Effect And Mechanism Of Chemotherapy And Immunotherapy For Lung Cancer With Inhibition Of Autophagy Potentiation By Fangchinoline

ObjectiveLung cancer remains the world’s leading causes of morbidity and mortality,and is one of the major problems threatening human life and health.Conventional therapies and promising immunotherapies have failed to achieve ideal clinical outcomes due to drug resistance.Therefore,it is urgent to find new strategies to overcome drug resistance and improve the effect of clinical drugs.Autophagy is a process of lysosomal hydrolases decompose and recycle damaged or excess organelles,which help cells cope with external pressure and maintain cell homeostasis.It is reported that protective autophagy induced by cisplatin during the treatment of lung cancer is one of the key mechanisms leading to drug resistance.In addition,tumor cells proliferate rapidly and are often in an autophagy-activated state due to nutrient deprivation,which can participate in the immune response of tumor cells and lead to immune tolerance by regulating antigen processing and presentation processes of MHC-I.Thus,finding drugs that effectively inhibit autophagy and revealing its mechanism of action become a new breakthrough in current adjuvant drug therapy for tumors.The combination therapy of active ingredients of Chinese herbs and traditional chemotherapeutics are widely used to overcome the multidrug resistance of cancer,which have achieved remarkable results.Fangchinoline has anti-inflammatory,antibacterial and other pharmacological activities,which is a bibenzyl isoquinoline alkaloid extracted from Fangchi.In recent years,it has been reported that fangchinoline has antitumor effects in a variety of tumors,but the mechanism is not clear.In our study,we explored that the effect and mechanism of fangchinoline on inhibition of autophagy in lung cancer cells,synergistic chemotherapy and immunotherapy in vivo and in vitro by means of cell biology experiment,molecular biology technique and immunology technique,so as to provide new ideas and theoretical basis for improving the therapeutic effect of patients and combining traditional Chinese medicine with modern medicine.Methods1.Immunofluorescence and Western blot were used to detect the effect of fangchinoline on autophagy of lung cancer cells.NCI-H1299/NCI-H1975 cells were transfected with mCherry-GFP-LC3 plasmid to observe the changes of autophagy flow.A stable strain of NCI-H1299-GFP-LC3 lung cancer cells was constructed,LysoBrite dye was used to label lysosomes,and the effect of fangchinoline on the fusion of autophagosome and lysosome in lung cancer cells was observed by Fluorescent confocal microscope.Lysosome fluorescence probe LysoTracker(red)assay and acridine orange(AO)lysosome staining assay were used to detect the effects of fangchinoline on lysosome acidification in lung cancer cells.2.Fangchinoline combined with cisplatin/paclitaxel were used to interfere NCI-H1299/NCI-H1975 lung cancer cells,and CCK8 assay was used to detect the effect of the combination on the proliferation of lung cancer cells;AnnexinV/PI assay was used to detect the effect of combination of two drugs on apoptosis of lung cancer cells.DCFH-DA staining was used to detect the effect of fangchinoline combined with cisplatin on ROS levels in lung cancer cells.CCK8 assay was used to determine whether ROS clearance could reverse the inhibitory effect of fangchinoline combined with cisplatin on lung cancer cell proliferation.3.The xenograft tumor model of nude mice for lung cancer was established to observe the effects of fangchinoline combined with cisplatin on tumor volume and tumor weight.4.LLC-OVA lung cancer cells were co-cultured with OT-Ⅰ mice spleen CD8+T lymphocytes.AnnexinV/PI assay was used to detect the killing effect of fangchinoline combined with anti-PD1 antibody on LLC-OVA cells.CD8+T lymphocytes of OT-Ⅰ mice and fangchinoline with treated LLC-OVA cells co-cultured,AnnexinV/PI,Hoechst/PI double staining,flow cytometry and ELISA were used to detect the killing effect of CD8+T lymphocytes on LLC-OVA cells.5.Western blot,immunofluorescence assay and flow cytometry were used to detect the changes of MHC-Ⅰ and LC3 expression in BEAS-2B and NCI-H1299,NCI-H1975 and A549 cells,as well as the membrane expression of MHC-Ⅰ on cell surface.LLC-OVA-ATG4BC74A cells were constructed by lentivirus transfection to detect the effects of autophagy inhibition on MHC-Ⅰ molecules and antigen presentation in lung cancer cells.And siRNA transfection assay was used to detect the effect of inhibition