Study On Mechanism Of Myricetin Inducing Apoptosis Of Colorectal Cancer SW620 Cells Through ROS-mediated AKT/GSK-3β Pathway

Objective:Colorectal cancer(CRC)is one of the common malignant tumors in the human gastrointestinal tract,is the world’s third largest cancer.Globally,about 4.2 percent of people will be diagnosed with colorectal Cancer during their lifetime,according to the National Cancer Institute.Over 1.3 million people worldwide are jeopardized by colorectal cancer each year,serious impact on patient health.In addition,cases caused by controllable risk factors such as unhealthy eating habits,excessive smoking and lack of exercise accounted for more than 50% of the entire cases in the past two decades.The mainstream means of treating most cases of colon cancer are still surgery and chemotherapy.And although these means in clinical treatment and application have acquireed good outcome,the five-year survival rate of advanced patients is only 13%.Therefore an urgent is needed to exploit novel drug with low toxicity,efficient and inexpensive.It is reported that many natural compounds or Chinese herbal medicines have been used in the treatment of cancer,such as Pristimerin,Costunolide,etc.Myricetin(Myr)is a kind of polyhydroxyl flavonoids extracted from bayberry bark,which has anti-tumor,anti-viral,anti-bacterial,anti-inflammatory,weight loss and other effects.Currently Myr has been indicated to restrain the growth of a variety of tumor cells,such as ovarian,liver,etc.However,the latent molecular mechanism of Myr in colorectal cancer remains unclear.Therefore,in this study,colorectal cancer SW620 cells were choosed to investigate the mechanism of Myr activated apoptosis,in the hope of providing a new theory for the application of Myr in the treatment of colorectal cancer,making it a lead compound for the treatment of colorectal cancer.Method:Colorectal cancer SW620 cells were handled using different concentrations of Myr and examined as follows:1.The morphology of SW620 cells were observed and photographed using an inverted microscope,and then the effects of Myr on the viability of SW620 cells were checked by the MTT means.2.SW620 cells were examined by flow cytometry for cell cycle distribution,progression of apoptosis and intracellular reactive oxygen species(ROS)accumulation using PI dyeing,annexin V-FITC and PI double dyeing and DCFH-DA treatment.After DAPI dyeing,the influence of Myr on SW620 cells were surveyed using an inverted fluorescence microscope.3.The expression alter of cycle related proteins cyclin A2 and p21,apoptosis related protein cleaved PARP,autophagy related protein beclin-1 and lc3 b,and Akt/GSK-3β pathway related proteins were proved by Western blot(WB).4.SW620 cells were stimulated by NAC(N-acetyl-L-cysteine,ROS scavenge)and3-MA(3-Methyladenine,autophagy inhibitor)in advance,and then handled with Myr.MTT means was used to indicate the influence of eliminating ROS and restraining autophagy on cell proliferation.WB experiment was used to detect the effect of eliminating ROS on autophagy proteins and AKT/GSK-3β pathway protein expression.Results:1.The MTT assay indicated that Myr in a concentration dependent manner markedly restrained the proliferation of SW620 cells.It was surveyed with an inverted microscope that the number of SW620 cell deaths was positively correlated with the Myr concentration.2.Flow cytometry results suggested that Myr mediated S phase arrest in SW620 cells.It was concentration gradient dependent.WB experiments certificated that Myr downregulated the expression levels of Cyclin A2 and upregulated p21 protein in SW620 cells.3.Flow cytometry results suggested that Myricetin could induce SW620 cell apoptosis.The results of DAPI staining indicated that the number of intense blue fluorescent dots inside SW620 cells continuously increased as the concentration of Myr increased.According to the WB results,Myricetin could upregulate the expression of apoptosis related protein Cleaved PARP in SW620 cells.4.Flow cytometry certified that with the increase of Myr concentration,intracellular ROS levels was rised.While MTT results indicated that addition of NAC pretreatment attenuated the extent of Myricetin inhibition on SW620 cell proliferation.5.According to the WB results,Myr could block the phosphorylation of Akt and GSK3β in the Akt/GSK3β pathway in SW620 cells,but the expression levels were upregulated after pretreatment with the addition of NAC.Whereas the expression of Akt and GSK3β did not change either before or after NAC pretreatment.6.WB assay revealed that with the increase of Myr concentration,the protein expressions of Beclin-1 and LC3B-Ⅱ were both rised,Whereas its expression was downregulated after after addition of NAC pretreatment.Then autophagy inhibitor 3-MA pretreatment attenuated the inhibition of SW620 cell proliferation by Myr.Conclusions:Based on the above results demonstrated that Myr could inhibit SW620 cell proliferation and induce cell apoptosis.Further studies concluded that Myr induced SW620 cells to arrests in S phase through downregulation of Cyclin A2 and upregulation of p21.Moreover,Myr could induce the increase of intracellular ROS,and downregulate the expression of p-AKT and p-GSK3β through ROS mediation,thereby inhibiting the AKT/GSK3β pathway that cells depend on for survival to induce cell apoptosis.Myr could also upregulate the expression of Beclin-1 and LC3B-Ⅱ through ROS mediation to induce cell autophagy and promoted SW620 cell apoptosis.This study indicated that Myr can be used as a potential lead compound,providing a novel strategy for the application of Myr in the treatment of colorectal cancer.