The Inhibitory Effect Of Berberine On Endometrial Carcinoma Cell And Its Mechanism

Objective:Endometrial cancer is a serious threat to women's health as reproductive tract malignant tumors,the incidence increased year by year in recent years.Surgery is the main treatment,by now it's lack of effective preventive measures.Exposed to estrogen for a long time without progesterone antagonist is thought to be the main cause of endometrial carcinoma,and high blood pressure,obesity,diabetes is regarded as the risk factors of endometrial cancer,called"trilogy of endometrial carcinoma".Along with the increase of obese women in our country,the endometrial cancer incidence is also increase,and shows younger trend,many childless women with endometrial cancer or precancerous lesion lose their uterus and fertility.Therefore,the chemical prevent of endometrial cancer is very important.Epidemiological data indicate that obesity is at least related to 40%endometrial carcinoma,the incidence of endometrial cancer is 2-3 times in obesity higher than that in the general population for increased level of endogenous estrogen.Exposure to estrogen for long-term can lead to endometrial hyperplasia and potential atypical hyperplasia.Diabetes will increase the incidence of endometrial cancer has been fully confirmed.The risk of suffering endometrial cancer in Diabetic women is higher than women without diabetes.Some scholars think that endometrial carcinoma can be regarded as a set of metabolic abnormalities syndrome,and changes in lifestyle,diet,strengthening exercise,control of metabolic diseases such as obesity,diabetes,high blood pressure helps to reduce the occurrence of endometrial carcinoma.Due to the high incidence of endometrial carcinoma,and lack of effective treatment measures,so looking for effective preventive measures to reduce the incidence of endometrial cancer is important.Using safe and effective drugs as chemical prevention are very promising research direction,especially for early endometrial lesions and those female patients who want to reserve the reproductive function.Cancer chemoprevention was coined in 1976 by Michael Sporn,now the national cancer institute(NCI)and several other institutions and individuals recognized is defined as the use of natural,synthetic or biological substances to prevent,slow or reverse the process of cancer development,strategies to reduce cancer incidence and mortality rates.Berberine is a drug with multiple targets,the application of berberine in the body can lead to the change of gene expression,thus promotes high-energy mediated catabolism.Both animal experiments and clinical studies of berberine on glucose metabolism,lipid metabolism,diabetes treatment,the role of regulating endothelial function and cardiovascular system are obtained the satisfactory results.In conclusion,obesity and diabetes are high risk factors of endometrial cancer,controlling obesity,diabetes and other metabolic diseases could help reduce the occurrence of endometrial cancer.Berberine has very good treatment effect on glucolipid metabolic disorder disease such as obesity and diabetes,so we can try to use berberine for chemical prevention and treatment of endometrial cancer.This topic proposed through the study of berberine inhibiting endometrial cancer cell proliferation,and further study of its mechanism of action,to discuss the possibility of berberine application in the treatment of endometrial cancer.Methods:1.Study population:RL95-2,HEC-1-A and AN3 CA cell lines of endometrial carcinoma.A total of 15 EC tissues and matched normal endometrial tissues around the lesion were collected.All specimens were taken from patients undergoing EC surgery in Shengjing Hospital affiliated to China Medical University.Immediately after operation,it was frozen in liquid nitrogen and stored at-80 C until it was used.5-6week old nude mice.2.Study methods:(1)The first part:People endometrial carcinoma RL95-2 and HEC-1-A cell lines were incubated in vitro,with different concentrations of berberine in the two cell lines.To detect endometrial cancer cell proliferation inhibition by MTS method,and the effects of berberine on endometrial cancer cell cycle by cycleflow cytometry test.Then detect the change of apoptosis related proteins caspase-3 and caspase-9 induced by berberine by western blotting,and JC-1 staining method is used to study the mitochondrial membrane potential changes in the cell before and after berberine effect.Glucose oxidase method and lactate oxidase method is used to detect the change in glucose consumption and lactic acid production in endometrial cancer cells after berberine effect.Western blotting is used to test the expression of p-AMPK,AMPK,p-mTOR and mTOR before and after berberine effect,and then to add AMPK specific inhibitors into the cell,again test the protein expression changes of p-AMPK,AMPK,p-mTOR and mTOR in the cell.