| Objective: Ovarian cancer is one of the three female genitalia malignant tumor, and has low early diagnosis, high metastasis rate and poor prognosis. So the pathogenesis of ovarian cancer still has many problems to be solved. There is increasing evidences suggesting that it is the hormonal environment of the normal ovarian surface epithelium (OSE) and ovarian epithelial cancer (OEC) cells that is associated with the development and progression of ovarian cancer. Exposure to excess follicle stimulating hormone (FSH), related to menopause, ovulation, or infertility therapy, has been implicated as an important possible risk factor for ovarian cancer. A number of studies have shown that FSH acting on FSH receptor in the cell surface play a role by activating of intracellular signal transduction pathways, but its specific mechanism of action is unknown. Phosphatidylinositols-kinase/protein kinase B (PI3K/AKT) signaling pathway play an important role in tumor occurrence, development, apoptosis, angiogenesis, metastasis and drug resistance, etc. PI3K/AKT pathway may be directly or indirectly affect the downstream molecules include cell proliferation and protein synthesis related factors and a number of apoptosis related factors. Nuclear transcription factor (NF-κB) as a center of signal transduction pathway, is one of important downstream molecule of AKT. Its abnormal activation may regulate cell proliferation and apoptosis related gene transcription, dissolved in extracellular matrix to promote tumor cell infiltration and metastasis. Therefore, this experiment mainly study the role of FSH on NF-κB through PI3K/AKT pathway and to regulate its expression, get the occurrence mechanism of epithelial ovarian cancer cell proliferation and invasion, and the malignant progression of ovarian cancer by blocking the pathway. This study will provide new experimental evidence for the pathogenesis of ovarian cancer and gene therapy.Methods: SKOV-3, 3AO cells were cultured at 37℃in RPMI-1640 in the tissue culture flask under a humidified 5%CO2 atmosphere, applying to the experiment when they entered the logarithmic phase .1 Light microscopy observation of cell morphological changes: different concentrations of FSH and PI3K/AKT inhibitor LY294002 were roled in SKOV-3, 3AO cells and to observe the growth of cell regularly in inverted phase contrast microscope and to photo.2 MTT method: adjusting the cell concentration of 1×105/ml, with 100μl per well cultured in 96-well plate. Then the cells were exposed in different concentrations of FSH (10, 20, 40, 80, 160 mIU/ml) when the cells entered logarithmic growth phase. At last MTT method detected the proliferation of cell growth in different times(12h, 24h, 48h, 72h).3 Western blot analyses: To observe FSHR, Akt1 / 2, p-AKT, NF-kappaB protein expression of SKOV-3, 3AO cells before and after exposed to FSH and LY294002 respectively or combined by western blot, as well as the different expression of NF-kappaB protein in the nucleus and cytoplasm.4 Transwell invasion method: adjusting the cell concentration of 1×105/ml, with 200μl cell suspension in the inner cup of the 24-well Corning chamber that had been coated with 40μl Matrigel and 500μl RMPI-1640 culture media supplemented with 10% heat-inactivated FBS in the outer cup. Then the cells were exposed in FSH 40mIU/ml and LY294002 10μmol/L respectively or combined until adherencing. After 48 h, cells that had invaded through the Matrigel were fixed, stained, then observed and counted under the light microscope.5 Application of SPSS13.0 statistical software for statistical analysis.Results:1 The appearance of SKOV-3, 3AO cells exposed to FSH were observed under inverted phase contrast microscope. As the concentration increased and the time extended, the shape of the cells began to change slightly longer, the growth rate and intensity increased significantly. After LY294002 treating 30 minutes, FSH was exposed to continue to effect, leading to little change in cell morphology, growth rate and intensity reduced. Exposing LY294002 alone, some cells were from the spindle into a round shape, and then began to shrink. The cell growth capacity diminished.2 After we treated SKOV-3, 3AO cells in different time (12h, 24h, 48h, and 72h ) and with FSH in different concentration(s10,20,40,80,160 mIU/ml), MTT results showed that SKOV-3 cells were in the fastest growing for 48 h and FSH in 40μmol/ml, the concentration compared with the other groups was significantly different (P<0.05), and between the time groups there was no differences (P>0.05). 3AO cells were for 24 h and in 40μmol/ml, and compared with the other concentrations and times has significant differences (p <0.05).3 Result of western blotting:①FSHR protein were expressed in SKOV-3, 3AO cell. After FSH and LY294002 alone or in combination treating the cells, there were no significant differences as compared with the control group (p> 0.05).