Brusatol去羟甲基化修饰抑制IDH1-α-KG-TET1-Nrf2信号通路逆转Ⅰ型子宫内膜癌孕激素耐药研究

Brusatol inhibits IDH1-α-KG-TET1-Nrf2 signaling pathway to reverse progesterone resistance in type Ⅰ endometrial carcinoma through demethylation modification

目的探讨中草药提取物鸦胆子苦醇Brusatol能否通过去羟甲基化修饰抑制异柠檬酸脱氢酶1(IDH1)-α-酮戊二酸(α-KG)-TET1-核转录因子E2相关因子(Nrf2)信号通路逆转I型子宫内膜癌孕激素耐药。方法将20只裸鼠随机分为4组,每组5只,采用人子宫内膜癌细胞系Ishikawa细胞建立裸鼠皮下移植瘤模型,分为空白对照组、Brusatol组、醋酸甲羟孕酮(MPA)组、Brusatol+MPA组,分别经腹腔注射0.1ml 0.9%氯化钠注射液、2 mg/kg Brusatol、100 mg/kg MPA、2 mg/kg Brusatol+100 mg/kg MPA,干预3次/周,共9次。用药干预3周后,处死成瘤裸鼠,取移植瘤测量肿瘤体积,计算肿瘤抑制率。采用Western blot法检测各组移植瘤组织IDH1-α-KG-TET1-Nrf2信号通路相关蛋白表达水平;采用CCK-8法检测各组Ishikawa细胞增殖能力;采用Dot blot法检测各组Ishikawa细胞TET1羟甲基化水平。结果Brusatol组、MPA组、Brusatol+MPA组裸鼠肿瘤抑制率分别为(30.24±1.52)%、(50.37±2.36)%、(72.56±3.82)%,Brusatol+MPA组>MPA组>Brusatol组>空白对照组(P<0.05)。Brusatol+MPA组移植瘤组织IDH1、α-KG、TET1、Nrf2蛋白表达水平均低于MPA组、Brusatol组、空白对照组(均P<0.05);MPA组、Brusatol组亦低于空白对照组(均P<0.05);MPA组与Brusatol组比较差异无统计学意义(均P>0.05)。Brusa-tol+MPA组Ishikawa细胞增殖能力均低于MPA组、Brusatol组、空白对照组(均P<0.05);MPA组、Brusatol组细胞增殖能力亦弱于空白对照组(均P<0.05);MPA组细胞增殖能力弱于Brusatol组(P<0.05)。Brusatol+MPA组Ishikawa细胞TET1羟甲基化水平均低于MPA组、Brusatol组、空白对照(均P<0.05);MPA组、Brusatol组TET1羟甲基化水平低于空白对照组(均P<0.05);MPA组TET1羟甲基化水平低于Brusatol组(P<0.05)。结论Brusatol能逆转I型子宫内膜癌的孕激素耐药性,其逆转机制可能与通过去羟甲基化修饰抑制IDH1-α-KG-TET1-Nrf2信号通路相关蛋白的表达有关。

Objective To investigate the effect of Brusatol on progesterone resistance of type I endometrial carcinoma and its mechanism.Methods Human endometrial carcinoma Ishikawa cells were inoculated subcutaneously in 20 nude mice to establish transplanted tumor model.The tumor-bearing nude mice were randomly divided into 4 groups with 5 mice in each group.The mice were injected intraperitoneally with 0.1 ml 0.9%sodium chloride(blank control group),2 mg/kg Bru-satol(Brusatol group),100 mg/kg medroxyprogesterone acetate(MPA)(MPA group)or 2 mg/kg Brusatol+100 mg/kg MPA(Brusatol+MPA group),3/wk for 3 wks,respectively.Then the tumor-bearing nude mice were sacrificed,the tumor volume was measured,and the tumor inhibition rate was calculated.Western blot was used to detect the expression of IDH1-α-KG-TET1-Nrf2 signaling pathway related proteins in endometrial cancer cells;CCK 8 method was used to detect the cell proliferation;DOT blot was used to analyze the TET1 hydroxymethylation levels of Ishikawa cells.Results The tumor inhi-bition rates of Brusatol group,MPA group and Brusatol+MPA group were(30.24±1.52)%,(50.37±2.36)%,(72.56±3.82)%,respectively.Compared to blank control group,the Brusatol+MPA group had the highest tumor inhibition rate,fol-lowed by MPA group and Brusatol group(P<0.05).The protein expression levels of IDH1,α-KG,TET1 and Nrf2 in Brusatol+MPA group were lower than those in MPA group,Brusatol group and blank control group(all P<0.05);MPA group and Bru-satol group were lower than those in the blank control group(all P<0.05);while there was no significant difference between MPA group and Brusatol group(P>0.05).The proliferation of Ishikawa cells in Brusatol+MPA group was lower than that in MPA group,Brusatol group and Blank control group(all P<0.05);cell proliferation in MPA group and Brusatol group was lower than that in the blank control group(all P<0.05);and the cell proliferation in MPA group was lower than that in Brusa-tol group(all P<0.05).TET1 hydroxymethylation level of Ishikawa cells in Brusatol+MPA group was lower than that in MPA group,Brusatol group and Blank control group(all P<0.05);the hydroxymethylation level in MPA group and Brusatol group was lower than that in the blank control group(all P<0.05);and the TET1 hydroxymethylation level in MPA group was lower than that in Brusatol group(all P<0.05).Conclusion Brusatol can reverse progesterone resistance in type I endometrial cancer,which may be related to the inhibition of IDH1-α-KG-TET1-Nrf2 signaling pathway related proteins through de-methylation modification.