(2)The second part:A total of 15 EC tissues and matched normal tissues were from Shengjing Hospital,Human EC cells(AN3 CA and HEC-1-A)were cultured.Transfection of miR-101 mimic or miR-101 inhibitor with scrambled miRNA as a negative control was performed with Lipofectamine?2000.RT-qPCR method was used to calculate differences in mRNA expression.Cell viability was assessed with Cell Counting Kit 8(CCK8,Beyotime,Shanghai,China).The viability of the cells was analyzed after incubation for 72h.Invasion and migration activity of EC cells were analyzed by using 24-well trans-well chambers.Protein isolation and western blot analysis was used to detect the expression of protein COX-2,and PGE2 levels released in the medium were determined by ELISA.Luciferase Reporter Assay,Following transfection of pGL3-Basic-pri-miR-101-2 promoter and a control vector pRL-SV40into EC cells,cells were treated with BBR.Dual-Luciferase Reporter Assay were performed to measure the firefly and Renilla luciferase activities.Tumor xenograft,HEC-1-A cells were subcutaneously implanted into the right flank of five six-week old nude mice,When the tumor volume was around 150-200 mm~3,the mice were randomly grouped into three groups with 6 mice per group.The selected mice were orally administrated with 0.5%MC(vehicle control),BBR(15 mg/kg,p.o.qd)or BBR(50 mg/kg,p.o.qd),respectively.Tumor volume of each group was calculated by caliper every 3 days.EC lung metastases mouse model were established by tail vein injections of HEC-1-A cells into nude mice.After 4 weeks treatment,the lungs were excised,fixed and paraffin embedded for counting of macroscopic lung metastases.Results:The first part:The MTS results show that the proliferation rate of two kinds of cell were significantly lower after treated by berberine,and the rendering is time and dose dependent.Flow cytometry results showed that the G1/G0 cell proportion increased significantly,while in S and G2/M stage cells proportion is significantly reduced after the effect of berberine on two kinds of cells.According to the results of western blotting,after the effect of berberine,the expression of cleaved-caspase-3 and cleaved-caspase-9 in RL95-2 cell is increased significantly,but in the HEC-1-A cell,there is no obvious difference was found between the above two kinds of protein expression.JC-1 staining results showed that the mitochondrial membrane potential were weakened in two kinds of cells after the effect of berberine.According to the results of glucose oxidase method and lactate oxidase method,glucose consumption and lactic acid production were significantly increased after the effect of berberine in two kinds of cells.According to the results of western blotting,during the effect of berberine on RL95-2 cell,the expression of p-AMPK increase gradually,and p-mTOR reduce gradually;And after adding specific inhibitors of AMPK in advance,the role of berberine activates AMPK is abate,but the expression of p-mTOR was no significant difference than that of pure berberine effect.The second part:Our findings revealed that BBR significantly inhibited cell proliferation in AN3 CA and HEC-1-A EC cells in both dose-and time-dependent manner.Similarly,colony formation assays also showed that BBR treatment dramatically suppressed the ability of colony formation in AN3 CA and HEC-1-A EC cells,In athymic mice model,oral administration of BBR at 50 mg/kg and 100 mg/kg significantly induced a tumor growth inhibition in tumor models.The effect of BBR on cell migration was assessed with a modified Boyden chamber assay.The results showed that BBR significantly repressed the migratory ability of AN3 CA and HEC-1-A EC cells.and BBR treatment caused a remarkable reduction of the invasion in AN3 CA and HEC-1-A EC cells.Next,we established a tail vein injection induced lung metastasis mouse models to evaluate the effect of BBR on EC cell metastasis.Administration of BBR resulted in a dramatic decrease in the number of metastatic nodules in a dose-dependent manner.We determined the protein levels of COX-2 in AN3 CA and HEC-1-A EC cells in response to treatment with BBR,BBR significantly inhibited the protein level of COX-2 in a concentration-dependent manner.We also assessed the protein level of PGE2 upon treatment with BBR with ELISA assay.The results showed that BBR dose dependently and remarkably inhibited PGE2 level.To further clarify whether COX-2 contributed to the action of BBR on tumor growth and metastasis in EC cells,HEC-1A cells were treated with BBR in the presence or absence of overexpression of COX-2.We demonstrated that overexpression COX-2 induced around 2.3-fold increase of cell survival while addition of BBR at 50?M abolished the effect of COX-2.BBR treatment wakened the effect of COX-2 on cell motility and invasion.miR-101 expression was significantly downregulated in EC tumor tissues relative to matched normal tissues,whereas COX-2 protein level was markedly upregulated.The regulation of COX-2 by miR-101 was also confirmed with Q-PCR and western blot assays.BBR dose dependently stimulated miR-101 expression.We found that BBR induced increase of luciferase activity while depletion of AP-1 binding sites diminished BBR induced luciferase activity.Furthermore,cotreatment of miR-101inhibitor and BBR abolished BBR-induced COX-2 increase in HEC-1-A cells.Conclusions:.Berberine can inhibit proliferation of human endometrial cancer RL95-2and HEC-1-A cell,and induce cell cycle arrest in two kinds of cells.Berberine exert its inhibitory effect in RL95-2 and HEC-1-A cell by inhibiting the mitochondrial function.After the Mitochondrial function are suppressed,both intracellular glucose consumption and lactic acid generation are increased.Berberine inhibit RL95-2 cell by inducing apoptosis through the mitochondrial pathway,but the inhibition of HEC-1-A cell is not achieved by apoptosis.The mechanism of Berberine inducing apoptosis in RL95-2 cells through mitochondrial way is to inhibit the core metabolic molecular mTOR,and the inhibition do not rely on AMPK pathway.BBR inhibits cell migration,invasion and metastasis in EC.BBR inhibited cell survival,migration and invasion mediated by COX-2/PGE-2 axis in EC cells.We conclude that miR-101 is involved in the regulation of COX-2 by BBR.