| Objective: (1) To investigate the regulatory effect of hCG on proliferation and apoptosis in human ovarian cancer 3AO cells in vitro. (2) To establish enzyme receptor assay (ERA) for LH/hCG receptor on intact culture cells, and to study the characteristics of LH/hCG receptor by ERA. (3) To study the effects of hCG on intracellular cyclic adenosine 3',5'-monophosphate (cAMP) and calmodulin (CaM) levels and protein kinase C (PKC) activity in 3AO cells. (4) In addition, to preliminarily analyze the mechanism of hCG signaling involved in proliferation and apoptosis.Methods: Ovarian cancer cell line 3AO was cultured in vitro. Two groups were set up: study group with hCG in different concentrations and control group without hCG. The growth rate of ovarian cancer cell line 3AO was assessed by 3-(4,5-dimethylthiazol-z-yl)-2,5-dipheny tertrazolium blue (MTT) colorimetric assay, and cell cycle kinetics was detected by flow cytometry (FCM). Apoptosis induced by cisplatin with or without hCG treatment was investigated by fluorescent microscope and FCM. ERA was applied to determine the maximum binding (Bmax) and affinity (Kd) of LH/hCG receptor on 3AO cells as well as on the ovarian cancer cells SKOV-3 as negative control. Intracellular cAMP and CaM levels, PKC activity wereexamined by radio-immunoassay, phosophodiesterase (PDE) assay and PepTag Assay for Non-Radioactive Detection Kit, respectively. To analyze further the mechanism of hCG signaling involved in growth-stimulation and apoptosis-inhibition, the proteins and mRNA expression of the insulin-like growth factor-1 (IGF-I) and bcl-2/bax system were measured by flow cytometric indirect immunofluorescent technique and quantitative reverse transcription polymerase chain reaction (RT-PCR).Results: (1) The growth rate of SAO cells obviously increased with hCG (0.1, lμg/ml) treated for 72 hours in a dose-dependent manner. But high concentrations slightly inhibited the proliferation of 3AO cells. The maximum magnitude of hCG stimulation was about 121% of control (P <0.01). The cell cycle was significantly changed after incubated with different concentrations of hCG for 24,48,72 hours, respectively. The cell rate of Stage G0/G1 of SAO with hCG decreased from 83.77% to 79.34%~70.22%, cell rate of Stage S did not changed much, and cell rate of Stage G2/M increased from 15.15% to 17.86%~29.14%. Meanwhile, hCG could obviously inhibit the apoptosis of SAO cells induced by cisplatin. The inhibition rate was about 10.4% (P <0.05). (2) The Bmax and Kd values of LH/hCG receptor were 142.4ng/mg protein and 16.142ng/ml, respectively. And the competitive inhibition ICso of unlabelled hCG against HRP-hCG binding was 3.584ug/ml. (3) After incubation with hCG, the intracellular cAMP level was increased by hCG in a dose- and time-dependent manner. On the other hand, the intracellular CaM level was not changed by hCG, and hCG could only slightly affect intracellular PKC activity in SAO cells. (4) In addition, hCG treatment increased the intracellular IGF-I level. But the proteins and mRNA expressionof bcl-2 and bax were not changed.Conclusions: (1) Our findings suggest that hCG not only could obviously stimulated the growth of 3AO cells but also could inhibit the cisplitin-induced apoptosis in vitro. (2) Competitive inhibition assay, saturation binding assay and scatchard analysis testify the expression of LH/hCG receptor. (3) hCG exerts its specific effects on 3AO cells mainly by activating adenylate cyclase(AC)/cAMP. And the other for phospholipase C/inositol phosphate /diacylglycerol isn't involved in hCG signaling. (4) hCG treated could affect the intracellular IGF-I level in 3AO cells. It is suggested that the effect of hCG on proliferation and apoptosis are due to via up-regulation of IGF-I intracellular expression, and hCG treatment has not effect on bcl-2 and bax expression in ovarian cancer cells. |
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