目的 研究替莫唑胺(TMZ)联合zeste基因增强子类同源物2(EZH2)抑制药GSK343对GH3细胞凋亡和侵袭的作用及机制.方法 使用不同浓度的TMZ处理细胞筛选TMZ处理剂量,并将GH3细胞随机分为空白组、TMZ低剂量组(200 µmol·L-1 TMZ)、TMZ 高剂量组(400µmol·L-1 TMZ)、GSK343 组(20 μmol·L-1 GSK343)、TMZ+GSK343 组(400 μmol·L-1TMZ 和 20 μmol·L 1 GSK343).药物处理48 h后用细胞计数试剂盒-8(CCK-8)实验检测细胞存活率,以原位末端标记(TUNEL)法和流式细胞术检测细胞凋亡情况,以Transwell实验检测细胞侵袭能力,以蛋白质印迹法检测细胞相关蛋白表达.结果 空白组、TMZ低剂量组、TMZ高剂量组、GSK343组、TMZ+GSK343组TUNEL阳性细胞率分别 为(4.31±0.71)％、(15.36±0.91)％、(22.26±2.13)％、(13.05±0.71)％和(34.55±3.75)％;细胞侵袭数分别为(247.67±27.23)、(183.00±20.66)、(152.11±8.82)、(182.89±18.24)和(116.11±12.73)个;B细胞淋巴瘤-2相关X蛋白(Bax)蛋白表达水平分别为0.44±0.05、0.58±0.06、0.81±0.07、0.66±0.06和1.03±0.06;上皮细胞钙黏蛋白(E-cadherin)表达水平分别为 0.33±0.05、0.57±0.05、0.84±0.12、0.59±0.07和1.00±0.12;波形蛋白(Vimentin)表达水平分别为 0.91±0.14、0.72±0.09、0.62±0.07、0.77±0.08和0.47±0.04;磷酸化磷脂酰化醇3-激酶(p-PI3K)蛋白表达水平分别为0.99±0.11、0.86±0.06、0.68±0.07、0.72±0.08 和 0.52±0.08;磷酸化的蛋白质激酶 B(p-AKT)蛋白表达水平分别为 1.01±0.06、0.71±0.07、0.57±0.05、0.80±0.07 和 0.43±0.04.TMZ 低高剂量组、TMZ 高剂量组、GSK343 组、TMZ+GSK343组上述指标分别与空白组比较,在统计学上差异均有统计学意义(均P＜0.05);TMZ+GSK343组上述指标分别与TMZ低、高剂量组和GSK343组比较,在统计学上差异均有统计学意义(均P＜0.05).结论 TMZ联合EZH2抑制药GSK343可显著抑制垂体瘤GH3细胞侵袭并诱导凋亡,这可能与调控PI3K/AKT通路有关.
Objective To investigate the effect of temozolomide(TMZ)combined with histone methyltransferase enhancer of Zeke 2(EZH2)inhibitor GSK343 on apoptosis and invasion of GH3 cells and its mechanism.Methods Different concentrations of TMZ treated cells were used to screen TMZ treated dose.GH3 cells were randomly divided into blank group,TMZ-L group(200 μmol·L-1 TMZ),TMZ-H group(400 μmol·L-1 TMZ),GSK343 group(20 μmol·L-1 GSK343)and TMZ+GSK343 group(400 μmol·L-1+20 μmol·L-1 GSK343).After 48 h of drug treatment,cell counting kit-8(CCK-8)assay was used to detect cell survival rate;terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)and flow cytometry were used to detect cell apoptosis;Transwell assay was used to detect cell invasion ability;Western blot assay was used to detect cell-related protein expression.Results TUNEL positive cell rates in blank group,TMZ-L group,TMZ-H group,GSK343 group and TMZ+GSK343 group were(4.31±0.71)％,(15.36±0.91)％,(22.26±2.13)％,(13.05±0.71)％and(34.55±3.75)％;cell invasion numbers were(247.67±27.23),(183.00±20.66),(152.11±8.82),(182.89±18.24)and(116.11±12.73)cells;the expression levels of Bax protein were 0.44±0.05,0.58±0.06,0.81±0.07,0.66±0.06 and 1.03±0.06;the expression of E-cadherin protein were 0.33±0.05,0.57±0.05,0.84±0.12,0.59±0.07 and 1.00±0.12;Vimentin protein expression levels were 0.91±0.14,0.72±0.09,0.62±0.07,0.77±0.08 and 0.47±0.04;phosphorylated phosphatidyl alcohol 3-kinase(p-PI3K)protein expression levels were 0.99±0.11,0.86±0.06,0.68±0.07,0.72±0.08 and 0.52±0.08;phosphorylated protein kinase B(p-AKT)protein expression levels were 1.01±0.06,0.71±0.07,0.57±0.05,0.80±0.07 and 0.43±0.04.TMZ-L group,TMZ-H group,GSK343 group and TMZ+GSK343 group had significant differences in the above indexes compared with blank group(all P＜0.05);TMZ+GSK343 group was compared with TMZ-L group TM2-H group or GSK343 group,and the above indexes were significantly difference(all P＜0.05).Conclusion TMZ combined with EZH2 inhibitor GSK343 can significantly inhibit GH3 cell invasion and induce apoptosis,which may be related to the regulation of PI3 K/AKT pathway.
张俊;路长宇;付佳;王静;王斌;梁剑峰
北京大学国际医院神经外科,北京 102206
药学
替莫唑胺;zeste基因增强子类同源物2;垂体瘤;细胞侵袭;细胞凋亡;上皮间质转化
temozolomide;histone methyltransferase enhancer of Zeke 2;pituitary tumor;cell invasion;cell apoptosis;epithelial mesenchymal transformation
《中国临床药理学杂志》 2024 (008)
1150-1154 / 5
吴阶平医学基金会临床科研专项基金资助项目(320.6750.2023-11-7)
10.13699/j.cnki.1001-6821.2024.08.013
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