of autophagy related receptors NBR1,NDP52,p62 and OPTN on the MHC-Ⅰ and the proportion of MHC-Ⅰ antigen peptide complex on lung cancer cell surface.Intervention of fangchinoline on human lung cancer cells NCI-H1299 and NCI-H1975.The changes of MHC-Ⅰ protein levels and MHC-Ⅰ molecules on cell surface were determined by Western Blot detection and flow cytometry.Fangchinoline was used to intervene LLC lung cancer cells and autophagy related indexes were detected by immunofluorescence and Westernblot.Flow cytometry was used to determine the effect of fangchinoline intervention on the ratio of MHC-Ⅰ molecules to MHC-Ⅰ antigen peptide complex on cell surface.6.C57BL/6J mouse lung cancer transplant tumor model was constructed to detect th e effect of fangchinoline base enhancing anti-PD1 antibody on the tumor and the vo lume of mouse lung cancer transplant tumor.HE staining observed cell morphology and immunohistochemistry detected infiltration of CD8+T lymphocytes.Flow cytomet ry was used to detect the effect of CD8+T lymphocytes activation in spleen of tumo r mice.Results1.Fangchinoline inhibited lysosome acidification in lung cancer cells and blocked late autophagy flow in lung cancer cells.2.Fangchinoline combined with cisplatin inhibits the clearance of damaged mitochondria in lung cancer cells,resulting in excessive accumulation of intracellular ROS to synergistic killing effect.3.The results of fangchinoline combined with cisplatin in xenograft tumor models in nude mice confirmed that fangchinoline combined with cisplatin could enhance the killing effect of NCI-H1299 cells by reducing the growth rate of tumor volume and tumor mass in tumor mice.4.The level of MHC-Ⅰ decreased after autophagy activation in lung cancer cells.After siRNA interfered with NBR1,NDP52,p62 and OPTN autophagy related receptors in human lung cancer cells,the inhibition of si-NDP52 function led to the increase of MHC-Ⅰmolecules on the surface of human lung cancer cells,and the proportion of antigen peptide complex on the surface of mouse lung cancer cells increased after si-NDP52 transfection,which confirmed that the excessive degradation of MHC-Ⅰ in lung cancer cells was related to autophagy receptor NDP52.The inhibition of autophagy by fangchinoline can enhance the level of MHC-Ⅰ on the surface of mouse lung cancer cells and increase the proportion of MHC-Ⅰ antigen peptide complex,thus up-regulating the anti-tumor immune killing effect of OT-Ⅰ mouse over CD8+T cells activated by lung cancer cell antigen presentation.5.The synergistic effect of fangchinoline combined with anti-PD1 antibody in vitro confirmed that the combination of the two drugs promoted the immune killing effect of CD8+T lymphocytes of OT-Ⅰ mice on LLC-OVA cells.In addition,in the co-culture of LLC-OVA cells and CD8+Tcells of OT-Ⅰ mice,the immune killing effect of CD8+T lymphocytes of OT-Ⅰ mice on lung cancer cells was significantly enhanced,and the proportion of Granzyme+T lymphocytes and the content of IFN-y and TNF-α factors in the supernatant of co-cultured cells were significantly increased.It is confirmed that fangchinoline can enhance the immune killing effect of CD8+T lymphocytes of OT-Ⅰ mice on lung cancer cells.6.The results of fangchinoline combined with anti-PD1 antibody in mouse LLC transplanted tumor model showed that fangchinoline enhanced the killing effect of immune checkpoint inhibitor on LLC cells,and the number tumor-Ⅰnfiltrating CD8+T and the proportion activation of spleen CD8+T cells in mice increased significantly,indicating that the killing effect of fangchinoline on LLC cells may be related to anti-tumor immune mechanism.Conclusion1.Fangchinoline blocks the late autophagy flux of lung cancer cells by inhibiting lysosomal acidification.2.Fangchinoline combined with cisplatin can synergically inhibit the proliferation and promote apoptosis of lung cancer cells.Fangchinoline can enhance the killing effect of cisplatin on xenograft tumor of lung cancer in nude mice,which is consistent with the results of in vitro experiments.3.The decrease of MHC-Ⅰ in autophagy activated lung cancer cells is related to the autophagy related receptor NDP52.Fangchinoline inhibits autophagy and enhances MHC-Ⅰexpression and formation of MHC-Ⅰ antigen peptide complex on lung cancer cell surface,and enhancing lung cancer cell antigen presentation and CD8+T cell activation.4.Fangchinoline combined with anti-PD1 antibody had a synergistic effect on mouse transplanted tumor,and up-regulated the number of tumor-infiltrating CD8+T and the activation of splenic CD8+T cells.