Study Of KLT Combined With Gefitinib On Human Lung Adenocarcinoma Cell Line NCL-H1975

Objective: Today, the incidence rate of lung cancer is located in the first place, In China. Since the early symptoms of lung cancer are more hidden, most patients have been diagnosed with advanced stage. Chemotherapy is the cornerstone of treatment of lung cancer, Chemotherapy have a greater adverse reactions, and chemotherapy has reached a plateau, with the advent of molecular targeted therapy, the treatment of advanced non-small cell lung cancer shift from the chemotherapy to the molecular targeted therapy with genotyping guidance. More commonly used is the epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) drugs such as Gefitinib, Erlotinib, Icotinib, etc. But with the widespread application of EGFR-TKI, drug resistance issues corresponding emerged, among the many resistance mechanisms, where T790 M mutation accounts for about 50% ~ 60% EGFR-TKI resistance. China’s traditional medicine has its special effects in anti-cancer therapy, so people began to explore using the traditional Chinese medicine combined with EGFR-TKI drugs, whether can solve the EGFR-TKI drug resistance problem. KLT injection is a traditional Chinese medicine, Yiyiren is its main ingredients. It has shown a strong inhibition and cytotoxicity to variety of tumor cell, especially for non-small cell lung cancer(NSCLC) cells. At present, studies have shown that PI3 K / Akt and MAPK / ERK signaling pathway play an important role in apoptosis, proliferation, invasion, and a series of biological processes in non-small cell lung cancer. In this study, the intervention by Gefitinib, KLT and Gefitinib combined with KLT against NCI-H1975 cell line(human lung adenocarcinoma with T790 M mutations). To explore the effect and mechanism of Gefitinib in combination with KLT on NCI-H1975 cell line(human lung adenocarcinoma with T790 M mutations), we apply a series of experimental methods: detected the rate of apoptosis,Akt gene expression in PI3K/Akt signaling pathway, MAPK1(ERK2) gene expression in MAPK / ERK signaling pathway and the expression of p-Akt protein and p-ERK protein in NCI-H1975 cells.Method:1 Cultured human lung adenocarcinoma NCI-H1975 cells, select the logarithmic growth phase cells for experiment. The experiment was divided into blank control group, Gefitinib group, KLT group, Gefitinib combined KLT group. The concentrations of gefitinib group were: 0.1μmol/L, 0.5μmol/L, 1μmol/L, 5μmol/L, 10μmol/L, 25μmol/L, 50μmol/L, the reaction time were: 24 h, 48 h, 72h; The concentrations of gefitinib group were: 5μl/ml, 10μl/ml, 20μl/ml, 40μl/ml, 80μl/ml, 100μl/ml, 160μl/ml, the reaction time were: 24 h, 48 h, 72h; applied MTT method to detect the inhibition rates of different concentrations and reaction times of each group, and describe the growth curve. Calculate the Gefitinib group IC30 and the KLT group IC50. 2 Determine the concentration of the combined group and the reaction time: According to the results of MTT, the Gefitinib group IC30=25μl/ml;the KLT group IC50=40μl/ml. Therefore, the combination group take Gefitinib 25μl/ml, take KLT 40μl/ml, detect the 48 h inhibition rates by MTT. 3 Took the logarithmic growth phase cells, groups set ibid. Extracted RNA after 48 hours, detected the amount, purity and concentration of total RNA. Applied RT-PCR to detect the Akt m RNA and MAPK1(ERK2) m RNA expression levels in each group. 4 Placed the logarithmic growth phase cells in six-well plates and cultured, groups set ibid. Extracted protein after 48 hours, detection of total protein concentration by BCA(bicinchonininc acid) method, and use Western-blot method to detect the p-Akt and p-ERK protein expression levels in each group. 5 Use IBM SPSS Statistics 22.0.0.0 statistical software for data processing.Results:1 MTT results showed:1.1 Compared with the control group, with increasing concentrations and the extension of reaction time of KLT group, the human lung adenocarcinoma NCI-H1975 cell inhibition rate gradually increased, the inhibition effect in a dose and time dependent manner; Gefitinib treated 24 hours group, with the drug concentration 0.1-5μmol/l, there is no significant inhibition on human lung adenocarcinoma NCI-H1975 cells. With the drug concentration 10-50μmol/l, it shown mildly inhibition on human lung adenocarcinoma NCI-H1975 cells. With Gefitinib 48 h and 72 h group, the inhibition rate gradually increased, the inhibition effect in a dose and time dependent manner. 1.2 Combined treatment group: Gefitinib combined KLT after 48 h culture the combined group inhibition rate of NCI-H1975 cell is higher than the monotherapy groups, it was statistically significant(P<0.05). 2 RT-PCR results showed that: Compared Gefitinib group, KLT group and Gefitinib combined KLT group with the control group, the m RNA expression levels of Akt and MAPK1(ERK2) genes are lower, the combination group was significantly lower than other groups, there was statistically significant(P<0.05). 3 Western-blot results showed that: Compared Gefitinib group, KLT group and Gefitinib combined KLT group with the control group, the protein expression levels of ERK are lower, the combination group was significantly lower than other groups, there was statistically significant(P<0.05).Conclusion:1 Both KLT and Gefitinib have inhibitory effect on human lung adenocarcinoma NCI-H1975 cell, and KLT combined with Gefitinib can enhance this inhibitory effect. 2 Both KLT and Gefitinib can down-regulating Akt and MAPK1(ERK2) gene and p-Akt and p-ERK protein expression levels, the Akt and MAPK1(ERK2) gene and p-Akt and p-ERK protein expression levels in combination group is lower than other group. 3 KLT could reverse the drug resistance of human lung adenocarcinoma NCI-H1975 cells,which probably is related with its down-regulation of Akt and MAPK1(ERK2) gene and p-Akt and p-ERK protein expression.