Mechanism Of Tetrandrine Inhibiting Autophagic Degradation And Sensitizing Vincristine To Inhibit Cell Proliferation In Gastric Cancer Cells

BackgroundRecent studies have shown that autophagy inhibitor can sensitize tumor chemotherapeutic drugs to inhibit tumor cell proliferation,but so far,most autophagy inhibitors have been used only as research tools,and few autophagy inhibitors applied in clinical practice,mainly chloroquine and its derivative hydroxychloroquine.It is important to find new autophagy inhibitors,study their mechanism of action,and develop new tumor chemotherapeutic drug sensitizers.Increasing studies have shown that the active ingredients extracted from our traditional Chinese medicine can be used as autophagy inhibitors with the advantages of high efficiency and low toxicity.Tetrandrine,an alkaloid extracted from the roots of traditional Chinese medicine Han Fangji,has been used clinically for many years,mainly for the treatment of hypertension,with less toxic side effects.We found that tetrandrine can inhibit the degradation of autophagy in a variety of tumor cells.Tetrandrine inhibits the fusion of autophagosomes-lysosomes and sensitizes vincristine to inhibit cell proliferation in gastric cancer cells.However,the mechanisms by which tetrandrine inhibits the fusion of autophagosomes-lysosomes and sensitizes vincristine to inhibit cell proliferation in gastric cancer cells remain unclear.In this study,we investigated the mechanisms of vincristine-inhibited autophagic degradation and sensitized vincristine to inhibit cell proliferation in gastric cancer cells.ObjectivesTo elucidate the mechanism by which tetrandrine inhibits autophagic degradation and sensitizes vincristine to inhibit cell proliferation in gastric cancer cells,to develop a novel sensitizer of tumor chemotherapy drugs,in order to provide a novel therapeutic strategy for treatment of gastric cancer.MethodsThe expression of autophagy-,apoptosis-and cell cycle-related proteins were detected by Western blot;The changes of cellular autophagic flow,co-localization of autophagosomes and lysosomes,and mitochondrial morphology were observed by laser confocal microscopy;The interactions between proteins were observed by immunoprecipitation;Cell proliferation was detected by MTT assay and crystal violet assay;Cell apoptosis and cell cycle were detected by flow cytometry;The changes of autophagosomes were observed by transmission electron microscopy.Results1.Tetrandrine inhibits autophagic degradation in gastric cancer cellsTetrandrine can inhibit autophagy degradation of various tumor cells,resulting in increased autophagosome and increased expression of autophago-related proteins P62 and LC3 B in gastric cancer SGC-7901/VCR cells,and its effect was similar to that of the autophagy inhibitor Bafilomycin A1,but opposite to that of the autophagy inducer Rapamycin.The double fluorescent LC3 plasmid transfection assay confirmed that tetrandrine blocked the autophagic degradation in gastric cancer SGC-7901/VCR cells.2.Tetrandrine inhibits autophagosome-lysosome fusion in gastric cancer cellsThe plasmid transfection assay and protein immunoprecipitation assay revealed that tetrandrine prevented the binding of SNAP29 to VAMP8,which in turn interfered with the formation of SNARE protein complex,inhibited autophagosome-lysosome fusion and suppressed autophagic degradation in gastric cancer SGC-7901/VCR cells.3.Tetrandrine sensitizes vincristine to inhibit cell proliferation in gastric cancer cellsMTT assay found that tetrandrine could sensitize vincristine to inhibit cell proliferation in gastric cancer SGC-7901/VCR cells and esophageal cancer EC-109/VCR cells,and sensitize doxorubicin to inhibit cell proliferation in gastric cancer SGC-7901/ADR cells and human myeloid leukemia K562/ADR cells.Flow cytometry and Western blot assay revealed that tetrandrine could sensitize vincristine to induce apoptosis in gastric cancer SGC-7901/VCR cells and activate the apoptotic pathway.Further study revealed that apoptosis of gastric cancer SGC-7901/VCR cells was closely associated with excessive accumulation of autophagosomes.4.Tetrandrine sensitizes vincristine affects MAPK signaling pathway and cell cycle in gastric cancer cellsWestern blot assay revealed that the combination of vincristine with tetrandrine increased phosphorylation of p38 and JNK and decreased phosphorylation of ERK in gastric cancer SGC-7901/VCR cells.Pretreatment of SGC-7901/VCR cells with P38 or JNK inhibitors attenuated the combination-induced apoptosis(P < 0.01),while pretreatment with ERK inhibitor enhanced the combination-induced apoptosis(P < 0.01).Flow cytometry and Western blot assays revealed that combined treatment with vincristine and tetrandrine resulted in S/G2 cell cycle arrest in SGC-7901/VCR cells.Conclusion1.Tetrandrine is an inhibitor of autophagy degradation of various tumor cells.It impedes the binding of SNAP29 to VAMP8 and interferes with the formation of SNARE protein complex,resulting in inhibiting the autophagosome-lysosome fusion in gastric cancer SGC-7901/VCR cells,leading to suppress the autophagic degradation,and culminating in the accumulation of autophagosome.2.As a sensitizer of many chemotherapeutic drugs for tumor,tetrandrine synergistically with vincristine led to the accumulation of a large number of autophagosomes in gastric cancer SGC-7901/VCR cells,activates p38 and JNK pathways of MAPK signaling pathway,inhibits ERK pathway,and activates the apoptotic pathway,and leads to mitochondrial damage,cell cycle arrest,apoptosis,results in inhibition of cell proliferation.