Kang Xiao-hong, Wang Li-fang, Cao Fei, Fan Fang-tian, Xu Zhen-ye
Department of Oncology, Longhua Hospital Affiliated to Shanghai University of TCM, Shanghai 200032, China.
Department of Oncology, Longhua Hospital Affiliated to Shanghai University of TCM, Shanghai 200032, China. Email:
Zhonghua Zhong Liu Za Zhi. 2013 Oct;35(10):732-6.
To observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.
The effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.
The MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.
HGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.
观察肿瘤微环境来源的肝细胞生长因子（HGF）和/或阿法替尼对人肺腺癌H1975细胞生长的影响，并探讨HGF诱导对阿法替尼原发性耐药的潜在机制。
采用MTT法评估HGF、转化生长因子-α（TGF-α）和阿法替尼对H1975细胞生长的影响。通过ELISA法测定正常人类胎儿肺成纤维细胞MRC-5细胞和人肺腺癌H1975细胞共培养或单独培养时的HGF浓度。采用蛋白质免疫印迹法检测用HGF和/或阿法替尼处理的H1975细胞中表皮生长因子受体（EGFR）和间质-上皮转化因子（Met）信号通路相关蛋白以及上皮-间质转化（EMT）标志物的表达。
MTT法显示，在有HGF存在时，H1975细胞对阿法替尼低敏。ELISA法显示，H1975细胞产生的HGF低于0.1 ng/2.0×10⁶个细胞，但MRC-5细胞培养48小时产生的HGF为（151.37±2.07）ng/2.0×10⁶个细胞。当H1975细胞与MRC-5细胞共培养72小时时，培养上清液中HGF浓度为（61.13±16.21）ng/ml。在有HGF存在时，H1975细胞中磷酸化Met（p-Met）、磷酸化蛋白激酶B（p-Akt）和磷酸化细胞外信号调节激酶（p-ERK）蛋白的表达明显上调。阿法替尼抑制磷酸化EGFR（p-EGFR），但不影响p-Met、p-Akt和p-ERK蛋白的表达。在有阿法替尼存在时，HGF上调波形蛋白的表达并下调E-钙黏蛋白的表达。
肿瘤微环境中基质细胞分泌的HGF可能通过激活Met/磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/Akt）和Met/丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK）信号通路，赋予H1975细胞对阿法替尼的耐药性，并参与上皮-间质转化过程。
