Kangai Injection Regulates Beclin 1-dependent Autophagy-apoptosis Interaction To Improve Cisplatin Resistance In A549/DDP Cell

Purpose:Kangai Injection（KAI）is a commonly used traditional Chinese medicine compound injection in clinical practice,with the function of tonifying qi and enhancing immunity.It is widely used to protect hematopoietic function,reduce expression of tumor-related cytokines,and can significantly improve the efficacy of chemotherapy,alleviate adverse reactions,and improve the quality of life of patients when used in combination with chemotherapy drugs.Currently,most research on Kangai Injection focuses on clinical efficacy evaluation,and its anti-tumor mechanism is not fully understood.This study used two different lung adenocarcinoma cell lines with different levels of drug resistance（A549 cells as parent cells and A549/DDP cells as cisplatin-resistant cell lines）to explore the mechanism of Kangai Injection in improving cisplatin resistance in non-small cell lung cancer through the autophagy-apoptosis interaction mechanism under the regulation of PI3K and Beclin 1 protein,providing scientific laboratory evidence for the clinical application of tonifying qi and enhancing immunity in adjunctive treatment of malignant tumors,and providing new ideas for the study of tumor resistance mechanisms.Materials and methods:1.In the online website analysis tool（Kaplan-Meier Plotter）that includes TCGA,GEO,and EGA databases,survival-related data analysis was conducted on 719 patients with lung adenocarcinoma to explore the correlation between BECN1 m RNA expression level（a key autophagy-regulating gene）and the survival period of patients with lung adenocarcinoma.2.The drug resistance of A549 cells and A549/DDP cells was compared using the CCK-8 assay.Dose-response curves for both cell types were fitted to determine the platinum IC₅₀ values,and the resistance index（RI）was calculated.The expression level of the key autophagy regulatory protein Beclin 1 was analyzed using the Western blot method to investigate its relationship with platinum resistance.3.Observe the morphological characteristics of A549 and A549/DDP cells using a high-content analysis system,and study the apoptosis-related morphological changes that occur in both cell types under 10μmol/L cisplatin intervention.4.Perform laser scanning confocal microscopy to compare the normal morphological features of A549 and A549/DDP cells,as well as the apoptosis-related morphological changes that occur in both cell types after treatment with 10μmol/L cisplatin.5.The Western blotting method was used to compare the expression changes of Beclin 1 protein,autophagy-related proteins（ATG7,LC3I,LC3II）,and apoptosis-related proteins（cleaved caspase-3,Bax,Bcl-2）,as well as changes in the Bax/Bcl-2 ratio in A549 and A549/DDP cells with different levels of resistance under cisplatin intervention.6.Compare the inhibitory effects of Kang’ai injection on cisplatin-resistant A549/DDP cells using the CCK-8 method,fit the IC₁₀,IC₂₀,and IC₃₀ values of Kang’ai injection,and observe the cell inhibition effects of gradient platinum combined with low,medium,and high concentrations of Kang’ai injection(low concentration:IC₁₀,medium concentration:IC₂₀,high concentration:IC₃₀).7.Using the WB method,detect the expression changes of autophagy-related proteins（ATG7,LC3Ⅰ,LC3Ⅱ）and apoptosis-related proteins（cleaved caspase-3,Bax,Bcl-2）in cisplatin-resistant A549/DDP cells treated with 10μmol/L cisplatin combined with low,medium,and high concentrations of Kang’ai injection,and evaluate the changes in the Bax/Bcl-2 ratio.8.Use flow cytometry to detect changes in the apoptosis rate of cisplatin-resistant A549/DDP cells treated with 10μmol/L cisplatin combined with low,medium,and high concentrations of Kang’ai injection.9.Using the WB method,detect the expression changes of autophagy-related proteins（ATG7,LC3Ⅰ,LC3Ⅱ）and apoptosis-related proteins（Bcl-2,Bax,cleaved caspase-3）in cisplatin-resistant A549/DDP cells treated with 10μmol/L cisplatin combined with high concentrations of Kang’ai injection under the action of autophagy and apoptosis inhibitors 3-MA and z-VAD-fma,and evaluate the role of autophagy and apoptosis in combination therapy by assessing the changes in the Bax/Bcl-2 ratio.10.Using the WB method,detect the changes in PI3K and Beclin 1 protein expression in cisplatin-resistant A549/DDP cells treated with 