瑞香狼毒提取液逆转EGFR-TKI耐药肺腺癌H1975细胞的作用及其机制

Reversal effect of extracts from stellera chamaejasme L on EGFR-TKI resistance in lung adenocarcinoma H1975 cells and its mechanism

目的:探讨瑞香狼毒(stellera chamaejasme L,SCL)乙醇提取液逆转表皮生长因子酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors,EGFR-TKI)耐药肺腺癌H1975细胞的作用及其机制。方法:用MTT法检测SCL、吉非替尼(gefitinib)及两药联用对H1975细胞的抑制率,细胞划痕实验检测药物对H1975细胞迁移的影响,流式细胞术测定药物对细胞凋亡的影响,Western blotting检测不同药物处理后各组肿瘤细胞凋亡相关蛋白Bcl-2及细胞通路关键分子p-EGFR的表达。观察SCL联用gefitinib后裸鼠移植瘤的体积和总生存期(OS),ELISA检测荷瘤裸鼠血清中Bcl-2、p-EGFR含量。结果 :SCL对H1975细胞IC50为(21.35±2.11)mg/ml,gefitinib的IC50为(11.21±1.68)μmol/L。SCL联用gefitinib后H1975细胞抑制率明显高于gefitinib(1μmol/L)和SCL(5 mg/ml)两组单独应用[(49.78±7.09)%vs(8.45±2.57%)、(12.88±3.64)%,均P<0.01],两药联用H1975细胞迁移指数明显低于gefitinib和SCL两组单独应用[(22.4±6.5)%vs(70.3±4.9)%、(67.1±10.5),均P<0.01],流式细胞仪检测显示两药联用细胞凋亡率明显高于两者单用[(51.68±6.56)%vs(9.88±2.71)%、(9.48±2.45)%,均P<0.01],Western blotting检测H1975细胞凋亡相关蛋白发现,联用组可明显降低Bcl-2和p-EGFR的表达(P<0.01)。抑制移植瘤实验显示,联用药组荷瘤小鼠肿瘤生长缓慢,OS明显延长(P<0.01)。结论:SCL可逆转肺腺癌H1975细胞对EGFR-TKI的耐药,其机制可能与降低EGFR的磷酸化、下调抗凋亡蛋白Bcl-2表达有关,从而为EGFR-TKI耐药肺腺癌治疗提供了新思路。

Objective： To investigate the reversal effects of the extracts from stellera chamaejasme L （SCL） on EG- FR-TKI （epidermal growth factor receptor tyrosine kinase inhibitors） resistance in lung adenocarcinoma H1975 cells, and to explore the underlying mechanism. Methods： The inhibitory effects of SCL, gefitinib, and SCL plus ge- fitinib on the proliferation of H1975 cells were evaluated by MTT assay; the effects on apoptosis of H1975 cells were determined by flow cytometry （ FACS）; Wound healing assay was performed to examine cell migration; and Western blotting was conducted to detect the expression of p-EGFR and apoptosis-related protein Bcl-2. Nude mice xenograft model was constructed, and the volume of xenografts as well as the overall survival （OS） was observed after treatment with SCL plus gefitinib. Moreover, ELISA was carried out to detect the serum levels of Bcl-2 and p-EGFR. Results： ICs0 of SCL and gefitinib on H1975 cells was （21.35±2.11 ） mg/ml and （11.21±1.68） μmol/L, re- spectively. Compared with single treatment of SCL（5 mg/ml） or Gefitnib（1 μmol/L）, combined treatment of SCL and gefitnib significantly inhibited the proliferation rate of H1975 cells （[49.78±7.09]% vs [12.88±3.641%, [8.45±2.57]%; all P〈0.01）, and inhibited the migration ofH1975 cells （[22.4±6.5]% vs [70.3±4.9]%, [67.1±10.51%; all P〈0.01）. FACS showed that the apoptosis rate in combined treatment group was obviously higher than that in single treatment group （[51.68±6.561% vs [9.88±2.711%, [9.48±2.45]%; all P〈0.01）. Western blotting showed the protein expressions of Bcl-2 and p-EGFR in H1975 cells were remarkably decreased after the combined treatment of SCL and gefitinib （P〈0.01）. In vivo xenograft experiment showed that combined treatment of SCL and gefitinib signifi- cantly inhibited progressive tumor growth and promoted the survival time compared with single treatment of SCL or gefitinib （all P〈0.01）. Conclusion： SCL might reverse the EGFR-TKI resistance in H1975 cells. The potential mech- anism might be related to phosphorylation of EGFR and down-regulation of Bcl-2 expression, which might provide new thoughts of treating lung adenocarcinoma.