The Studies Of PA-MSHA Incombination With Gefitninb In Human Non-small Cell Lung Cancer Cell Lines With Different Genotypes

Part I. Studies on the Effects of PA-MSHA in combination with Gefitninb in vitro in different genotype Human NSCLC CellsThe incidence of lung cancer was rising in recent years, it has been the first cause of death of malignant tumor in worldwide. In developed countries, the5-year survival rate is only about10%-15%, the less in our country. Approximately80%of lung cancer is non-small cell lung cancer (NSCLC), many lung cancer patients were asymptomatic, when they came to the clinic, the disease was always the advanced stage. If the advanced NSCLC patients had no treatments, the median overall survival was only4-5months,1-year survival rate was less than10%. The first line treatment of advanced NSCLC is platinum based doublet regimen, the median OS was about1year, but the quality of life of patients was interfered by the adverse events of chemotherapy. Epidermal growth factor receptor (EGFR) played a critical role in the development of NSCLC, EGFR-tyrosine kinase inhibitors (TKIs), such as Gefitinib and Erlotinib were widely used in advanced NSCLC. But unfortunately, not all NSCLC patients were effective to EGFR-TKIs, in the end; all patients will be resistant to TKIs. Therefore, we need more effective, less toxic, economic agents. Investigator has been going in for the studies of targeting therapy of lung cancer and drug resistance. Until now, we didn’t find an optimal agent to reverse the drug resistance of EGFR-TKIs.PA-MSHA (Pseudomonas aeruginosa, Mannose-sensitive hemagglutination) is a type of therapeutic biological product approved in China for adjuvant treatment of patients with malignant tumors. This product is made from an inactivated mutant strain of Pseudomonas aeruginosa (PA-MSHA) that is characterized by rich mannose-sensitive hemagglutination pili (type1fimbriae). Although PA-MSHA has been successfully used in clinical cancer therapy for many years, its detailed mechanism of action remains unclear. In recent studies, PA-MSHA has been shown to directly inhibit tumor cell proliferation in vitro and induce apoptosis in human hepatocarcinoma, nasopharyngeal cancer and breast cancer cells. Interestingly, Liu’s in-depth study demonstrated that the mannose-mediated EGFR signaling pathway was involved in the apoptosis of breast cancer cells (MDA-MB-231HM and MDA-MB-468) induced by PA-MSHA. These results imply the potential therapeutic value of PA-MSHA in tumors typically associated with EGFR over-expression and mutations.In this part of study, we chose3different genotype NSCLC cell lines, PA-MSHA alone, Gefitninb alone, PA-MSHA in combination with Gefitinib were administered. MTT, flow cytometry, westernblot were performed to analyze the cell growth, cell cycle redistribution, apoptosis, EGFR signal transduction. These data suggest that PA-MSHA has the potential to inhibit proliferation and induce apoptosis and down-regulate EGFR signal pathway in NSCLC cells. Furthermore, PA-MSHA in combination with Gefitinib has more potent effect in cell growth inhibition, induction of apoptosis, cell cycle redistribution and down-regulating EGFR signal pathway. These data provide mechanistic details for the potential application of PA-MSHA-based therapies for the treatment of different NSCLC genotypes and PA-MSHA will be a potential agent to reverse the drug resistance of EGFR-TKIs in NSCLC. Part Ⅱ. Studies on the Effects of PA-MSHA in combination with Gefitninb in vivo in different genotype Human NSCLC CellsRecently, the prognosis of advance NSCLC is poor, the main problem of treatment failure is drug resistance to EGFR-TKIs, the mechanism of EGFR-TKIs resistance remains unclear. Many preclinical and clinical studies have been done to reverse the drug resistance, but a lot of limitations still remains. PA-MSHA is made from an inactivated mutant strain of Pseudomonas aeruginosa (PA-MSHA) that is characterized by rich mannose-sensitive hemagglutination pili. PA-MSHA has been successfully used in clinical cancer therapy for many years and the toxicities are very low and former studies have suggested the inhibition of EGFR signal pathway. In our study, in vitro, PA-MSHA was able to inhibit the cell growth in3different genotype NSCLC cell lines, redistribute the cells to sub-G1phase, induce apoptosis and down-regulate EGFR signal pathway. When in combination with Gefitinib, some synergistic effects were observed. Our data showed PA-MSHA may be a EGFR signal pathway inhibitor which is different from EGFR-TKIs or monoclonal antibody, and able to reverse the drug resistance of EGFR-TKIs, but the anti-tumor effect in vivo and the mechanism remains unclear, and the dose of clinical use to augment the anti-tumor effects need to be clarified. Therefore, we created nude mice model of different genotypes of NSCLC, PA-MSHA alone, Gefitninb alone, PA-MSHA in combination with Gefitinib were administered, the tumor growth, weight of mice, EGFR signal pathway related protein expression were observed. Thus, we are able to find the mechanism and target of PA-MSHA and clarify the possibility and mechanism of reversing the drug resistance of EGFR-TKIs.In this study, we create3different genotypes of NSCLC nude mice model, including EGFR-TKIs primary resistance, acquired resistance, and sensitive cells. We found that PA-MSHA was able to inhibit the tumor growth in vivo, especially for the primary resistance, acquired resistance cell lines. Moreover, we observed that the expression of EGFR signal pathway phosphorylated proteins such as p-Akt,p-ERK were down-regulated. Otherwise, we used RNA interference tech to block the binding of PA-MSHA and EGFR, the results showed that inhibition of EGFR signal pathway due to PA-MSHA was blocked. Therefore, the mechanism of PA-MSHA inhibits EGFR signal pathway in NSCLC was clarified.In summary, PA-MSHA was able to inhibit the tumor growth and proliferation, induce the apoptosis, redistribute the cell cycle, and down-regulate EGFR signal pathway, when in combination with Gefitinib, the EGFR-TKIs resistance was reversed. Our study first claimed PA-MSHA may have a potential to be targeting therapy in NSCLC. Recently, the personalized treatment to NSCLC has been a hot spot of cancer therapy. Our data will be a new theoretical base and treatment to NSCLC, especially for the EGFR-TKI resistant tumors.