舒尼替尼对EGFR T790M突变非小细胞肺癌H1975细胞增殖抑制作用及其机制

Antiproliferative effects and mechanism of sunitinib on EGFR T790M mutation in human non-small cell lung cancer cell line H1975

目的探讨舒尼替尼(sunitinib)对EGFR T790M突变的非小细胞肺癌H1975细胞的生长抑制作用及其可能的机制。方法应用MTT法检测sunitinib作用H1975细胞的增殖抑制作用;荧光染色法观察sunitinib诱导细胞凋亡形态学的变化;流式细胞术检测细胞周期的变化,Western blot分析Bcl-2蛋白水平的变化。结果在0.75～24μmol/L浓度范围内,sunitinib明显抑制H1975细胞增殖,且具有浓度和时间依赖性,24、48、72 h的半数抑制浓度(IC50)分别为(11.70±1.49)、(6.49±0.70)、(3.54±0.28)μmol/L。荧光显微镜观察发现sunitinib能够诱导细胞出现核固缩、染色质凝集等典型的凋亡形态学改变。流式细胞仪检测细胞周期显示,sunitinib阻滞H1975细胞于G0/G1期。Western blot结果显示sunitinib能够下调Bcl-2蛋白的表达水平。结论 sunitinib对EGFR T790M突变的H1975细胞具有抑制增殖、改变细胞周期分布和诱导细胞凋亡的作用。

Objective To research the inhibitory effects and mechanism of sunitinib on acquired epidermal growth factor receptor tyrosine kinase inhibitors （EGFR TKIs ）-resistant in human non-small cell lung cancer cell line H1975 with EGFR T790M mutation in vitro. Methods The inhibitory effects on H1975 cells exposed to sunitinib were evaluated by MTT method. The morphological changes of apoptosis induced by sunitinib were observed by fluorescence staining under the fluorescence microscope. Cell cycle was detected by flow cytometry. The Bcl-2 protein expression levels were measured by Western blot. Results The MTT assay showed sunitinib had significantly antiproliferative effects with dose-dependent and time-dependent at the concentration ranged from 0.75 to 24 μmol/L. The IC50 values of sunitinib on H1975 cells were （ 11.70 ± 1.49）, （6.49 ±0. 70） and （3.54±0.28 ）μmol/L at 24,48,72 h,respectively. Typical apoptotic morphologic changes were observed by fluorescence microscope, such as nuclear condensation and chromatin condensation. Flow cytometry assay showed that cell cycle was arrested at the Go/G1 phase on H1975 cells. Western blot assay indicated that sunitinib down-regulated the protein level of Bcl-2. Conclusion Sunitinib can exert inhibitory effects on acquired EGFR TKIs-resistant in human non-small cell lung cancer cell line H1975 with EGFR T790M mutation, and it also can change the distribution of cell cycle and induce apoptosis.