10μmol/L cisplatin combined with low,medium,and high concentrations of Kang’ai injection.Results:1.The Kaplan-Meier Plotter online tool showed that in patients with lung adenocarcinoma,high expression of BECN1 m RNA was significantly associated with increased survival time compared to those with low expression（P<0.001）,and the expression level of BECN1 m RNA was positively correlated with patient prognosis.2.The protein expression level of Beclin 1 was higher in A549/DDP cells resistant to cisplatin than in cisplatin-sensitive A549 cells（P<0.05）.3.Platinum intervention upregulated the expression levels of autophagy-related proteins Beclin1,ATG7,LC3Ⅰ,and LC3Ⅱin both A549 and A549/DDP cells（A549:P<0.001（Beclin 1）,P<0.001（ATG7）,P<0.01（LC3Ⅰ）,P<0.01（LC3Ⅱ）;A549/DDP:P<0.001（Beclin 1）,P<0.05（ATG7）,P<0.01（LC3Ⅰ）,P<0.01（LC3Ⅱ））.In A549 cells,the expression of Bax and cleaved caspase-3 increased（P<0.001（Bax）,P<0.01（cleaved caspase-3））,while Bcl-2 expression decreased（P<0.01）,and the Bax/Bcl-2 ratio increased（P<0.01）.In A549/DDP cells,the expression of Bax and Bcl-2 increased（P<0.01（Bax）,P<0.05（Bcl-2））,but the Bax/Bcl-2 ratio decreased（P<0.05）.4.The combination of Kang’ai injection and cisplatin significantly inhibited the proliferation activity of A549/DDP cells（P<0.01（low concentration of Kang’ai injection combined with cisplatin）,P<0.001（medium concentration of Kang’ai injection combined with cisplatin）,P<0.001（high concentration of Kang’ai injection combined with cisplatin））.Compared with the cisplatin group,the combination of Kang’ai injection and cisplatin upregulated the expression levels of ATG7（P<0.05（low concentration of Kang’ai injection combined with cisplatin）,P<0.05（medium concentration of Kang’ai injection combined with cisplatin）,P<0.01（high concentration of Kang’ai injection combined with cisplatin））,LC3Ⅰ（P<0.01（low concentration of Kang’ai injection combined with cisplatin）,P<0.001（medium concentration of Kang’ai injection combined with cisplatin）,P<0.001（high concentration of Kang’ai injection combined with cisplatin））,and LC3Ⅱ（P<0.01（low concentration of Kang’ai injection combined with cisplatin）,P<0.001（medium concentration of Kang’ai injection combined with cisplatin）,P<0.001（high concentration of Kang’ai injection combined with cisplatin））,and the upregulation levels were positively correlated with the concentration of Kang’ai injection.Bax expression was downregulated in the cisplatin+low concentration of Kang’ai injection group（P<0.05）and upregulated in the cisplatin+medium and high concentration of Kang’ai injection groups（P<0.01）;Bcl-2 expression was downregulated in the cisplatin+low and medium concentration of Kang’ai injection groups（P<0.05）and upregulated in the cisplatin+high concentration of Kang’ai injection group（P<0.05）;cleaved caspase-3 expression and the Bax/Bcl-2 ratio were upregulated in all three cisplatin+Kang’ai injection combination groups（cleaved caspase-3 expression level:P<0.05（low concentration of Kang’ai injection combined with cisplatin）,P<0.01（medium concentration of Kang’ai injection combined with cisplatin）,P<0.01（high concentration of Kang’ai injection combined with cisplatin）;Bax/Bcl-2 ratio:P<0.05（low concentration of Kang’ai injection combined with cisplatin）,P<0.001（medium concentration of Kang’ai injection combined with cisplatin）,P<0.05（high concentration of Kang’ai injection combined with cisplatin））,and the upregulation of cleaved caspase-3expression was positively correlated with the concentration of Kang’ai injection.5.Beclin 1 expression was downregulated in the cisplatin+low concentration of Kang’ai injection group（P<0.001）and upregulated in the cisplatin+medium and high concentration of Kang’ai injection groups（P<0.001）,with the cisplatin+high concentration of Kang’ai injection group showing the most significant upregulation（P<0.001）.The combination of Kang’ai injection and cisplatin could induce downregulation of PI3K,a protein that inhibits autophagy,and this effect was dose-dependent（P<0.001）.Conclusion:Kang’ai injection in combination with cisplatin downregulated the expression of autophagy inhibitor PI3K protein and upregulated the expression of autophagy inducer Beclin 1 protein,activating Beclin 1-mediated autophagy-apoptosis crosstalk,inducing autophagic cell death and apoptosis in cisplatin-resistant A549/DDP cells,effectively improving the cisplatin resistance of A549/DDP cells.