中国全科医学2017，Vol.20Issue(9)：1076-1083,8.DOI:10.3969/j.issn.1007-9572.2017.09.011
肝细胞生长因子诱导敏感非小细胞肺癌细胞对厄洛替尼耐药及机制研究
Mechanism of Erlotinib Resistance Induced by Hepatocyte Growth Factor in Sensitive Non - small - cell Lung Cancer Cells in Vivo
摘要
Abstract
Objective To study the mechanism of erlotinib resistance induced by hepatocyte growth factor (HGF)in non - small - cell lung cancer (NSCLC)cells and to investigate whether c - Met and its downstream signaling pathway proteins participate in the HGF - induced erlotinib resistance of NSCLC cells with different genetypes in vivo. Methods This study was conducted from January 2014 to January 2015. We selected NSCLC cell lines with different EGFR genes (PC - 9:EGFR -mutation type,sensitive;H292:EGFR - wild type,sensitive)and human embryonic lung fibroblasts MRC - 5. The HGF level in cell culture supernatants secreted by PC - 9,H292 and MRC - 5 cells was quantified by ELISA. PC - 9 and H292 cells were induced by MRC - 5 cell culture supernatant;the expressions of c - Met and its downstream signaling pathway proteins were examined by Western blotting. Fifty - six female and SPF BALB/ c nude mice were randomly divided into 8 groups,with each group containing 7. In building the PC - 9 cell - induced model,the control group (group C)and erlotinib treatment group (group E)were subcutaneously inoculated PC - 9 cell suspension and MRC - 5 induced group (group H),MRC - 5 and erlotinib treatment group (group HE)were inoculated PC - 9 + MRC - 5 cell suspension subcutaneously. In establishing the H292 cell - induced model,group C and group E were inoculated H292 cell suspension subcutaneously while group H and group HE were inoculated H292 + MRC - 5 cell suspension subcutaneously. When the tumor diameter reached 4 mm in all groups,group C and group H were lavaged with 0. 9% sodium chloride solution while group E and group HE were lavaged with erlotinib, respectively,in both built models. Mice were euthanized at after dosing. Comparisons of the weight of transplanted tumor were made between all groups in models induced by PC - 9 and H292 cells. The expressions of c - Met and its downstream signaling pathway proteins in transplanted tumor tissues in nude mice were determined via immunohistochemistry. Results The concentration of HGF was not detected in PC - 9 and H292 cell culture supernatants,while that in the supernatant of MRC - 5 cells was (1262 ± 90)pg/ ml. Western blotting results showed that HGF in the MRC - 5 cell culture supernatant could stimulate the expression of p - Met,p - Akt,p - Stat3 and phosphorylated extracellular regulated protein kinase 1 / 2 (p - Erk1 / 2)in PC- 9 and H292 cells. The weight of transplanted tumor in group E was less than that in group C (P < 0. 05)in PC - 9 cell -induced model;the weight of transplanted tumor in group HE was less than that in group H,but was greater than that in group E (P < 0. 05)in PC - 9 cell - induced model. The weight of transplanted tumor in group E was less than in group C (P < 0. 05)in H292 cell - induced model;the weight of transplanted tumor in group HE was less than that in group H,whereas it was greater than that in group E (P < 0. 05)in H292 cell - induced model. c - Met and p - Met were localized in the cell membrane and cytoplasm,respectively. In both models,expressions of c - Met did not differ significantly between groups of C,H,E,and HE (P > 0. 05);group H had higher expressions of p - Met than group C (P < 0. 05),and group HE had higher expressions of p -Met than group E (P < 0. 05). Stat3 was localized in cytoplasm and p - Stat3 in nucleus. In both models,no distinct differences in the expressions of Stat3 were noted between groups of C,H,E and HE (P > 0. 05);group H had higher expressions of Stat3 than group C (P < 0. 05),and group HE showed higher expressions of Stat3 than group E (P < 0. 05). Akt and p - Akt were located in cytoplasm. In both models,the differences in the expressions of Akt were not prominent between groups of C,H,E and HE (P > 0. 05);expressions of p - Akt in group H were higher than those in group C (P < 0. 05);expressions of p - Akt were found to be higher in group HE than in group E (P < 0. 05). Erk1 / 2 was localized in cytoplasm and p - Erk1 / 2 was localized in nucleus. In both models,expressions of Erk1 / 2 demonstrated no significant differences between groups of C,H,E and HE (P > 0. 05);the expressions of p - Erk1 / 2 in H group were higher than those in group C (P < 0. 05);expressions of p- Erk1 / 2 in group HE were higher than those in group E (P < 0. 05). Conclusion HGF secreted by MRC - 5 cell induces Erlotinib resistance in PC - 9 and H292 cells in sensitive NSCLC cells. The HGF - induced activation of c - Met and its downstream signaling pathway proteins phosphorylation may be an important mechanism of Erlotinib resistance in sensitive NSCLC cells with different genetypes.关键词
癌,非小细胞肺/肝细胞生长因子/抗药性,肿瘤Key words
Carcinoma/non - small - cell lung/Hepatocyte growth factor/Drug resistance/neoplasm分类
医药卫生引用本文复制引用
崔青松,朴红梅,崔晶刚,王富佳,安昌善..肝细胞生长因子诱导敏感非小细胞肺癌细胞对厄洛替尼耐药及机制研究[J].中国全科医学,2017,20(9):1076-1083,8.基金项目
国家自然科学基金资助项目(81160291) （81160291）
