The Effects And Mechanisms Of P-Akt, C-Met Expression Of A549Cells Treated With KLT And Gefitinib

Objective: Non-small cell lung cancer (NSCLC) is one of the commonmalignant tumors nowadays, it is the first national cancer cause of death inmajority countries. In recent years, the emergence of new molecular targeteddrugs epidermal growth factor receptor-tyrosine kinase inhibitors(EGFR-TKIs) brought the dawn for the treatment of NSCLC. Gefitinib is anorally active, selective EGFR-TKIs, which has become a mature moleculartargeted drug in the treatment of lung cancer. Clinical studies have shown thatthe total efficiency of gefitinib in the treatment of various types of NSCLC isonly10%to20%,so people hope to combine gefitinib with other medicines toenhance the anti-tumor effects. KLT injection is a traditional Chinesemedicine, made of Yiyiren oil extracted from the Chinese medicine Yiyiren,studies have shown that it has strong killing and inhibition effects to varioustumor cells. Small sample clinical studies found that KLT combined withchemotherapy can improve the treatment effect of advanced NSCLCsignificantly. There are many studies proved that PI3K/Akt signaling pathwayplay an important role in the development of NSCLC. P-Akt, presence of Aktphosphorylation, can be detected in50%-70%of NSCLC. This indicates thatthe activation of the PI3K/Akt signaling pathway is very common in NSCLC.C-Met protein is encoded by an original oncogene c-Met, has a tyrosinekinase activity of growth factor receptors, specifically binds with thehepatocyte growth factor (Hepatocyte Growth Factor, HGF), activate a seriesof downstream signal transduction pathways, regulate the proliferation,vitality, migration, angiogenesis, invasion, and morphogenesis of cells. Thechange of c-Met signaling can promote tumorigenesis and development. Theresults show that the secondary overexpression of c-Met is related to theacquired resistance of gefitinib. This experiment applied a series of experimental methods, detected the rate of apoptosis, the mRNA and proteinexpression of p-Akt and c-Met in A549cells treated with KLT and gefitinib, toexplore the effects to A549cells and its mechanism.Method:1Cultured lung adenocarcinoma A549cells in vitro, selected thelogarithmic growth phase cells. The experiment was divided into blank controlgroup, gefitinib group, KLT group, gefitinib and KLT group. Theconcentrations of gefitinib group were:0.1,0.5,1,5,10,20,40μmol/L, the actiontimes were:24h,48h,72h; The concentrations of KLT group were:5,10,20,40,80,100,160μl/ml, the action times were:24h,48h,72h; Combinedtreatment group: chose IC50for KLT, the peak concentration of thisexperiment for gefitinib; applied MTT method to detect the inhibition rates ofdifferent concentrations and action times of each group.2According to the results of MTT, KLT IC50=80μl/ml, chose thisconcentration for KLT and the highest concentration40μmol/L in thisexperiment for gefitinib. After48-hour treatment of two medicines together,applied flow cytometry to detect cell growth inhibitions in each group.3Placed the logarithmic growth phase cells in six-well plates andcultured, groups set ibid. The morphological changes were observed under aninverted microscope. SP staining was applied to detect the changes of p-Akt,c-Met protein expressions in each group.4Took the logarithmic growth phase cells, groups set ibid. ExtractedRNA after48hours, detected the amount, purity and integrity of total RNA.Applied RT-PCR to detect the c-Met mRNA expression levels in each group.5The statistics were processed by SPSS18.0, used normality test. P<0.05indicated significant difference, P<0.01represented a very significantdifference.Results:1MTT results showed:1.1Compared with the control group, with the increase of concentration andaction time of KLT, the inhibition rate of A549cells gradually increased, the inhibition effect depended on dose and time.1.2Combined treatment group: after48h, the inhibition rate of the two drugswas higher than KLT group and gefitinib group, it was statistically significant(P <0.05);1.3Compared with the control group, gefitinib group showed certaininhibitory effect to A549cells, but not obvious.2Flow cytometry results showed:2.1After48hours, the rate of apoptosis of KLT group，gefitinib group andcombined treatment group were significantly higher than the control group, itwas statistically significant (P <0.05);2.2Apoptosis rate of combined group was significantly higher than KLTgroup and gefitinib group, it was statistically significant (P <0.05);3Immunohistochemistry results showed:After48hours, the rates of p-Akt and c-Met protein expression in KLT groupwere lower than control group, P<0.05. The effects in gefitinib group were notsignificant, P>0.05.The rates of p-Akt and c-Met protein expression incombined group was significantly lower than other groups, P<0.05.4RT-PCR results show:4.1The c-Met mRNA expression levels in KLT group were lower than controlgroup,but the levels in gefitinib group were not changed significantly;4.2The c-Met mRNA expression levels in two-drug combination group waslower than control group and gefitinib group significantly.Conclusion:1Within a certain range, with the drug concentration and action timeincreased, the inhibition rate of KLT increased, and showed adose-time-dependent effect; the inhibition rate of combined group was higherthan other groups after48hours; the two-drug combination may have certainsynergies.2KLT can induced apoptosis in tumor cells after48hours，apoptosis rateof combined group was higher than any other groups (P<0.05).3Compared with KLT group, the KLT and gefitinib group lowered the levels of transcription and protein of p-Akt and c-Met obviously, it wasspeculated that the KLT may play an anti-tumor effect through the PI3K/Aktsignaling pathway, and may regulate the resistance of gefitinib.