Effect Of KLT And Gefitinib On The Expression Of FAS,AKT2and Their Gene Products In Human Lung Cancer A549Cell Line

Objective:The incidence and mortality of lung cancer are highest in malignant tumors,meanwhile the treatment effect of advanced non-small cell lung cancer (NSCLC) is not satisfactory. Gefitinib is an epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), which can effectively inhibit tumor cell growth, invasion, metastasis and tumor angiogenesis.Following the expanding clinical application of EGFR-TKI in recent years, EGFR-TKI has become the first-line treatment in advanced NSCLC in EGFR mutation positive patients.However, the problem of drug resistance of EGFR-TKI is a serious one which results in a decrease in curative effect. Therefore? seeking ways to increase therapeutic effect and even reverse drug resistance of EGFR-TKI has become a research hotspot. Some studies have shown that Kanglaite injection,as a traditional Chinese drug on anti-tumor,combined with chemotherapy can obviously increase therapeutic effect of advanced NSCLC, display synergistic action and sensitization and even reverse drug resistance. Many clinical investigations show that the persistent activation of PI3K/AKT pathway,which leads to inhibition of tumor cell apoptosis is an important mechanism of bad therapeutic effect of gefitinib. In this experiment MTT method, flow cytology, immunohistochemistry, reverse transcription polymerase chain reaction (RT PCR) method were used to detect the cell apoptosis rate, FAS、AKT2mRNA and their corresponding proteins to investigate the apoptosis induction effect and possible mechanism of gefitinib combined with Kanglaite on A549cells.Methods:1Human lung cancer A549cell line were maintained in vitro, we selected the logarithmic growing cells to make test, using MTT assay to investigate the inhibitive effect rate.Experiments were divided into control group, Kanglaite group,Gefitinib group and Kanglaite combined Gefitinib group. The concentration of Gefitinib groups was0.1umol/L、0.5umol/L、 lumol/L、5umol/L、10umol/L、20umol/L、40umol/L.Kanglaite groups was5uMnl、1Oul/ml、20ul/ml、40ul/ml、80ul/ml、100ul/ml、160ul/ml、The combinative groups of Kanglaite and Gefitinib,chose concentration with inhibition ratio50%(IC50) in Kanglaite as the work concentration for experiment. Each concentration respectively interfered A549cells24hours,48hours,72hours.2Flow cytometry(FCM) was employed to investigate the cell apoptosis and cycle of the combinative groups, the concentration of Kanglaite was80ul/ml, Gefitinib was40umol/L.3S-P Immunohistochemistry were used to detect the protien express of AKT2and FAS. A549cell were interfered by every group48hours, the concentration of Kanglaite was80ul/ml, Gefitinib was40umol/L4The alternations of mRNA expression levels of AKT2and FAS were detected by RT-PCR.Results:1MTT Results1.1The cell inhibition rates were measured by MTT when Kanglaite single-agent affecting human lung adenocarcinoma cell line A549.It was found that Kanglaite significantly inhibited the growth of human lung adenocarcinoma cell line A549, and the inhibition showed dose-and time-dependent. There were significant differences between all the inhibition effects of different concentrations and hours,significant differences were considered at P<0.05.1.2The cell inhibition rates were measured by MTT when Gefitinib single-agent affecting A549cells.It was found that, gefitinib were used to treat A549cells line for24h, with the increase of drug concentration (0.1、0.5、1、5、10、20、40umol/L), the inhibitory rate increased slowly,P>0.05, no significant difference were found.when the time more than24hours, significant differences were considered at P<0.05. Each drug concentration with treatment time (24h,48h,72h), the inhibitory rate increased slowly, P>0.05, no significant difference were found. The Gefitinib single drug to inhibit the proliferation of hela cells may have no obvious concentration-and time-dependent.1.3The cell inhibition rates were measured by MTT when Kanglaite combine Gefitinib.It was found that the combining inhibition effects were significantly exceeding the separated Kanglaite or Gefitinib, significant differences were considered at P<0.05. With the increase of drug concentration the inhibitory rate increased, significant differences were considered at P<0.05.Each drug concentration with treatment time (24h,48h,72h), the inhibitory rate increased, P<0.05. The inhibition showed dose-and time-dependent.2Flow cytometry results showed that:The cell apoptotic rates of combination significantly exceeded the separated Kanglaite or Gefitinib, significant differences were considered at P<0.05;the cell apoptotic rates of Kanglaite group and Gefitinib+Kanglaite group were significantly different of the control group(P<0.05), but gefitinib group no significant difference (P>0.05).3Immunohistochemical detection results show that, the proteins expression rate of FAS in each experimental group were:Kanglaite(50%), Gefitinib+Kanglaite(58.33%), which reduced significantly compared with the control group(P<0.05), Gefitinib(16.67%), which no significant difference compared with the control group(P>0.05), when Kanglaite combined with Gefitinib, the proteins expression rate of FAS increased, which were significant differences with the Gefitinib (P<0.05)The proteins expression rate of AKT2in every group were: Kanglaite(33.33%),Gefitinib+Kanglaite(16.67%), significant differences were considered at P<0.05, Gefitinib (66.67%), no significant difference compared with the control group(P>0.05); the Gefitinib group reduced significantly compared with the combined group(P<0.05).4According to RT-PCR analysis of FAS、AKT2mRNA. Compared with the control group, statistical analysis showed that the expression levels of FASmRNA of Kanglaite group and the combined group were significantly increased (P<0.05), thus AKT2mRNA reducing (P<0.05); Compared with gefitinib, the expression levels of AKT2mRNA of the combined group were reduced (P<0.05), FAS protein increasing (P<0.05).Conclusion:1Separated Kanglaite could inhibit the proliferation of A549cells effectively, the inhibition showed dose-and time-dependent.2Separated Gefitinib have a slight inhibition of A549cells.3Compared with the effects of Kanglaite or Gefitinib separately at high concentrations,combination of Kanglaite and Gefitinib at low concentrations proved to be much more effitive in the inhibition of the proliferation and induction of the apoptosis of A549cell lines.4When Kanglaite combined with Gefitinib interfered A549cells48hours, the expression of FASmRNA and proteins upregulated,AKT2mRNA and proteins downregulated,the results show that, KLT combined with gefitinib could affect the PI3K/AKT pathway, Thereby affecting the phosphorylation of AKT2,which can increase the expression of FAS protein, promote FAS and its receptor FASL binding, and ultimately Induce cell apoptosis.