②A fter treating SKOV-3 cells with FSH and LY294002 alone, compared with the control group showed that p-AKT protein expression increased in FSH group, and the expression decreased in LY294002 group (p <0.05). In FSH/LY294002 combined treatement, the expression decreased compared with the control group (P<0.05). And AKT1 / 2 protein expression has no significant differences among the four groups (P>0.05). NF-κB total protein expression in the three experimental groups as compared with the control group, there was no statistical significance (P>0.05); but FSH group has more expression than LY294002 group (P<0.05). NF-κB protein expression in the nucleus of FSH group, compared with the control group increased (P<0.05), while the expression of LY294002 and FSH/LY294002 group decreased (P>0.05); and the expression of FSH group more than LY294002 group, FSH /LY294002 group (P<0.05). The latter two showed no difference (P>0.05). NF-κB protein expression in the cytoplasm of three experimental groups as compared with the control group had increased (p> 0.05). Analyzed statistically the ratio of the nucleus and the cytoplasm in the control group, FSH group, FSH/LY294002 group and LY294002 group, the four groups were significantly different (P<0.05). Which, FSH significantly larger than FSH/LY294002 group and LY294002 group, LY294002 group was significantly less than the control group and the FSH group (P<0.05), while there were no differences between the other groups (P>0.05).③p-AKT protein expression in 3AO cells, the three experimental groups compared with control group were statistically different (P<0.05). Which, p-AKT protein expression of FSH group was significantly more than the other three groups (p <0.05); LY294002 group, FSH/LY294002 group expressed significantly lower than the control group (P<0.05). Among the four groups AKT1/2 protein expression had on difference (P>0.05). NF-κB protein expression of total extracted protein, FSH group was significantly less than the control group and LY294002 group (P<0.05), while the other groups and between the four groups showed no significant difference (P>0.05). NF-κB protein expression of the nucleus in the four groups have more significant differences (p <0.05), which, the expression of FSH group increased significantly compared with the other three groups (P<0.05); while the expression of LY294002 group, FSH/LY294002 group decreased compared with the control group, but no statistically significant difference (P>0.05), and no difference between the two groups (P>0.05); NF-κB protein expression of the cytoplasm in FSH group were more than the control group and LY294002 group, FSH/LY294002 group, but between the latter two groups was no significant difference (P>0.05). Analyzed statistically the ratio of the nucleus and the cytoplasm in the control group, FSH group, FSH/LY294002 group and LY294002 group, the four groups were significantly different (P<0.05). Which, FSH group was significantly higher than the other three groups (P<0.05), FSH/LY294002 group was significantly more than the control group (P<0.05), while the FSH/LY294002 group and LY294002 group had no significant difference (p> 0.05).4 The results of Transwell invasion method: Treating the two kinds of cells with FSH, the number of cells invading through the Matrigel were more than the control group (P<0.05). LY294002 treatment alone group and combined of the two drugs group also had a few cells invading through. And the cells were significantly less than FSH group (P<0.05), but compared the two groups had no significant differences (P>0.05).Conclusion:1 Follicle-stimulating hormone has the effect of inducing proliferation on epithelial ovarian cancer cell line SKOV-3, 3AO. That suggests that the peak concentration may be the strongest proliferative concentration.2 FSH receptor exist in both of SKOV-3, 3AO cells. That FSH acts on SKOV-3, 3AO cells could excite PI3K/Akt pathway. So p-AKT protein expression increase. Thereby, inducing its downstream molecule NF -κB protein expression increase in the nucleus, promoted cell proliferation and invasion ability.3 PI3K/Akt inhibitor LY294002 acts on SKOV-3, 3AO cells and then given to FSH stimulation, p-AKT activity is inhibited, protein expression is significantly reduced, and thus NF-κB protein expression in the nucleus is reduced, inhibiting cell proliferation, reducing the invasive ability of tumor cells.4 Follicle-stimulating hormone may be through PI3K/Akt pathway to promote ovarian cancer cell line SKOV-3, 3AO in the expression of NF-κB, then achieve on promoting the proliferation and invasion of ovarian cancer cells.
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