Investigation Of The Synergersitic Cytotoxicity And Mechanism Of Small Molecular Tyrosine Kinase Inhibitors AG825 Or AG1478 Combine With Cisplatin On NSCLC Cell Lines

Objective: The present study was conducted to observe HER2 and EGFR expression in 5 NSCLC cell lines A549, H322, H358, H460, H1299, and to investigate the relationship between HER2 expression in 5 NSCLC cell lines and cisplatin IC50. the antiproliferative effects AGainst 5 NSCLC cell lines was evaluated With specific inhibitors of the EGFR-associated tyrosine kinase AG825 and AG1478. We also showed that coadministration of AG825 or AG1478 will enhance the efficacy of cytotoxic AGent cisplatin. Moreover, we investigated the mechanism.Methods: (1) five NSCLC cell lines A549, H322, H460, H1299 were cultured in vitro. Western blot analyses were performed to detect the expression of HER2 and EGFR of the five NSCLC cell lines. (2) Growth inhibition of five NSCLC cell lines by AG825, AG1478, cisplatin paclitaxel wwre determined by MTT assay. (3) Synergistic effects of AG825 plus cisplatin or AG1478 plus cisplatin in five NSCLC cell lines were determined by MTT assay. (4) cultured H322, HI299 cells were divided into six groups : control, cisplatin, AG825, AG1478, AG825+cisplatin, AG1478+cisplatin, then treated for 96h. The contration of cisplatin, AG825, AG1478 were 0.1μm, 40μm,5μm, respectively. The effects of AG825, AG1478 combine cisplatin on the proliferation of H322, H1299 cell lines were determined by cell proliferation assay in vitro. (5) The effects of AG825, AG1478 combine cisplatin on the cell cycle of H322, HI299 cell lines were determined by flow cytometry. (6) The effects of AG825, AG1478 combine cisplatin on the apoptosisand necrosis of H322, HI299 cell lines were determined by using Annexin V-EGFP apoptosis kit. (7) The effects of AG825, AG1478 combine cisplatin on the expression of EGFR , phosphorylated ERK, total ERK of H322, HI299 cell lines were determined by Western blot analysis.Results: (1) The HER2 expression levels of five NSCLC cell lines were different. The cell line H322 was classified as the high HER2 expressor, and the cell lines H460, HI299 were classified as the low HER2 expressors. The EGFR expression levels of five NSCLC cell lines were different. The cell line A549, H322 were classified as the high EGFR expressors, and the cell lines H460 was classified as the low HER2 expressor. (2) HER2 specific small molecular tyrosine kinase inhibitor Tyrphostin AG825 had significant growth inhibitive effect on five NSCLC cell lines A549, H322, H358, H460, H1299, and this growth inhibitive effect was not correlated with the HER2 expression level. EGFR specific small molecular tyrosine kinase inhibitor Tyrphostin AG1478 had significant growth inhibitive effect on five NSCLC cell lines A549, H322, H358, H460, H1299, and this growth inhibitive effect was not correlated with the EGFR expression level. Paclitaxel had significant growth inhibitive effect on five NSCLC cell lines A549, H322, H358, H460, H1299 even at very low contration (0.1nm). The IC50 (half inhibition concentration) of cisplatin in five NSCLC cell lines A549, H322, H358, H460, H1299 was 7.31 μm, 16.29μm,12.88μm, 4.513μm,1.769nm, respectively. There was a significant correlation between the IC50 value of cisplatin and the level of HER2 expression in the panel of 5 NSCLC cell lines.(P<0.01). (3) AG825 combine cisplatin had Synergistic growth inhibitive effects on 5 NSCLC cell lines A549, H322, H358, H460, HI299, especially at low cisplatin concentration, and this synergistic growth inhibitive effect was not correlated with the HER2 expression level. AG825 plus cisplatin had the most potential synergistic growthcorrelated with the IC50 value of cisplatin. This finding indicate that overexpression of HER2 is a marker for intrinsic cisplatin resistance in NSCLC cell lines. (2) HER2 specific small molecular tyrosine kinase inhibitor Tyrphostin AG825 had significant growth inhibitive effect on five NSCLC cell lines, and this growth inhibitive effect was not correlated with the HER2 expression level. EGFR specific small molecular tyrosine kinase inhibitor Tyrphostin AG1478 had significant growth inhibitive effect on five NSCLC...