Study On The Pharmacological Mechanism Of Moracin N Against Lung Tumor Through Apoptosis And Autophagy Induction

Cancer is the second leading cause of death.Lung cancer is the most common cancer with the highest incidence and the highest mortality among cancer around the world,which is a serious threat to human health.Lung adenocarcinoma represents most of lung cancer.Clinically,the treatment of lung cancer includes surgical resection,radiotherapy,chemotherapy,targeted treatment,immunotherapy and so on.However,due to the difficulties in early diagnosis,the side effects of radiotherapy and chemotherapy,the emergence of tumor resistance and the dark side of emerging therapies,the treatment of lung adenocarcinoma is still insufficient.Mulberry is a traditional Chinese herb with its leaves and roots can be used as medicine to treat lung diseases such as pulmonary nodules.Moracin N(MAN)is a small molecular polyphenol extracted from mulberry leaves,which has antiviral and antioxidant effects.However,its anti-tumor effect is rarely reported.In this paper,the inhibition effect of MAN on ten human cancer cell lines was evaluated by MTT assay and clony formation assay.To explore the pharmacological mechanism of MAN against lung adenocarcinoma,wound healing assay,cell cycle analysis,apoptosis rate detecting,morphology of cells and organelle,mitochondrial membrane potential detecting,reactive oxygen species test,immunofluorescence,Western blot,as well as introduction of autophagy inhibitor and RNA interference were applied.The results are as follows:1.MAN inhibited the growth and proliferation of cancer cells.MTT assay showed that MAN could inhibit the viability of cancer cells in a dose-depedent and time-dependent manner.Clony formation assay showed that MAN could inhibit the proferation of A549 and PC9 cells.2.MAN induced mitochondrial apoptosis and cytotoxic autophagy through ROS accumulation.Wound healing assay showed that MAN could inhibit A549 migration.Besides,MAN induced mitochondria fragmentation,loss of mitochondrial membrane potential and release of cyt c and eventially activated A549 mitochondrial apoptosis through cyt c/caspase-9/caspase-3 pathway.What's more,MAN could induce autophagy via AKT/mTOR pathway inhibition and promote the fusion of autophagy and lysosome.Autophagy inhibitors and Atg5 protein knowdown significantly reduced MAN cytotoxicity,indicating that autophagy can promote MAN induced cell death.3.MAN inhibited PC9 cells migration and activated mitochondrial apoptosis and cytotoxic autophagy cell death and induced endoplasmic reticulum stress.MAN could reverse the EMT process of PC9 cells to inhibit migration.Besides,MAN regulated Bcl-2 family proteins and induced PC9 cells apoptosis.The down-regulation of p-mTOR indicated that PC9 cells autophagy activation was related to mTOR pathway.In addition,the up regulation of GRP78 protein resvealed that MAN induced ER stress in PC9 cells and may induce apoptosis by PERK/eIF2?/CHOP pathway.Our results show that MAN inhibits NSCLCs proliferation by activating mitochondrial apoptosis and cytotoxic autophagy,which provides a theoretical basis for the development of new drugs for lung cancer treatment.