| Objective:In order to find the effective Chinese medicine monomer to treat epithelial ovarian cancer,we verified that quercetin have the effect to inhibit the invasion and metastasis of epithelial ovarian cancer cells.We searched the prevention molecular mechanism of quercetin which inhibits the invasion and metastasis of epithelial ovarian cancer and found a scientific basis for Chinese medicine treatment of epithelial ovarian cancer recurrence.Method:(1)Methylthiazolyldiphenyl-tetrazolium bromide(MTT)was used to test the inhibition effect of quercetin in different concentrations on SKOV-3 cell,to calculate the inhibition rate and 50%inhibiting concentration;(2)Morphologic changes on quercetin were detected after 48h treatment on SKOV-3 cells by inverted microscope.We verified that quercetin can reverse the phenotype transformation of SKOV-3 cells from Epithelial to mesenchymal.(3)Wound healing assay was used to detect the ability of cell migration of SKOV-3 cells after the treatment of quercetin by inverted microscope.We verified that quercetin can inhibit the migration of SKOV-3 cell.(4)Transwell experiment was used to determine whether quercetin affects cell migration and invasion of SKOV-3 cells.The migration ability of transwell cells was detected without Matrigel,by counting cells which through the polycarbonate membrane to evaluate cells migration ability;The invasion ability of transwell cells was detected with Matrigel,by counting cells which through the polycarbonate membrane to evaluate cell invasiveness;(5)Gene expression profiling analyzed by Agilent mouse whole-genome chip,were performed in Shanghai KangCheng bio-tech Corporation.Differentially expressed genes were determined via the fold change and p values.(6)RT-PCR were used to verify the microarray results.(7)Immunohistochemical was used to detect the expression of lysine specific demethylase(LSD1)in normal ovarian epithelial tissues,cystadenoma,borderline cystadenoma,and cystadenocarcinoma.Next,the correlations between expression of LSD 1 and clinicopathological features was assessed in 96 cases of serous cystadenocarcinoma and 36 cases of mucinous cystadenocarcinoma.(8)Real-time quantitative PCR(quantitative realtime PCR,qPCR)and Western blot(WB)were used to detect LSD1(act as genic modifiers of EMT transcription factor)mRNA and protein levels respectively.EMT marker such as E-cadherin?N-cadherin?Vimentin moleculars were also observed after 48h treatment with quercetin.Result:(1)MTT assay results showed that at the same time comparing with the control group,incubation with quercetin beyond a certain range of concentration in SKOV3 cells for 24,48,72 hours decreased the cell viability significantly(p<0.05)and showed a concentration-dependent and time-dependent manner(p<0.05).The IC50 of quercetin in SKOV3 cells on 24h,48h and 72h were calculated to be 155.12 ?mol/L,67.13 ?mol/L and 34.89 ?mol/L,respectively.(2)Our data demonstrated that SCOV-3 cells appeared in mesenchymal-like cell morphology after 48h treatment with quercetin.Wound healing assay indicated that quercetin can effectively inhibit the migration and invasion of SCOV-3 cells.(3)Microarray results showed that compared with the control group,there are 3385 differentially expressed genes(Ratio,>2),the expression of 1806 genes decreased(53.35%),expression of 1579 genes raised(46.64%).Among the decreased genes,LSD1 is related to tumor invasion and metastasis.LSD1 may be the key point of the inhibitory effects of quercetin on epithelial ovarian cancer.The results of quantitative PCR were consistent with the results of previous microarray detection.(4)Immunohistochemical results showed that the expression of LSD1 was gradually increased from benign cystadenoma to borderline cystadenoma and to cystadenocarcinoma.The positive ratio of LSD1 expression was respectively as 50%in normal ovarian epithelial tissues,72%in serous cystadenoma,73%in mucinous cystadenoma,82%in borderline serous cystadenoma,83%in borderline mucinous cystadenoma,94%in serous cystadenocarcinoma and 92%in mucinous cystadenocarcinoma.LSD1 expression levels were associated with International Federation of Gynecology and Obstetrics stage and lymphatic metastasis in both serous and mucinous cystadenocarcinoma samples.Kaplan-Meier curves suggested that overall survival time of patients with high LSD1 expression was significantly shorter than that of patients with low LSD1 expression.Multivariate Cox proportional hazard regression indicated that higher LSD1 expression was a significant independent predictor of poor survival of EOC patients(p=0.016).(5)The expressions of LSD1 mRNA and proteins were all decreased markedly in the SCOV-3 cells,and SCOV-3 cells appeared with high expression of epithelial marker(E-cadherin)and low expression of mesenchymal marker(Vimentin)and neural marker(N-cadherin)both in mRNA and proteins after 48h and 72h treatment with quercetin.Conclusion:This study shows that:LSD1 plays an important role in the invasion of quercetin against ovarian cancer.LSD 1 may be involved in carcinogenesis and progression in epithelial ovarian cancer,and becoming a clinical molecular marker to predict the poor clinical prognosis,and has certain therapeutic potential of epithelial ovarian cancer.Quercetin as the traditional Chinese medicine monomer can effectively inhibit the growth and metastasis of epithelial ovarian cancer cells.The mechanism of inhibitory effect of quercetin on epithelial ovarian cancer may be related with the inhibition of quercetin on LSD 1 which control of EMT process.Quercetin can inhibit epithelial ovarian cancer migration and invasion.This research can provide a molecular basis for the TCM clinical prevention and treatment of epithelial ovarian cancer. |
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