Combined Inhibition Of IGF-1R Andnf-κB Enhances The Effects Of Gefitinib In EGFR-TK Inhibitors Resistant Lung Cancer Cells Lines

Part One IGF-1R siRNA enhances the effects of gefitinib in H1650 and H1975: Non small cell lung cancer cell lines with acquried resistance to EGFR-TK inhibitorsPurpose:H1975 and H1650 are non small cell lung cancer (NSCLC) cells lines which display resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). We investigated the effect of silencing insulin like growth factor 1 receptor (IGF-1R) on the sensitivity of H1650 and H1975 to Gefitinib by siRNA considering that IGF1R signaling has been involved in the development of EGFR-TKIs acquired resistance in lung cancer.Methods:Three human NSCLC cell lines with EGFR mutations of HCC827, H1975 and H1650 were used for this part of experiment. Cell viability and proliferative activity were assessed by MTT. The expression of EGFR- and IGFR-related proteins was examined by western blots.Results:H1650 and H1975 cells were much more resistant to gefitinib than HCC827 cells, respectively. In spite of gefitinib treatment, phosphatase and tensin homolog loss and sustained phosphorylation of Akt were evident in H1650 and H1975 cells, but not in HCC827 cells. When the tumor cells were treated with gefitinib, IGF-1R phosphorylation, which was decreased in HCC827 cells, maintained in H1650 and H1975 cells. Combined treatment with IGF1R siRNA enhanced growth inhibition and apoptosis induced by gefitinib, and down-regulated phosphorylated Akt, EGFR and IGF1R.Conclusion:Combined inhibition of IGF1R signaling by si RNA enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the aquired resistance to EGFR-TKIs in lung cancer. Part Two Combined inhibition of IGF-1R and NF-κB enhances the effects of gefitinib in EGFR-TK inhibitors resistant lung cancer cells line H1650Purpose:We have previously demonstrated that IGF-1R siRNA is a crucial regulator in mediating gefitinib-induced cell death, which upregulates apoptosis-related molecules. Nevertheless, the clinical trials that evaluated the addition of IGF-1R inhibition to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in lung cancer have not demonstrated improvements in outcomes. There is increasing evidence suggests that NF-κB pathway is a determinant of sensitivity of non-small cell lung cancer (NSCLC) cells to EGFR-TKIs. Since the interactions of IGF-1R and NF-κB pathways were demonstrated in types of cells and tissues, we try to explore the effect of combined inhibition of NF-κB and IGF-1R on EGFR-TKIs resistant lung cancer cell line H1650.Methods:The expression of IGF-1R in H1650 was silenced using an IGF-1R specific siRNA. PDTC was taken as NF-κB specifitic inhibitor in this study. Cell viability was assessed by MTT. The apoptosis of cells was measured and analysed by flow cytometry. The expression and activity of EGFR-, IGFR-and NF-κB-related proteins were detected by western blots and EMSA.Results:We found that H1650, with resistance to Gefitinib exhibited a high level activity of both IGF-1R and NF-κB. The DNA binding activity of NF-κB in H1650 was partly reduced by the silencing of IGF-1R, suggesting the activation of NF-κB was not totally IGF-1R dependent in lung cancer. In addition, the silencing of IGF-1R expression together with NF-κB inhibition made the lung cancer cells more sensitive to gefitinib-mediated apoptosis than it did alone, suggesting the involvement of both IGF-1R and NF-κB pathways in gefitinib acquired resistance in H1650 cells.Conclusion:These results suggest that NF-κB is potentially involved in promoting gefitinib resistance even when the IGF-1R pathway was inhibited in lung cancer cell line H1650. Our findings also suggest that NF-κB activation was partially IGF-1R dependent, thus, combined inhibition of both NF-κB and IGF-1R may provide an effective way to overcome EGFR-TKIs resistance.