The Effect Of Silibiniin On Sensitivity Of Ovarian Carcinoma Cells To Paclitaxel And Invasiveness Ability

PARTâ… THE SURVIVIN EXPRESSION IN OVARIAN CARCINOMA AND THEIR CLINICAL SIGNIFICANCEObjective:To examine the expression of survivin in ovarian carcinoma tissues and to explore their clinical significantMethods:Quantitative real-time PCR and immunohistochemistry were used to examine the mRNA and protein expressions of survivin in ovarian carcinoma tissues (n=65).Normal ovary tissues(n=11)serve as the control.Results:1.The mRNA expression of survivin in normal ovary and ovarian carcinoma tissue:No survivin mRNA was expressed in all normal ovary specimens while 54 cases with positive survivin mRNA expression in 65 ovarian carcinoma tissues(83%). In tissues with positive expression of survivin,we also found that the survivin mRNA expression levels correlated with the clinic stages,histological grade,lymphonode metastasis(P＜0.05),but negative to histotype(P＞0.05).2.The protein expression of survivin in normal ovary and ovarian carcinoma tissue: The subcellular localization of survivin in ovarian carcinoma cell:localized in nucleus only,localized in nucleus only,simultaneous expression in nucleus and cytoplasm. There were no survivin mRNA was expressed in all normal ovary specimens while 51 cases with positive survivin protein expression in 65 ovarian carcinoma tissues(78%). In tissues with positive expression of survivin protein,we found there was no significant relationship between the clinic stages,histological grade(P＞0.05), histotype,lymphonode metastasis and the subcellular localization of survivin in ovarian carcinoma tissues.Conclusions:1.The survivin mRNA expression levels correlated with the clinic stages,histological grade,lymphonode metastasis.2.No significant relationship between the clinic stages,histological grade(P＞0.05), histotype,lymphonode metastasis and the subcellular localization of survivin in ovarian carcinoma tissues.3.The mRNA expression levels of survivin may be indicators of the progression of ovarian carcinoma patients.PARTâ…¡EFFECT OF SILIBININ ON THE SENSITIZATION OF OVARIAN CARCINOMA CELL TO PACLITAXELObjective:To explore the effect and mechanism of Silibinin on the sensitization of ovarian carcinoma cell to Paclitaxel.Methods:1.A2780 and A2780/taxol cells were treated with Silibinin and Paclitaxel, either alone or in combination.Cell viability was evaluated by 3,4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide(MTT)assay.2.FCM(flow cytometric)was used to detect the cell cycle distribution and apoptosis of A2780 and A2780/taxol cells after Silibinin or/and Paclitaxel treatment for 24 hours.3.After treated with Silibinin and or Paclitaxel for 72 hours,protein and mRNA levels influenced by the treatment were studied by Western blots and Quantitative real-time PCR.Results:1.The cell growth inhibition effect of Silibinin on A2780 or A2780/taxol cells:Treatment of A2780 or A2780/taxol cells with different doses of Silibinin(50, 75,100,125,150μmol/L)resulted in 13.21%,25.35%,39.16%,42.72%,44.12%and 12.13%,28.47%,40.63%,43.41%,45.38%growth inhibition.Silibinin inhibit A2780 and A2780/taxol cells cell growth.2.The cell growth inhibition effect of Silibinin combination with Paclitaxel on A2780 or A2780/taxol cells:Paclitaxel alone at 100nmol/L dose produced 46%cell growth inhibition,whereas a combination with 50,100,150μmol/L Silibinin resulted in 53.52%,62.34%,63.47%(P＜0.001-0.01)growth inhibition in A2780.Similarly, compared to 52%growth inhibition at 1000nmol/L Paclitaxel,addition of 50,100, 150μmol/L doses of Silibinin caused 62.16%,71.69%,82.82%growth inhibition in A2780/taxol(P＜0.001-0.05).The combination treatment of Silibinin with Paclitaxel resulted in a stronger cell growth