibit the proliferation and invasion of U251/TR cells and co-reverse the drug resistance of U251/TR cells, whose mechanism is not related to the changes of MGMT gene promoter methylation.
