| Ovarian cancer is the leading cause of death from gynecological malignancies. Despite significant advances in surgery and chemotherapy, the molecular basis of ovarian carcinogenesis remains poorly understood and chemotherapy resistance of ovarian cancer is difficult to resolve, which limit our ability to treat this disease and lead to the relatively low five-year survival rates.A common feature of cancer is a high rate of aerobic glycolysis, which not only provide ATP for the tumor cells' high bioenergetic demands but also provide precursors for the synthesis of macromolecules, such as nucleotides, proteins, and lipids. Therefore, malignant cells often have abnormal activities of many enzymes, which are involved in the glucose metabolism. Among them, glycogen synthase kinase-3β (GSK-3β), except from inhibiting glycogen synthase and promoting glycolysis, has multiple functions including regulating activities of transcription factors and structure proteins. Thus, several researches have shown that GSK-3β plays some role in tumorigenesis, however the conclusions are different. And there're little reports about GSK-3β and ovarian cancer. So we tried to study the effect of GSK.-3β on the proliferation of human ovarian cancer cells both in vitro and in vivo. Though four methods, that is, cell growth curve, BrdU incorporation assay, colony formation assay and tumorigenic ability in nude mice, we analyzed the short-term and long-term effect of GSK-3β on ovarian cancer cells. Then we tried to explain the mechanism by cell cycle assay. At the same time, we found that increasing GSK-3β activity can sensitize ovarian cancer cells to chemotherapy. At last, we studied the regulation of GSK-3β by estrogen and progestin. Our results suggested GSK-3β could promote ovarian cancer cell proliferation and sensitize ovarian cancer cells to chemotherapy, estrogen and progestin could regulate GSK-3β activity.Our researches are composed of 3 parts: (1) GSK-3β promotes proliferation of ovarian cancer cells; (2) GSK-3β regulates ovarian cancer cell cycle progression and sensitizes ovarian cancer cells to chemotherapy; (3) Regulation of estrogen andprogestin to GSK-3β.Part 1 GSK-3β promotes proliferation of ovarian cancer cellsObjective To investigate the effect of glycogen synthase kinase-3β (GSK-3β) on the proliferation of human ovarian cancer cells.Methods Two human ovarian cancer cell lines SKOV3 and ES-2 were analysed for the expression of GSK-3β and phosphorylated GSK-3β at Ser 9 (pGSK-3βSer9) by western blot analysis. Cell growth curve analysis done by cell count was used to investigate the effect of GSK-3β inhibitors on the growth of SKOV3 and ES-2. Four plasmids, namely GSK-3βS9A, GID5-6, GID5-6LP and the control vector, were cotransfected respectively with the green fluorescent protein (GFP) into SKOV3 cells by electroporation, and then BrdU incorporation assay was adopted to analyse the role of GSK-3β activity in the proliferation of ovarian cancer cells. After transfection, G418 was added to the medium to select those stably transfected cells, which were used to investigate the long term effect of GSK-3β activity change on the proliferation of ovarian cancer cells by colony formation assay. SKOV3 cells, treated with NaCl or LiCl for 30 min, were then subcutaneously injected into the right flank of athymic nude mice (5 × 106 cells/mouse). Three weeks after implantation, mice were euthanized and tumor growth was measured by tumor diameters with a vernier caliper and by tumor weight.Results Both SKOV3 and ES-2 cells expressed GSK-3β, though the expression level of pGSK-3βSer9 was lower in SKOV3 than in ES-2 cells. GSK-3β inhibitors attenuated the growth of SKOV3 and ES-2 cells. Transfection with GSK-3βS9A to upregulate the GSK-3β activity resulted in the increase of BrdU incorporation in SKOV3 cells, compared with the control vector. On the contrary, transfection with GID5-6 to downregulate GSK-3β activity decreased the BrdU incorporation in