inhibition as compared to single agent.3.The effects of Silibinin on the sensitization of A2780 and A2780/taxol cells to Paclitaxel:The IC₅₀of Paclitaxel in A2780 and A2780/taxol cells with increasing concentrations of Silibinin 0,50,75,100,125,150μmol/L respectively is 138.15, 96.23,80.61,59.19,58.32,57.18 and 699.06,498.68,354.42,200.89,110.15, 64.33(nmol/L).Silibinin enhanced the sensitivity of A2780 and A2780/taxol cells to Paclitaxel in dose-dependent manner.4.The effect of Silibinin and Paclitaxel compounds combination on A2780 and A2780/taxol cells apoptosis and cycle progression:The apoptosis rates of A2780 or A2780/taxol cells after treated with Silibinin(150μmol/L)or/and Paclitaxel (100nmol/L)for 48 hours were 12.23%,14.12%,39.37%or 5.56%,11.82%,23.41% respectively(P＜0.001).Silibinin/Paclitaxel combination significantly increased cell apoptosis compared to either of two drugs alone.A2780/taxol cells demonstrated resistance for Paclitaxel-induced G₂/M arrest.The accumulation of cells in the G₂/M phase had been also observed in A2780/taxol were exposed to 100 nmol/L Paclitaxel in the presence of 150μmol/L Silibinin.5.The gene and proteins expression of A2780 and A2780/taxol cells after treatment with Silibinin and Paclitaxel:The combination of Silibinin and Paclitaxel substantially decreased the expression of survivin and enhanced caspase-3 activation in A2780 or A2780/taxol for 72h.Fourthermore the expression of P-gp,MDR-1 were suppressed in A2780/taxol cells.No significant difference was found in the expression change of Bcl-2.Conclusions:1.Silibinin inhibits ovarian carcinoma cells growth.2.Silibinin enhances sensitivity of ovarian carcinoma cells to Paclitaxel and the mechanism associated with down-regulated the expression of survivin,P-gp and MDR-1.PARTâ…¢EFFECT OF SILIBININ ON INVASIVENESS ABILITY OF HUMAN OVARIAN CANCER CELLSObjective:To study the effect and mechanism of Silibinin on invasiveness ability of A2780 and A2780/taxol cells.Methods:1.Cell viability was evaluated by 3,4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide(MTT)assay after A2780 and A2780/taxol cells were treated with Silibinin for 24h.2.The invasive ability was tested by transwell matrigel invasion assays after A2780 and A2780/taxol cells were treated with Silibinin for 24h.3.Quantitative real-time PCR and Western blot were used to detect the expression of MMP-2,MMP-9,MDR-1 and P-gp in A2780 and A2780/taxol cells after treated with Silibinin for 24h.Results:1.A2780/taxol cells demonstrated a two-fold increase in invasiveness ability compared to the parental cells(A2780).2.The cell growth inhibition effect of Silibinin on A2780 and A2780/taxol:No significant cytotoxicity to A2780 and A2780/taxol cells after treated Silibinin at 50, 75,100μmol/L for 24h..3.The effect of Silibinin on invasiveness ability of A2780 and A2780/taxol cells:The inhibition rates of invasiveness ability in A2780 or A2780/taxol cells after treated with Silibinin(50,75,100μmol/L)for 24h is 18.24%,36.72%,53.66%and 20.99%, 39.45%,56.50%respectively.Silibinin inhibit the invasiveness ability of A2780 and A2780/taxol cells in dose-dependent manner.4.The expression of MMP-2,MMP-9,MDR-1,P-gp in A2780 and A2780/taxol cell after treatment of Silibinin:Silibinin(50,75,100μmol/L)inhibited the expression of MMP-2,MMP-9 in dose-dependent manner in A2780 and A2780/taxol cell for 24h. Fourthermore the expression of P-gp,MDR-1 were suppressed by Silibinin in A2780/taxol cells.Conclusions:1.These results suggest that A2780/taxol cells demonstrated a twofold increase in invasiveness compared to the A2780 cells.2.Silibinin represents a potential anti-metastatic agent suppressing invasion of A2780 and A2780/taxol through inhibition of MMP-2,MMP-9,MDR-1 and P-gp expression.