ABSTRACT
Objective To explore the role of integrin β1 and relevant signaling pathway in acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib in non-small cell lung cancer (NSCLC). Methods Human lung adenocarcinoma cell line PC-9 and gefitinib-resistant PC-9/G cell lines were used in the present study. Western blotting analysis was used to examine the expression of integrin β1, Akt and phospho-Akt protein. The inhibitory effects of gefitinib and/or phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and extracellular regulated protein kinases (ERK) inhibitor PD98059 on cellular proliferation were tested by MTT assay. Cell apoptosis was analyzed by Annexin V/PI and TUNEL method. Results Overexpression of integrin β1 was observed in PC-9/G cell line. Silencing integrin β1 by RNAi method inhibited the proliferation and promoted apoptosis of PC-9/G cells. The inhibitory effect of gefitinib against Akt phosphorylation in PC-9/G cells was weaker than that in PC-9 cells. Knockdown of integrin β1 with RNAi decreased the phosphorylation level of Akt. ERK inhibitor PD98059 failed to restore the sensitivity of PC-9/G cells to gefitinib. PI3K inhibitor LY294002 could restore the sensitivity of PC-9/G cells to gefitinib. Conclusion It is suggested that overexpressed integrin β1 can activate the downstream signaling pathways through PI3K, which may be an important mechanism for resistance to EGFR-TKIs.
ABSTRACT
<p><b>OBJECTIVE</b>To explore the sensitivity of tumor cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs and provide preclinical basis of available chemotherapy regimens after failure of molecular targeted therapy.</p><p><b>METHODS</b>Human lung adenocarcinoma cell lines PC9 and PC9/G with acquired resistance to gefitinib were cultured in vitro. The sensitivity to chemotherapeutic drugs and inhibition rate of cell proliferation was determined by MTT assay. Effects of drugs on apoptosis and expression of P-170 were determined by flow cytometry. Difference of gene expression profile between PC9 and PC9/G cells was analyzed by DNA microarray. Western blot was used to test the expression of Akt, phospho-Akt and integrin beta1.</p><p><b>RESULTS</b>The resistance index of PC9/G cells to cisplatin was about 5.4-fold compared with that of PC9 cells. LY294002 may significantly elevate the sensitivity of PC9/G cells to cisplatin (P < 0.05). PC9/G cells were more sensitive to docetaxel than PC9 cells. No significant difference of sensitivity to pemetrexed was found between these two cell lines. Expression level of P-170 in PC9/G cells was lower than that in PC9 cells. In PC9/G cells, the expression of integrin beta1 and DNA healing gene was high and expression of gene during mitosis was low. The level of expression of Akt, phospho-Akt and integrin beta1 in PC9/G cells was higher than that in PC9 cells.</p><p><b>CONCLUSION</b>In PC9/G cells, a cell line with acquired resistance to gefitinib, over-expression of PI3K, integrin and DNA restoration gene and continuous activation of PI3K is found to be correlated with resistance to cisplatin. Docetaxel or pemetrexed is a more reasonable choice than cisplatin for treatment of NSCLC patients who failed to respond to EGFR-TKI.</p>