SKOV3 cells, compared with GID5-6LP, which is a control vector of GID5-6. Stable transfection with GSK-3βS9A increased the colony number while stable transfection with GID5-6 decreased the colony number, compared with each control vector. Compared with NaCl, LiCl inhibited the SKOV3 cell growth in vivo.Conclusion GSK-3β could promote the proliferation of ovarian cancer cells. So inhibition of GSK-3β might be a potential theraputic target for ovarian cancer.Part 2 GSK-3β regulates ovarian cancer cell cycle progression and sensitizes ovarian cancer cells to chemotherapyObjective To investigate the mechanism of the enhancement effect of GSK-3β on the ovarian cancer cell proliferation and to evaluate whether the increase of GSK-3β activity could sensitize ovarian cancer cells to chemotherapy.Methods The plasmid GSK-3βS9A, which could increase GSK-3β activity , and the plasmid GID5-6, which could inhibit GSK-3β activity, and their control vectors were cotransfected with the green fluorescent protein (GFP) into SKOV3 cells by electroporation respectively. Cell cycle distribution of the transfected cells was analyzed by FACS. The expression level of cell cycle protein cyclin D1 of the transfected cells was detected by western blot analysis. The appropriate concentration of taxol for the sensitivity experiment was determined by PI staining and counting the red cells (that is the dead cells) with a fluorescence microscope. SKOV3 cells, transfected with GSK-3βS9A or the control vector, were then incubated with the appropriate concentration of taxol for 24 h. After that, the sensitivity of the SKOV3 cells to taxol was detected by PI staining and counting the dead cells with the fluorescence microscope.Results Increasing the activity of GSK-3β by transfection with GSK-3βS9A plasmid made 45.5±1.7% of SKOV3 cells enter S phase, while transfection with the control vector made 37.5±2.9% of SKOV3 cells enter S phase; in contrast, transfection with GID5-6 to inhibit GSK-3β activity led to only 26.8±2.1% of SKOV3 cell into S phase, and transfection with the control plasmid resulted in 36.3±1.5% of SKOV3 cells into S phase (P<0.05). Transfection with GSK-3βS9A increased the expression level of cyclin Dl in SKOV3 cells, while with GID5-6 decreased the expression level, compared with the control plasmids respectively. The ideal concentration of taxol was 0.01 μmol/L. After transfection with GSK-3βS9A or the control plasmid and incubation with 0.01 μmol/L taxol for 24 h, PI staining showed that 27.2±4.1% of SKOV3 cells transfected with GSK-3βS9A were dead, while 12.8±0.8% of SKOV3 cells transfected with the control plasmid were dead (P<0.01).Conclusion GSK-3β could promote ovarian cancer cells proliferation through the mechanism of up-regulating cyclin Dl expression, thus prompting cells to enter S phase. Increasing GSK-3β activity could sensitize ovarian cancer cells to taxol.Part 3 Regulation of estrogen and progestin to GSK-3βObjective To investigate the regulation of GSK-3β by estrogen and progestin in ovarian cancer cells.Methods SKOV3 cells were pretreated with phenol red-free RPMI-1640 plus 10% charcoal-stripped serum for 3 days. Then cells were treated with 10-8 mol/L, 10-7mol/L, or 10-6mol/L 17-β estradiol or MPA. After 48 h, cells were lysed and the expression of pGSK-3βSer9 and GSK-3β were detected by western blot. And SKOV3 cells were treated with 10-6mol/L 17-β estradiol or MPA, and the expression of pGSK-3βSer9 and GSK-3β were detected by western blot at 24 h, 48 h or 72 h treatment.Results 17-β estradiol could down-regulate the expression level of pGSK-3βSer9, while MPA up-regulated the expression level, both in dose and time dependent manner. And the expression level of GSK-3β remained unchanged.Conclusion Estrogen could increase the activity of GSK-3β and MPA could decrease the activity of GSK-3β in ovarian cancer cells through the regulation of the expression level of pGSK-3βSer9.
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