Study On The Apoptosis And Autophagy Of Colon Cancer Cells Induced By Myricetin Via Inhibiting PI3K/Akt/mTOR Signalling

BACKGROUND and PURPOSEAccording to the American Cancer Society,1.8 million people will be diagnosed with colon cancer in the United States in 2018,and more than 800,000 people will die from colon cancer.According to data released by the National Cancer Center of China,the incidence of intestinal cancer continues to rise,which is the third most common malignant tumor in China,which seriously threatens national health.Although the clinical treatment of colon cancer has been developed,the five-year survival rate of patients with colon cancer is still at a low level.Natural products play a pivotal role in the history of human cancer treatment.A large number of natural compounds represented by paclitaxel,camptothecin and podophyllotoxin have been used in the clinical treatment of tumors.Finding substances with anticancer activity from natural products has become a research hotspot.Myricetin was originally a natural pigment extracted from the bark of bayberry,which has anti-inflammatory,regulates physiological activities,inhibits tumor cell growth and even antibacterial action Studies have shown that myricetin can inhibit the growth of a variety of human tumor cells,including colon cancer.Previous studies have shown that the mechanism by which myricetin inhibits the growth of human tumor cells includes inhibition of tumor cell cycle,induction of tumor cell apoptosis,inhibition of tumor angiogenesis and metastasis,and affecting tumor cell signaling pathways.In this paper,the mechanism of apoptosis induced by myricetin in human colon cancer cells was studied.The relationship between autophagy and apoptosis of human colon cancer cells induced by myricetin was also discussed.METHODS1.Myricetin inhibited the proliferation of four human colon cancer cells,HCT116,SW620,SW480 and HT-29.In this paper,the MTT method was used to observe the inhibition of myricetin on the proliferation of the above four human colon cancer cells,in order to screen the suitable experimental cell lines and experimental conditions(including drug concentration and administration time).2.To test the effects of myricetin on the apoptosis of two human colon cancer cells,HCT116 and SW620.The changes of nucleic acid after apoptosis was detected by Hoechst 33342 staining.Proportion of apoptosis induced by myricetin was detected using flow cytometry.The expression of apoptosis-related proteins Bax and Bcl-2 in cells treated with myricetin was detected by Western blotting.3.To investigate the effects of myricetin on autophagy of two human colon cancer cells,HCT116 and SW620.The formation of autophagosomes in colon cancer cells treated with myricetin was detected by colorimetric electron microscopy.The expression of autophagy-related proteins LC3-?/?-actin and Beclin-1 in the cells treated with myricetin was detected by Western blotting.4.Apoptosis and autophagy of two human colon cancer cells,HCT116 and SW620 were induced by treating 3-MA and myricetin.Two colon cancer cells were pretreated with autophagy inhibitor 3-MA,and the proliferation of two human colon cancer cells was detected by MTT assay.The expression of apoptosis-related proteins Bax and Bcl-2 in the cells treated with myricetin was detected by Western blotting.5.To investigate the effects of myricetin on the PI3K/Akt/mTOR pathways in two human colon cancer cells,HCT1 16 and SW620.The expression levels of p-PI3K,p-Akt,p-mTOR,PI3K,Akt and mTOR in the cells treated with myricetin were detectedby Western blotting.RESULTS 1.Myricetin can significantly inhibit the proliferation of four human colon cancer cells,HCT116,SW620,SW480 and HT-29.Among them,the inhibition rate of HCT116 and S W620 human colon cancer cells were the highest.And the best inhibition effects could be achieved by treatment with 100 ?mol/L myricetin for 48 h.2.Myricetin can induce apoptosis of two human colon cancer cells,HCT116 and S W620.Myricetin up-regulates the expression of the apoptosis-related protein Bax and down-regulates the expression of Bcl-2 protein in a dose-dependent manner.3.Myricetin can induce autophagy in two human colon cancer cells,HCT116 and SW620.Myricetin increased the ratio of LC3-?/?-actin protein expression and the expression level of Beclin-1 protein in colon cancer cells in a dose-dependent manner.4.The use of 3-MA can inhibit the autophagy of colon cancer cells induced by myricetin and increase the apoptosis of colon cancer cells.5.Myricetin can inhibit the expression levels of p-PI3K,p-Akt and p-mTOR,but has no effect on the expression levels of PI3K,AKT and mTOR proteins.CONCLUSIONThis study demonstrates that myricetin could induce apoptosis and autophagy in HCT116 and SW620 human colon cancer cells by inhibiting the PI3K/Akt/mTOR pathway.Inhibition of autophagy by autophagy inhibitors 3-MA can increase myricetin-induced colon cancer cell apoptosis.SIGNIFICANCEThis study demonstrates for the first time that myricetin could induce autophagy of human colon cancer cells,and inhibition of this autophagy can increase the apoptosis of human colon cancer cells induced by myricetin.This suggests that myricetin may be a candidate for colon cancer chemotherapy through its own anti-tumor effects or in combination with autophagy inhibitors.Our study might lay a foundation for further exploration of the application of myricetin in colon cancer.