Study Of Synergism Between Cisplatin And Gefitinib In Gefitinib Resistant Non-small Cell Lung Cancer

Background:The incidence and mortality of lung cancer are the most in malignant tumors in the world, and the non-small cell lung cancer (NSCLC) accounts for about 80% of the total number of lung cancer. The platinum based chemotherapy is the mainly applied in the treatment of advanced NSCLC, however, the advent of EGFR TKI has dramatically influenced the therapy strategies for lung cancer and greatly improves the survival of patients with EGFR sensitive mutations. But acquired drug resistance is the most difficult problem needs to be overcome after EGFR-TKI treatment, in addition, some patients with EGFR wild type also could benefit from EGFR-TKI therapy. Therefore, for the patients with EGFR-TKI resistance and EGFR wild type, whether combining platinum based chemotherapy with EGFR-TKI can enhance the sensitivity of EGFR-TKI or chemotherapy to prolong the survival of those patients, clinical trials shows opposite conclusions. Although, large clinical studies have suggested that EGFR-TKI combined with platinum based chemotherapy does not exist synergism, however, some reports indicated that some patients with EGFR-TKI resistance could benefit from the combination therapy of EGFR-TKI and platinum based chemotherapy, which indicated that the synergistic effect of combination of EGFR-TKI and platinum based chemotherapy is selective. There is a part of sensitive populations, which we are unable to target accurately due to the shortage of current molecular typing. The mechanism of synergistic between platinum based chemotherapy and EGFR-TKI is poorly understood, especially, the mechanism of interaction of cisplatin which is the core drug of chemotherapy and EGFR-TKI. Therefore, the combined effects and the mechanism of interaction between gefitinib and cisplatin will be studied in EGFR-TKI resistant NSCLC.Methods and results:We selected three NSCLC cell lines (H1299, A549 and HI975) as the study models, PCR amplification and gene sequencing were used to obtain the sequence of exons 18-21 of EGFR and exons 2-3 of K-ras. We found that HI299 cells contained the wild-type EGFR and K-Ras genes, A549 cells possessed an exon 2 mutation (G12S) of K-Ras and wild-type EGFR, the H1975 cell line possessed a sensitive mutation (exon 21 point mutation, L858R) and a resistance-associated mutation in exon 20 (T790M) of EGFR and wild-type K-ras. MTT and AV/PI flow cytometry were used to determine the viability of cells after treatment of gefitinib and cisplatin alone, and it was found that three cell lines were resistant to gefitinib and insensitive to cisplatin. We next examined the antiproliferative effect of cisplatin/gefitinib combination by MTT. The combination index (CI) was calculated by Chou-Talalay Method. H1299 cell line showed synergistic interactions (CI<1), while H1975 cell line was antagonistic (CI>1). However, A549 cell line showed antagonistic interactions (CI>1) when the concentration of gefitinib and cisplatin was low and synergistic (CI<1) when high-dose gefitinib and cisplatin were combined. In addition, the results of MTT,3D cell culture models and AV/PI flow cytometry showed that gefitinib pretreatment enhanced anti-proliferation and apotosis of cisplatin in H1299 and A549 cells, but not in H1975. Animal model experiments also verified cisplatin combinded with gefitinib exists synergistic anti-tumor effect on H1299 and A549 tumors, but no synergistic effect on H1975 tumors. We next examined the caspase-3 and the activation of caspase-3 after gefitinib pretreatment followed by cisplatin treatment in three cell lines through Western blotting. Cleaved caspase-3 was detected only in H1299 and A549 cell lines after gefitinib pretreatment followed by cisplatin, but not in H1975 cells. The results indicated that the synergism of gefitinib and cisplatin might be dependent on the selective sensibilization of cisplatin induced by gefitinib.Then, we studied the mechanism of sensibilization of cisplatin induced by gefitinib. PCR amplification and gene sequencing were used again to observe the sequence of exons 18-21 of EGFR and exons 2-3 of K-ras of orthotopic xenograft tumors. We found that cisplatin had no effect on EGFR and K-ras gene status. Western blot results showed that the the effect of combination of cisplatin and gefitinib on the expression and activation of EGFR/Akt/Erk/PTEN/BRCA1/ERCC1 protiens on H1299, A549 and H1975 cells was on difference, however, the phosphorylation of DNA-PK could be inhibited by gefitinib in H1299 cells and A549 cells, but not in H1975 cells. Cell viability was determined by MTT method, it showed that when the DNA-PK activity of H1299 and A549 cells was inhibited by NU7441 (DNA-PK inhibitor), the sensitizing effect of cisplatin induced by gefitinib was gone, however, it had no effect on H1975 cells. The data of co-immunoprecipitation and immunofluorescence showed that the binding interaction of EGFR and DNA-PK occurs in NSCLC, and the interaction could be inhibited by gefitinib through selectively preventing EGFR translocating into nucleus only in H1299 cells and A549 cells, but not in H1975 cells. The data of western blot verified that the expression of nucleus EGFR was decreased by gefitinib treatment on H1299 and A549 cells, but not in H1975 cells.Conclusions:Synergistic effect between gefitinib and ciaplatin in part of EGFR-TKI resistant NSCLC cells. The synergism of gefitinib and cisplatin might be dependent on the selective sensibilization of cisplatin induced by gefitinib. The combination of gefitinib and cisplatin is synergistic or not is determined by whether gefitinib can block EGFR entering the nucleus to inhibit the activity of DNA-PK. The single factor of EGFR or K-ras gene status is insufficient to effectively select the part of patients. Only after fully understanding the mechanism of selective inhibition of EGFR into nucleus by gefitinib might be able to find out the selective factors which can predict the effectiveness of the combination therapy. This study provides a new idea to explore the selective factors of the effectiveness of the combination of platinum-containing chemotherapy and EGFR-TKI.