The Effect And Mechanism Of Celastrus Orbiculatus Extract On The Inhibition Of Colorectal Cancer By Targeting The CSN6 Protein

IntroductionThe incidence of colorectal cancer ranked the third of the commom cancer worldwidly,and next to gastric cancer and esophageal cancer.In Shang hai,the incidence of colorectal cancer is 23.5/100,000 that also ranked the third of all kinds of the tumor and the mortality rate is 11.8/100,000.The main symptoms of colorectal cancer are diarrhea,constipation,lump,hematochezia,anemia,intestinal obstruction.On terminal cancer,cancer cells spread to the liver,lungs and abdominal cavity.The primary diagnosis of colorectal cancer includes digital rectal examination,X-ray,endoscopy and biopsy pathology examination,and so on.About half of patients with colorectal cancer receive about 5 year survival.Surgical removal and radiation therapy is the main treatment of colorectal cancer,but the effect of therapy alone is not good.An increasing number of botanical compounds are applied in clinical treatment of cancer in order to prevent recurrence and metastasis of carcinoma,relieve patients’ pain,improve the quality of life.Newman has reported that about 73%of the anticancer drugs is belong to or based on the natural drugs during 1981 to 2006.For its antitumor effects,such as the multiple targets,multiple links and multi-channel,natural drugs become the hot topics in the study of cancer.Celastrus orbiculatus Thunb(Celastraceae),a traditional Chinese medicinal herb,which is widely distributed in native,and its stems,roots,leaves and fruits have been used as a folk medicine against cancer diseases.A variety of ingredients have been separated from its stem,root,leave and fruit by modern technology,such as the terpene,triterpene,flavonoids,organic acids,steroids,tannin,etc.The effect of the ethyl acetate extract(COE),such as anti-tumor,anti-inflammatory,antiviral,antibacterial and antioxidant,has been widely confirmed.Previous studies focused its effect on the proliferation,apoptosis,invasion and metastasis and angiogenesis of tumor cells,but poorly focused on the autophagy.The study showed that COE has significant effect against proliferation of HT-29 cells and inducing apoptosis.We also found that the COE can reduce expression level of CSN6.CSN6,the sixth subunits of CSN,plays an important role in the development of mammals.Deletion of CSN6 will result in the death of the embryo,p53,p21 and CSN6 appear overexpression.The main function of CSN6 is to regulate the activity of ubiquitin ligase(ubiquitin-ligase enzyme,E3)in the process of the ubiquitination.However,the role of CSN6 on the apoptosis and autophagy in colorectal cancer is not very clear.Therefore,this experiment intends to study whether the CSN6 is involved in regulating the apoptosis and autophagy induced by COE in HT-29 cells.This study includes three parts,as follows:The first part:The expression of CSN6 and Myc in colorectal cancer and its clinical significanceObjective:To study the expression of CSN6 and Myc in colorectal carcinoma and adjacent tissue to carcinoma,analyze the relationship between CSN6 and Myc during the occurrence,development and metastasis of colorectal cancer.Methods:To detect the level of the Mrna,protein of CSN6 and Myc in colorectal cancer and adjacent tissue to carcinoma by RT-PCR and IHC,48 cases of colorectal carcinoma confirmed by pathology from January 2012 to December 2014 were collected from Subei Hospital affiliated to YZU.To analyze the data with the X2 test or Fisher’s exact probability method,and all the data were performed statistically by SPSS 19.0 software.Results:IHC showed that the positive reaction of Myc and CSN6 was brown and yellow,mainly in the cytoplasm or nucleus.CSN6 positive expression rate was 43%(21/48)in colorectal carcinoma and 10.4%(5/48)in the adjacent tissues.Overexpression rate of Myc is about 83.3%,higher than that in the adjacent tissues with significant difference(P<0.05).qPCR showed that the expression of CSN6 mRNA and Myc mRNA in colorectal carcinoma was significantly higher than that in the adjacent tissues(P<0.05).The expression of CSN6 mRNA and Myc mRNA was positively correlated in colorectal carcinoma(p<0.05).Conclusion:(1)The expressions of CSN6 mRNA,Myc mRNA in colorectal cancer are more than in adjacent tissue to carcinoma.The expression of CSN6 mRNA and Myc mRNA was positively correlated in colorectal carcinoma(p<0.05).