Effect And Mechanism Of Ionizing Radiation On Drug Resistance Caused By T790M Mutation In NSCLC Cell Lines

Background and PurposeAlthough epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) produce an initially dramatic response in lung cancer patients who harboring an active mutation in the EGFR gene, development of acquired resistance is almost inevitable. A secondary mutation of threonine790(T790M) is associated with half of the cases of acquired resistance approximataly. Currently, there are no approved therapies available for ues in the acquired resistance patients caused by T790M mutation. Radiotherapy is an important means for NSCLC. Theoretically, rapid eradication of radiotherapy would decrease the chance of drug resistance during effective systemic treatment At the same time, more and more retrospective analysis showed that:integration of radiotherapy during TKI treatment would decrease the drug resistance and prolong progression-tree survival (PFS) and overall survival (OS). This study investigated whether the addition of radiotherapy with gefitinib could overcome the drug resistance caused by T790M mutation.MethodsWe study the effects of the combination of radiotherapy and gefitinib in two NSCLC cell lines (including H3255had the L858R replacement in the exon21of the EGFR and H1975had L858R+T790M double mutations in the EGFR). The effects of two means on EGFR signaling, proliferation, apoptosis, contents of the T790M mutation were evaluated.Results1、Effect of T790M mutation on clonogenic survivalIn the clonogenic cell survival assay, the survival fractions of the HI975cell line were same with the H3255cell line,which were treated by the X-ray at different doses. The differences were not statistically significant (p>0.05). These results suggest that T790M mutation has no effect on the radiosensitivity of NSCLC cell lines.2、Drug tolerance of H1975and H3255cell lines on the MTT assayWe examined the drug tolerance effects of different treatment groups using the MTT assay in NSCLC cell lines H1975(L858R+T790M) and H3255(L858R). we observed that the H3255cell line was sensitive to treatment with gefitinib. By contrast, the H1975cell line was approximately85.9-flod less sensitive to gefitinib. The drug tolerance dropped to39.2times after X-ray irradiation at2.5doses. These results suggest that T790M mutation plays a great role in the drug tolerance. Even more important is that radiotherapy could decrease the chance of drug resistance caused by T790M mutation.3、The potential mechanism of reducing the resistance by radiotherapyThe real-time quantitative PCR results suggest that the T790M mutation decreased after radiotherapy.The cell morphology were irregular with more nuclear fragmentations and floating dead cells in the combination treatment group.The combination means resulted in a greater induction of apoptosis in EGFR TKI-resistant NSCLC cell line(H1975)(P<0.05); The expressions of phosphorylated epidermal growth factor receptor (P-EGFR), phosphorylated protein kinase B (P-AKT) were significantly decreased by combination of radiotherapy and gefitinib. ConclusionEGFR-mutation lung cancers are amenable to local-therapy, the addition of radiotherapy to EGFR-TKI is a promising strategy to overcome T790M-mediated drug resistance.