Ailanthone Suppresses Non-small Cell Lung Cancer Cell Growth By Triggering Autophagy-dependent Ferroptosis

The incidence and mortality of lung cancer are among the highest in the world.Non-small cell lung cancer（NSCLC）accounts for about 85%of lung cancer,with high incidence,insidious onset,and poor prognosis.At present,the main clinical treatment methods of NSCLC include surgery,radiotherapy,chemotherapy,targeted therapy and immunotherapy.However,in most cases,the clinical effect is not satisfactory.Treatment methods such as surgery,radiotherapy,and chemotherapy have their own limitations,and drug resistance remains a major clinical problem.Compared with these therapies,Chinese herbal medicines and natural products usually have less side effects and wide applicability,which makes them more suitable for cancer treatment.Therefore,it is necessary to explore the mechanism of the occurrence and development of NSCLC and find low toxicity and high efficiency drugs for the treatment of lung cancer.Studies have shown that Ailanthone（AIL）is a traditional Chinese medicine component extracted from Ailanthus altissima.It is a low-toxic and efficient therapeutic drug that can inhibit the growth of various tumor cells,such as leukemia,liver cancer,and breast cancer.Autophagy is a highly conserved and regulated catabolic process in organisms.Autophagic lysosomes can degrade damaged cytoplasmic structures and invading pathogens,thus playing an important role in human health and diseases.Studies have shown that autophagy can inhibit tumor growth in the development of cancer.Ferroptosis is a type of regulatory cell death distinct from autophagy,which is characterized by accumulation of iron,accumulation of reactive oxygen species,increase of lipid peroxides and decrease of antioxidant capacity.It has been found that ferroptosis can inhibit the growth of lung cancer,breast cancer,and other cells.At present,there is increasing evidence that the occurrence of ferroptosis depends on the activation of autophagy and can be regulated through the AMPK/mTOR/p70S6K pathway.In this study,the inhibitory effect of AIL on cell growth was detected in NSCLC cell lines A549 and HCC827.The autophagy and ferroptosis induced by drug administration in each group were observed.The rescue experiment after adding autophagy/ferroptosis inhibitors confirmed that AIL induced ferroptosis through autophagy,thereby inhibiting the growth of NSCLC cell.In addition,we preliminarily explored whether the potential mechanism of AIL was related to the regulation of AMPK/mTOR/p70S6K autophagy pathway.This study is expected to provide a new direction for the treatment of NSCLC and provide a theoretical basis for the anti-tumor effect of AIL.Objective:To investigate whether AIL inhibits the growth of NSCLC cells through autophagy-dependent ferroptosis,and to explore the effect of AIL on the AMPK/mTOR/p70S6K signaling pathway.Methods:1.MTT assay was used to detect the cell viability of human normal lung epithelial cell BEAS-2B cells and five NSCLC cells（A549,HCC827,H1975,H226,H157 cells）after treatment with different concentrations（0,0.2,1,2,10,20,40μM）of AIL for 24 h,and the two cells with the greatest impact on cell viability were found for subsequent experiments.2.MTT,colony formation assay and Ed U assay were used to detect the proliferation of A549 cells and HCC827 cells under different concentrations of AIL（0,0.2,1,2μM）.3.The expression of ferroptosis-related proteins was detected by ferroptosis detection kit and Western blotting to observe whether AIL induced ferroptosis in NSCLC cells.4.After AIL and ferroptosis inhibitor（1μM Fer-1）were added to each group of cells,the expression of ferroptosis-related proteins was detected by ferroptosis detection kit and Western blotting,and the ferroptosis effect of AIL-induced NSCLC cells was observed by recovery experiment.5.After adding AIL and ferroptosis inhibitor（1μM Fer-1）to each group of cells,MTT,colony formation assay and Ed U assay were used to detect cell proliferation,and rescue experiments confirmed that AIL inhibited the growth of NSCLC cells through ferroptosis.6.The expression of autophagy-related proteins（p62,ATG5,Beclin1,LC3B）was detected by immunofluorescence and Western blotting to observe whether AIL caused autophagy in NSCLC cells.7.After adding AIL and autophagy inhibitor（200 n M Baf-A1）to each group of cells,the expression of ferroptosis-related proteins was detected by ferroptosis detection kit and Western blotting.The rescue experiment showed whether AIL could induce autophagy-dependent ferroptosis in NSCLC cells.8.After adding AIL and autophagy inhibitor（200 n M Baf-A1）to each group of cells,MTT,colony formation assay and Ed U assay were used to detect cell proliferation.Rescue experiments confirmed that AIL inhibited the growth of NSCLC cells through autophagy-dependent ferroptosis.9.After treatment with different concentrations of AIL（0,0.2,1,2μM）or mTOR activator（2μM MHY1485）,the expression of key proteins and downstream proteins in the AMPK/mTOR/p70S6K pathway was detected by Western blotting..Results:1.The IC₅₀value of AIL on BEAS-2B cells was（219.902±163.321）μM.AIL could inhibit the viability of five NSCLC cells（A549,HCC827,H1975,H226,H157 cells）,and the inhibitory effect on the viability of NSCLC cells increased with the increase of drug concentration.Among them,AIL had the most obvious inhibitory effect on A549 cells and HCC827 cells（F=39.290,P<0.001）.The IC₅₀value of A549 cells was（1.026±0.203）μM,and the IC₅₀value of HCC827 cells was（0.625±0.338）μM.2.AIL could significantly inhibit the proliferation and clone formation of A549 cells and HCC827 cells in a concentration-dependent and time-dependent manner.3.AIL could induce ferroptosis in A549 cells and HCC827 cells,and by adding Fer-1,it was verified that AIL inhibited NSCLC cell growth by inducing ferroptosis.4.AIL could promote autophagy in A549 cells and HCC827 cells.It was observed that the average fluorescence intensity of LC3B gradually increased and the average fluorescence intensity of p62 gradually decreased in immunofluorescence experiments.In the Western blot experiment,the expression of p62 protein decreased,and the expression of ATG5,Beclin1 and LC3B protein gradually increased.5.AIL inhibited the growth of NSCLC cells through autophagy-dependent ferroptosis:Baf-A1 partially reduced the promoting effect of AIL on ferroptosis,and Baf-A1 partially rescued the inhibitory effect of AIL on the proliferation of NSCLC cells.6.Different concentrations of AIL regulated the AMPK/mTOR/p70S6K pathway,and MHY1485 partially recovery the expression of AMPK/mTOR/p70S6K pathway and downstream proteins by AIL.Conclusion:1.AIL inhibited the growth of NSCLC cells by inducing autophagy-dependent ferroptosis.2.AIL may regulate AMPK/mTOR/p70S6K signaling pathway.AIL may regulate the classical signaling pathway AMPK/mTOR/p70S6K pathway of autophagy and affect the expression of downstream ferroptosis proteins.