Lovastatin Overcomes Gefitinib Resistance In Human Non-small Cell Lung Cancer PC9 Cell Line In Vitro

ObjectiveGefitinib, a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR), is used to treat the advanced stage NSCLC who had previously received platinum-based chemotherapy presently. Several studies have confirmed that Gefitinib for non-smoking patients with adenocarcinoma of the highly sensitive and Gefitinib have some features such as small side effects, similar efficacy with chemotherapy, the patient with good compliance and so on, so some scholars have suggested that for no smoking or less smoking older women patients with adenocarcinoma, may be considered as first-line treatment. However, targeted drug also exists in primary and secondary drug resistance problem. Currently the mechanism of resistance are the T790M secondary mutations theory, non-T790M secondary mutations and so on. But some studies have shown that Gefitinib resistance were related to the drug transport, EGFR gene amplification and signaling pathways change, non-single mechanism can fully explain the resistance.Lovastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor. Overseas research has shown that Lovastatin can inhibit EGFR / K-Ras and downstream of the Raf / MEK / ERK and PI3K/AKT pathway activity. Presently non-small cell lung cancer patients with Gefitinib-resistant how to select the next step of treatment is still not conclusive. Overseas research has shown that in vitro experiments, Lovastatin can induce Gefitinib resistant cell line with K-ras mutant apoptosis. At the present day Lovastatin can overcome domestic resistance to Gefitinib is not yet reported in the literature.In our research,we aimed to investigate the influence of Lovastatin combined with Gefitinib to cell apoptosis and related protein expression of non-small cell lung cancer cell line PC9 cell which is induced by Gefitinib in vitro acquired drug resistance.MethodsPC9 cell lines by Tongji University Affiliated Shanghai Pulmonary Hospital Professor Zhou Caicun as gifts, has been verified in the absence of drug resistance in medium can be maintained over 1 year. According to different experimental approach is divided into: (1) control group: (2) Gefitinib treatment group: Gefitinib diluted final concentration: 1μmmol / L; (3) Lovastatin treatment group: dilution final concentration of Lovastatin: 5μmmol / L; (4) Gefitinib combined Lovastatin treatment group: diluted Gefitinib and Lovastatin final concentrations: 1μmmol / L, 5μmmol / L. Treated with different drugs on the PC9 cell proliferation was tested by WST-1. Apoptosis were observed by flow cytometry.The modality of Apoptosis were observed by Hoechst33342.Related signaling pathway protein expression changing were detected by Western blot.ResultsIn human NSCLC cell lines, Lovastatin combined with Gefitinib compared to Gefitinib alone showed significantly promote cell growth inhibition and cytotoxicity by Hoechst 33342 staining , by Hoechst 33342 staining the cells were observed under inverted phase contrast microscope showed that two-drug combination group treated cells were irregular, and also can see the occurrence of condensation nuclei, fragmentation, and more floating dead cells. WST-1 assay showed that the two-drug combination group than in the Gefitinib group of large inhibitory effect on cell proliferation. Detected by flow cytometry showed that the control group of PC9 cells apoptosis rates was 2.5%, Gefitinib group and Lovastatin group apoptosis rates were 5.5% and 9.4%, the apoptotic rate of the two drugs combined were 65.5%. Two-drug combination group compared with the control group was statistically significant between the two (P <0.01); two-drug combination group compared with alone Gefitinib or Lovastatin was statistically significant (P <0. 01), suggesting that Lovastatin significantly induce apoptosis in PC9 cells. The expressions of p-EGFR, p-AKT, p-ERK were decreased significantly by Lovastatin combined with Gefitinib treatment.ConclusionIn vitro induction of acquired Gefitinib resistance in NSCLC cell line PC9, Lovastatin can overcome Gefitinib resistance, both drugs showed a great synergistic effect, the final results suggest that the two non-drug combination in Gefitinib resistance non-small cell lung cancer therapy may have great potential.