The Studies On Induction Of Apoptosis In Human Ovarian Cancer CoC1 Cells Through Inhibition Of CK2a By 6, 8-ditrifluoromethyl-7-acetoxychrysin, A Novel Chrysin Derivative

Objective: To investigate the effect and mechanism of 6,8-ditrifluoromethyl- 7-acetoxychrysin(dFMAChR),a novel chrysin derivative on induction of proliferation inhibition and apoptosis of human ovarian cancer(CoC1) cell line in vitro , and provide a molecular mechanism of this effect so as to find a new activity chemical entity for tumor chemotherapy.Methods: Human ovarian cancer (CoC1) cells were cultured in vitro. MTT assay was used to determine the effect of dFMAChR on the proliferation of CoC1 cells. The trypan blue exclusion method was used to count the number of viable and dead cells, then the survival rate and growth curve were achieved.Clone formation using soft agar was used to test inhibition of clone formation in CoC1 cells by dFMAChR. Fluorescence after AO/EB staining was used to observed the morphologic changes of apoptosis induced by dFMAChR in CoC1 cell line. DNA agarose gel electrophoresis was used to test apotosis induced by dFMAChR in CoC1 cell line. Flow cytometry (FCM) using PI staining was used to analyse the apoptosis change treated with dFMAChR.Western blot was used to analyze expression of CK2α,β-Catenin and NF-κB(p65) protein in CoC1 cells , and to explore a potential molecular mechanism of the anti-ovarian cancer action of dFMAChR. Results: The MTT assay showed that the proliferation inhibitory rate was 11.9%±1.15%,19.2%±2.05%,39.9%±2.24%,48.5%±3.37%,58.8%±2.74% after CoC1 cell was treated with dFMAChR at various concentration of 0.3,1.0,3.0,10.0,30.0μM for 24h, respectively dFMAChR significantly suppressed proliferation of CoC1 cell line, in a dose-dependant manner ,and its IC50 was 11.8μM.When the cell viability was determined by the trypan blue exclusion method, the results showed dFMAChR at various concentration of 3.0, 10.0, 30.0μM had inhibitory growth effect to CoC1 cell,in a dose-dependent manner. dFMAChR at 10μM could prolong CoC1 cell multiple time from 28.5h to 32.6h. The clone formation using soft agar assay showed that the inhibitory rate of clone formation was 31.2%±2.07%,48.6%±3.28%,68.5%±4.46% respectively after treatment with dFMAChR at various concentration of 3.0,10.0,30.0μM. Typical morphologic changes of apoptosis in CoC1 cell could be observed after treatment with dFMAChR by fluorescence microscope using AO/EB fluorescence staining. DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with dFMAChR at 10μM for 48h. Flow cytometry(FCM) analysis after PI stainning indicated that the apoptosis rate of CoC1 cells was 4.70%±1.02%,10.74%±1.03%,35.12%±3.13% respectively after treatment with dFMAChR at the concentrations of 3.0,10.0 and 30.0μM for 24h.Western-blot analysis indicated that exposure to dFMAChR at 0.3,3.0,30.0μM for 24h,the protein level of CK2α,β-Catenin and NF-κB(p65) was down-regulated by 13.2%±1.42%,27.4%±2.30%,41.7 %±2.0%;12.4 %±4.30%,26.4 %±2.11%,37.4 %±1.70%;8 %±1.62%,26.5%±3.43%,40.5%±3.16% respectively in comparision with the control group. After treatment with dFMAChR at 30.0μM for 4,8 and 24h, the protein level of CK2α,β-Catenin and NF-κB(p65) in CoC1 cells was down-regulated 8.3 %±3.02%,30 %±3.48%,49.7 %±1.76%;36.3 %±4.30%,45.3 %±2.11%,56.6 %±1.70%;32.8%±0.98%,48.2%±0.90%,50.3%±1.38% in comparision with the control group respectively. Conclusion:1) 6,8-ditrifluoromethyl-7-acetoxychrysin posses the inhibitory effect of the proliferation and growth of CoC1 cells,in a dose-dependent manner.2) 6,8-ditrifluoromethyl-7-acetoxychrysin can significantly induce apoptosis of CoC1 cells.3)The inhibition of growth and induction of apoptosis of CoC1 cells by 6, 8-ditrifluoromethy-7-acetoychrysin are associated with downregulation of CK2α,β-Catenin and NF-κB (p65) protein level expression.