The Research In Inhibitory Effect And Mechanism Of Baicalin On Human Ovarian Cancer Cell Line SKOV3

Ovarian malignant tumor,also known as ovarian cancer,is one of the most common malignant tumors of female reproductive organs.although the incidence of ovarian cancer ranks third not matching for cervical cancer and uterine cancer,its mortality takes the first place in the female reproductive system tumor mortality rate.with surgery and chemotherapy and radiotherapy for the side effect is so severe,which also restrictes the treatment of ovarian cancer.The recurrence and metastasis of the disease,the problems that is difficult to find,the difficulty in diagnose make ovarian cancer have a low cure rate and a high mortality rate.With the further development of Chinese medicine and Chinese medicine,treating the disease when gently、anal ysi sing the stage of disease、the overall concept have sunk into people’s hearts,especially Chinese medicine and traditional Chinese medicine having many targets and less or no side effect on anti tumor have the advantages of more and more attention at home and abroad.Therefore,looking for safe and reliable antitumor compound Chinese medicine or the monomer composition of traditional Chinese medicine is of great significance for the future development of the anti tumor.This paper is divided into two parts:the first part is literature review,mainly introducing the treatment of ovarian cancer and summarizing the main pharmacological effects of Scutellaria’s active ingredient concentration baicalin;the second part is the experimental research,including the effects of Baicalin on human ovarian cancer SKOV3 cells in proliferation、the effects of Baicalin on the ovarian cancer SKOV3 cells in migration、the effects of Baicalin of human ovarian cancer SKOV3 cells in invasion and the molecular mechanism of Baicalin’s inhibition on human ovarian cancer SKOV3 cellsMethods:Experiment 1:Human ovarian cancer SKOV3 cells are cultured in Petri dish,logarithmic growth of the cells are plated uniformly in 24 well plate.After cultured adherently for 24 hours,the cells are treatmented by different concentrations of Baicalin,each concentration having three parallel holes.After treatment with Baicalin for 48 hours,the cell proliferation assay kit is used to determine whether Baicalin could inhibit the growth of ovarian cancer cells and the minimal inhibitory concentration of Baicalin.Experiment 2:Human ovarian cancer SKOV3 cells are cultured in Petri dish,logarithmic growth of the cells are plated uniformly in 6 well plate which are divided into experimental groups and control groups,each group having three parallel wells.After cultured adherently for 24 hours,in one hole’s inside long side and short side along the 6 well plate evenly scratch 3 parallel lines,which showing a man-made "cell wound".Serum free medium is added in every well.The minimum effective concentration of Baicalin in Experiment one is added to the dosing group in each well.After cultured for 48h,the wound healing Level in 0 hours,6 hours,12 hours,24 hours,48 hours are observed respectively,to the degree of 12h,24h and 48h cells during after dosing,analysising Baicalin’s inhibition in migration of ovarian cancer cells.Experiment 3:Human ovarian cancer SKOV3 cells are cultured in Petri dish,the logarithmic growth cells diluted by serum-free medium are uniformly plated on the membrane already covered with Matrigel matrix in Transwell small chamber of 24 wells which the lower chamber is added conventional medium.The lowest and effective concentration of Baicalin in the Experiment one are added in the lower and higher chamber respectively and incubated for 48 hours in the incubator.By observing the invasion degree of cells by microscope,we can analysis wether Baicalin can inhibit the invasion ability of ovarian cancer cells.Experiment 4:Using protein immunoblot technology(Western Blot,WB)by detecting related signaling pathway proteins of AKT、ERK、STAT3、NF-κB explores the molecular mechanism of Baicalin inhibiting human ovarian cancer SKOV3 cells.Results:Experiment 1:The final concentration of Baicalin are divided into 0μM,2μM,10μM,5μM,20μM,40μM.Compared with 0μM,the final concentration of Baicalin’s inhibition in 2μM,5μM groups are not obvious,which has no statistical significance(P>0.05).Compared with OμM,the final concentration of Baicalin’s inhibition in 10μM,20μM,40μM groups are obvious,which has statistical significance(P<0.05).The final concentration of Baicalin in lOμM,20μM groups have a significant statistical difference(P<0.01);the final concentration of Baicalin in 40μM groups has a particular significant difference(P<0.001).So we will continue the next experiments all by baicalin on 10μM.Experiment 2:Compared with the control group,the final concentration of Baicalin group in 10μM has a inhibition trend in wound area’s healing,but has no statistical significance(p>0.05);Compared with the control group,the final concentration of Baicalin group in 1μM has a inhibition trend in wound area’s healing,and has statistical significance(p<0.05);Compared with the control group,the final concentration of Baicalin group in lOμM can distinctly inhibite the healing of wound area,and has statistical significance(p<0.001);Compared with the control group,the final concentration of Baicalin group in 10μM has a inhibition trend in wound area’s healing,and has statistical significance(p<0.05).Experiment 3:The number of invasion cell in the final concentration of 10μM Baicalin group is less than the control group,which has statistical significance(P<0.05).Experiment 4:Compared with the control group,the final concentration of lOμM Baicalin group can reduce the level of protein expression of p-AKT,p-ERK,p-STAT3,p-p65,P-IκB,Bmi1，Vimentin,Oct4,Sox2 in human ovarian cancer SKOV3 cells.Conclusion:Firstly,Baicalin on 5μM to 40μM can inhibit the proliferation of human ovarian cancer SKOV3 cells,and in a certain range is proportiona to dosage.Secongdly,Baicalin on 10μM can inhibit the migration of human ovarian cancer SKOV3 cells,and the most inhibitory effect is in the 24th hours.Thirdly,Baicalin on 10μM can inhibit the invasion ability of human ovarian cancer SKOV3 cells.Fourth,the abnormal activation of AKT,ERK,STAT3,NF-κB signaling pathway,induce epithelial mesenchymal transition of human ovarian cancer SKOV3 cells,thereby affects ovarian cancer stem cells,making the expression of ovarian cancer stem cells surface marker like Bmi1，Oct4,Sox2 and Vimentin protein have an abnormal increasing.Fifth,Baicalin,by inhibiting the protein expression level of p-AKT,p-ERK,p-STAT3,p-p65,p-IκB,can reverse the effects of epithelial mesenchymal transition,and affect the affection of ovarian cancer stem cells.Significance:This study provides an experimental theoretical foundation for the treatment of ovarian cancer in active ingredient of traditional Chinese medicine,and provides the research direction for ovarian cancer in metastasis,recurrence and drug resistance.