(2)The expressions of CSN6,Myc protein in colorectal cancer are more than that in adjacent tissue to carcinoma.The second part:The study of apoptosis and autophagy inducd by COE in human colon cancer cell line HT-29Objective:To investigate the mechanism of COE inhibiting proliferation of human colorectal cancer cell line HT-29 from the relationship between autophagy and apoptosis.Methods:HT-29 cells were treated with different concentrations of COE in vitro.And then examine the cell survival rate by MTT method,the cell morphology by electron microscopy(TEM),cell apoptosis and cell cycle by flow cytometry(Annexin V,FITC-PI double staining and PI staining respectively),WB examine the expression of some proteins associated with apoptosis and autophagy induced by COE in HT-29 cells.For further studying the effect of autophagy induced by COE on apoptosis,HT-29 cells treated with 80 mg/1 COE combined rapamycin(autophagy activation agent)and 3-MA(autophagy inhibitor)respectively,and then exam the proliferation of HT-29 cells by MTT.Results:The COE can inhibit the proliferation of HT-29 cells in the time and dose-dependent manner,upon treatment with 149.65±0.49 mg/1 of COE for 24 h,the cell viability reduced to 50%.With the increase of concentration of COE,TEM showed the autophagy and apoptosis increased obviously.The cell cycle also stayed in G0G1 phase in HT-29 cells treated with COE.Fluorescence microscopy revealed that AVO formation induced by 80 mg/1 COE was reduced in the presence of 3-MA and increased in the presence of rapamycin,additionally,WB analysis showed that the level of LC3-II went up in the presence of rapamycin and down in the presence of 3-MA upon treatment with 80 mg/1 COE.Conclusion:Autophagy and apoptosis induced by COE synergize to inhibit growth in HT-29 cells.The third part:The molecular mechanisms of apoptosis and autophagy induced by COE in HT-29 cellsObjective:To determine whether the Akt-CSN6-Myc signaling axis is involved in COE-induced apoptosis and autophagy in HT-29 cells.Methods:At first,examine the expressions of Akt,p-Akt,CSN6,Myc,and p53 by western blot,and the level of CSN6 mRNA by qRT-PCR.For studying the impact of CSN6 on COE-induced apoptosis and autophagy in HT-29 cells,CSN6 overexpression plasmid was successfully constructed and HT-29-MIGR1-CSN6 cell lines were obtained.HT-29 and HT-29-MIGR1-CSN6 cells were treated with 80mg/L COE for 24h,WB was used to detect the expressions associated with CSN6-Myc signaling axis,and cell survival rate was detected by MTT assay.To further study the impact of Akt kinase on CSN6-Myc signaling axis,the plasmid of Akt overexpression and HT-29-MIGRl-Akt cell were obtained,HT-29-MIGRl-Akt group and Akt kinase inhibition group(treated with PI3K inhibitor LY294002)were treated with 80mg/l COE for 24 h,WB examine the expression of p-Akt,CSN6,Myc,and p53.In addition,explore the effect of PI3K-Akt-mTOR signaling pathways on apoptosis and autophagy induced by COE in HT-29 cells.Results:The levels of Akt-Thr308,CSN6,and Myc protein reduced,and the expression of p53 increased compared with control group,but the level of CSN6 mRNA was not significantly altered after treatment with COE(P>0.05),the overexpression of CSN6 weakened the effect of COE treatment on HT-29 cells,indicated that COE can down-regulate the expression of CSN6 protein.Compared with control group,the expression of Akt,p-Akt,CSN6 and Myc were significantly increased,the expression of p53 decreased in HT-29-MIGRl-Akt group,akt kinase inhibitor group showed the opposite results,but COE could decrease the expression of p-Akt,CSN6,Myc protein and increased the expression of p53 protein in HT-29-MIGR1-Akt cells,indicated that COE can inhibit the phosphorylation of Akt,which down-regulate the level of CSN6 protein.In addition,COE-treatment decreased the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin(mTOR)and p70 ribosomal protein S6 kinase(p70S6K).Conclusion:COE-induced apoptosis and autophagy is involved in the Akt-CSN6-Myc signaling axis in HT-29 cells.The inhibition of PI3K-Akt-mTOR signaling pathway is also one of the mechanisms that COE suppresses colorectal cance.