Preliminary Study Of Apatinib Combined With Tanshinone Ⅱ In The Treatment Of Non-Small Cell Lung Cancer

Objective:Lung cancer is one of the most leading causes of cancer-related death in China,which seriously threatens human health and imposes a huge burden on society Tanshinone ⅡA is the main active ingredient extracted from the traditional Chinese medicine Salvia miltiorrhiza,it has a variety of pharmacological activity and less toxic side effects including anti-cancer.However,the study of Tanshinone ⅡA in non-small cell lung cancer has not been fully elucidated.For patients with advanced lung cancer,molecular targeted therapy is considered as a new hopeful treatment,and the traditional molecular targeted drug gefitinib could achieve curative effect.However,long-term application will lead to drug resistance.Therefore,it is particularly important to find new treatment options.Apatinib is a traditional drug for the treatment of chemotherapy-refractory gastric cancer,which binds to the ATP-binding site of VEGFR-2 competitively to block downstream signaling,thereby inhibiting the formation of tumor new blood vessels.The results of clinical trials in recent years indicate that the objective effect of Apatinib on non-small cell lung cancer is also obvious.In this paper,human non-small cell lung cancer(NSCLC)A549 cell line and H1299 cell line were used as research materials,we compare the two cell lines changes by using Apatinib and Tanshinone ⅡA alone,in combination and sequentially.Proliferation inhibition,promotion of apoptosis and changes in the expression level of apoptotic proteins,preliminary discussion of the relevant mechanism of action,provide a theoretical basis for the combination therapy of Apatinib and Tanshinone ⅡA.Methods:Experiment 1:Study on the morphology and cell viability of A549 cells and H1299 cells under the use of single-agent,combination and sequential administration of Apatinib and Tanshinone IIA.The final concentration of Apatinib and Tanshinone IIA was set to:0 μM,5μM,10 μM,20 μM,40 μM And 80 μM,5 sub-wells were set in each group,and the inoculated 96-well plates were placed in an incubator at 37 ℃C 5%CO2 for 48 h,and then CCK8 was detected.The detection of cell viability was carried out with each groupExperiment 2:Flow cytometry was used to detect the apoptosis of A549 and H1299 cells after different treatments.A549 cells and H1299 cells were cultured and drug-interfered.The apoptosis rate was detected by Annexin FITC/Pl double-stained cells according to the kit instructions.Each kind of cells was divided into 6 groups negative control group,Apatinib group,Tanshinone IIA group,combination group,Tanshinone IIA sequential Apatinib group and Apatinib sequential Tanshinone IIA groupExperiment 3:RT-qPCR was used to detect the changes of Bcl-2 and Bax mRNA levels after different treatments.A549 cells and H1299 cells were cultured,and the RNA was extracted after drug intervention.The cDNA was synthesized and detected by real-time PCR.The groups of cells were in the same manner as in Experiment 2Experiment 4:Western blotting was used to detect the changes of Bcl-2 and Bax protein levels after drug treatments.A549 cells and H1299 cells were cultured,and the total protein was extracted after drug extraction.The protein concentration was detected by BCA method and subjected to SDS-PAGE electrophoresis.The groups of cells were in the same manner as in Experiment 2Experiment 5:Detection of the changes of Caspase3 enzyme activity.Cultured A549 cells and H1299 cells,did the drug intervention and extraction of total protein of each group,used Bradford method to detect protein concentration,according to the reaction system,detected of pNA yield,thereby obtained Caspase3 enzyme activity The groups of cells were in the same manner as in Experiment 2Results:1.CCK8 results showed that the singular use of Apatinib or Tanshinone IIA with different concentrations could effectively inhibit the proliferation of A549 and H1299 cells in a dose-dependent and time-dependent manner compared with the normal culture group.The combination of two drugs could further inhibit cell proliferation,the difference was statistically significant(P<0.01)2.The detection of apoptotic rate of cancer cells after the different interventions.The combination group can increase the apoptotic rate compared with the single drug group and the sequential group,but it was not conducive to increase the apoptosis rate after changing the order of administration.Also,the combination therapy had a greater effect on H1299 cells than A549,the difference was statistically significant(P<0.05)3.The ratio of Bax/Bcl-2 after drug treatment was detected by real-time PCR.The combination group can increase the level of Bax and decrease the level of Bcl-2 compared with the single drug group and the sequential group with the significant difference(P<0.05)4.Western blotting was used to detect the expression of apoptotic proteins after drug treatment.The combination group can increase the level of Bax and decrease the level of Bcl-2 compared with the single drug group and the sequential group5.The results of Caspase3 enzyme activity after drug treatments showed:The combination group can increase Caspase3 activity mostly compared with the single drug group and the sequential group with the significant difference(P<0.05)Conclusion:The conbination of Apatinib and Tanshinone ⅡA promotes apoptosis of lung cancer cells,which may be related to the regulation of Bax/Bcl-2 balance and Caspase3 pathway.Apatinib combined with Tanshinone ⅡA may become a new drug combination for clinical treatment of lung cancer.