Eicosapentaenoic acid enhances sensitivity of glioma cell line U87 to temozolomide via inhibiting MGMT expression
10.3760/cma.j.issn.1671-8925.2018.09.003
- VernacularTitle:二十碳五烯酸通过抑制MGMT表达提高胶质瘤U87细胞对替莫唑胺的化疗敏感性
- Author:
Tianzao HUANG
1
;
Xiangrong CHEN
;
Chubin LIU
;
Yanlei CAO
;
Weipeng HU
Author Information
1. 福建医科大学附属第二医院神经外科
- Keywords:
Glioma;
Eicosapentaenoic acid;
Temozolomide;
O-6-methlguanine-DNA methyltransferase;
Nuclear factor-κB
- From:
Chinese Journal of Neuromedicine
2018;17(9):879-885
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the effect ofeicosapentaenoic acid (EPA) on sensitivity of glioma cell line U87 to temozolomide (TMZ) and its underlying mechanism.Methods (1) U87 cells were routinely cultured in vitro,and 0,25,50,100 and 200 μmol/L EPA was given to these cells for 12,24,and 48 h;MTT assay was used to detect the cell viability.(2) U87 cells were randomly divided into control group,EPA group,TMZ group and EPA+TMZ group (concentrations of EPA and TMZ were 25 and 100 mol/L;EPA pretreatment for 12,24 and 48 h was given;TMZ was given for 24 h);MTT assay was used to evaluate the inhibition ratio of cell proliferation.(3) U87 cells were randomly divided into control group Ⅰ,EPA group Ⅰ,TMZ group Ⅰ and EPA+TMZ group Ⅰ (concentrations of EPA and TMZ were 25 and 100 mol/L;EPA pretreatment for 6 h was given;TMZ was given for 24 h);the apoptotic ratio was examined by fiow eytometry (FCM);Western blotting was used to detect the protein expressions ofcleavedcaspase-3,Bax,O-6-methlguanine-DNAmethyltransferase (MGMT),nuclear factor (NF)-κB signaling pathway related protein p65,and NF-κB inhibitor IKBα;immunofluorescent staining was employed to detect the MGMT and NF-κB p65 expressions;methylated specific PCR (MSP)was used to detect the MGMT gene promoter methylation.Results (1) The 50,100 and 200 μmol/L EPA caused concentration-dependent and time-dependent proliferation inhibition of U87 cells.(2) The inhibition ratio of cell proliferation in EPA+TMZ group was significantly higher as compared with that in the TMZ group (P＜0.05).(3) As compared with that in the TMZ group Ⅰ (34.58％±4.35％),the apoptotic ratio of U87 cells in the EPA+TMZ group Ⅰ was significantly increased (53.28％±5.05％,P＜0.05);Western blotting showed that as compared with those in TMZ group Ⅰ,the protein expressions of activated caspase-3,Bax and IKBα were significantly increased,and MGMT and NF-κB p65 protein expressions were significantly decreased in EPA+TMZ group Ⅰ (P＜0.05);immunofluorescent staining indicated that the MGMT and NF-κB p65 protein expressions in EPA+TMZ group Ⅰ were significantly lower than those in TMZ group Ⅰ (P＜0.05);the MGMT gene promoter methylaion in EPA+TMZ group Ⅰ was higher than that in TMZ group Ⅰ.Conclusion EPA enhances the sensitivity of glioma cell line U87 to TMZ,which may inhibit the MGMT expression by NF-κB dependent pathway.
