The Research On Effect Of Different Administration Sequences Of Docetaxel, Cetuximab And Bevacizumab In KRAS Wild And Mutant Type Lung Cancer Cell Lines

Background and Purpose:Lung cancer is the mainly cause of cancer death in the world, and the morbidity and mortality are significantly increased. Lung cancer usually can be divided into two categories: non small cell lung cancer and small cell lung cancer, which is based on the difference of the biological behavior of lung cancer, and non small cell lung cancer accounts for 80% ~ 85% of all the lung cancer cases. Most of the patients are diagnosed with non small cell lung cancer has occurred locally advanced or metastatic. For such patients are usually given chemotherapy treatment, but the study found that chemotherapy can not change the long-term outcomes of lung cancer, and the efficacy of chemotherapy may have reached a plateau. In recent years, along with the increase of awareness of tumor molecular biology and gene level, the research and clinical application of molecular targeted therapy has become a hot point.Clinical study of cetuximab combined with chemotherapy as first-line treatment of advanced non small cell lung cancer(FLEX) showed that the chemotherapy plus cetuximab compared with chemotherapy alone can prolong overall survival(OS). In treatment field of advanced non small cell lung cancer, a number of clinical studies( E4599, AVAIL, SAIL) all showed that bevacizumab combined with chemotherapy can prolong the overall survival(OS) and progression free survival(PFS) of patients with advanced non small cell lung cancer.Recent studies have shown that different administration sequences of tyrosine kinase inhibitor(TKI) and cytotoxic drugs will have a definite effect on anti tumor effect, so people pay more attention to the best sequence of the application of cytotoxic drugs and molecular targeting drugs. But at present, the research on different administration sequences of cetuximab, bevacizumab and cytotoxic drugs is few, and there is no domestic and foreign similar research has been reported.Our study is a basic research on different administration sequences of cetuximab, bevacizumab and docetaxel. We select the lung cancer cell line A549 with mutant KRAS and H1299 with wild type KRAS as the object of this research. The two cell lines were treated with different sequential administration of docetaxel, cetuximab and bevacizumab, then apoptosis related protein, cell cycle arrest and cell apoptosis were test after different sequential administration. We should observe the antitumors effect of docetaxel combined with cetuximab then bevacizumab and docetaxel combined with bevacizumab then cetuximab in lung cancer cell lines with different types of KRAS, then determine the best sequence of cetuximab and bevacizumab, and provide a theory evidence for clinical rational use of drugs. Methods:We selected the lung cancer cell line A549(KRAS mutant type) and H1299(KRAS wild type) to culture, and the two cell lines were divided into 4 groups according to the design of experiment, which included the control group,the single drug group(TXT, C225, Bev), the combination of two drugs group(TXT+Bev, TXT+C225) and the combination of two drugs, then single drug sequential group(TXT+C225â†'Bev, TXT+ Bevâ†'C225). We used MTT method to detect the effect of different medication orders on the growth of two cell lines, and flow cytometry(FCM) to detect the effect of different medication orders on cell cycle and cell apoptosis. In addition, we determined the content of apoptosis related proteins using Western-blot method. Results:1. The different administration sequences of docetaxel, cetuximab and bevacizumab all can inhibit the growth of lung cancer cell line A549 and H1299. However, docetaxel combined with cetuximab then bevacizumab(TXT+ C225â†'Bev) and docetaxel combined with bevacizumab then cetuximab(TXT+ Bevâ†'C225) can effect the growth of A549 and H1299 more obviously than single drug group and combination of two drugs group.2. Docetaxel can produce significant G2/M phase arrest; cetuximab and bevacizumab can arrest the cell in G0/G1 phase. The combination of two drugs group(TXT+Bev, TXT+C225) increased G2/M phase arrest than the control group; while the combination of two drugs, then single drug sequential group(TXT+C225â†' Bev, TXT+ Bevâ†' C225) had no obvious change in cell cycle compared with combination of two drugs group.3. Docetaxel combined with cetuximab then bevacizumab(TXT+C225â†'Bev) and docetaxel combined with bevacizumab then cetuximab(TXT+ Bevâ†'C225) can effect the cell apoptosis more obviously than the single drug group and two drug combination group.4. The different administration sequences of docetaxel, cetuximab and bevacizumab all can promote apoptosis in A549 and H1299, the ratio of Bcl-2/Bax were lower than those in the control group. However, the combination of two drugs, then single drug sequential group(TXT+C225â†' Bev, TXT+ Bevâ†' C225) influenced the expression of apoptosis related proteins more significant. Conclusion:Docetaxel combined with cetuximab then bevacizumab(TXT+C225â†'Bev) and docetaxel combined with bevacizumab then cetuximab(TXT+ Bevâ†'C225) can effect the growth of A549 and H1299, cell apoptosis and content of apoptosis related proteins more obviously than the single drug group and two drug combination group, but there were no significant differences in antitumors effect.