Involvement of human chorionic gonadotropin in regulating vasculogenic mimicry and hypoxia-inducible factor-1α expression in ovarian cancer cells

Abstract

Background: Human chorionic gonadotropin (hCG) can play a crucial role in angiogenesis. In the present study, we focused on hCG to gain insight into its potential effects on vasculogenic mimicry (VM) in ovarian cancer cells.

Methods: Ovarian cancer OVCAR-3 cells were incubated with different concentrations of recombinant hCG in 3-dimensional cultures. VM was identified by morphological observations and vascular endothelial cell marker detection in OVCAR-3 cells. Expression of hCG, hypoxia-inducible factor-1α (HIF-1α), and the endothelial cell markers CD31, VEGF, and factor VIII were detected by reverse transcription polymerase chain reaction and western blotting. The effect of hCG on endothelial cell-marker expression in ovarian cancer cells was further explored using small interfering RNA (siRNA) and plasmid-based approaches.

Results: Incubation of OVCAR-3 cells with recombinant hCG induced vessel-like network formation, which was accompanied by significant elevation of vascular marker expression. Attenuation of hCG expression by siRNA in OVCAR-3 cells suppressed the expression of endothelial cell markers and HIF-1α by tumour cells. Overexpression of hCG in OVCAR-3 cells resulted in increased expression of endothelial cell markers and HIF-1α.

Conclusions: HCG was crucial for changing the phenotype of OVCAR-3 cells to endothelial-like cells. The effect of hCG induction on VM in ovarian cancer cells is potentially associated with HIF-1α.

Keywords: Human chorionic gonadotropin; Hypoxia inducible factor-1α; Ovarian cancer; Vasculogenic mimicry.

Fig. 1

Expression of vascular cell markers and hCG in and morphological flexibility of hCG-treated OVCAR-3 cells. a, b Expression levels of vascular markers CD31, VEGF, factor VIII, hCG, and HIF-1α were determined in OVCAR-3 cells exposed to 50, 500, or 5000 mU/ml hCG for 7 days. HCG treatment stimulated the expression of vascular markers and HIF-1α in OVCAR-3 cells in a dose-dependent manner. a The mRNA levels were analysed by RT-PCR. b Protein levels were detected by western blotting. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content was quantified for 3 independent replicates and the data are presented as the mean ± SD. The data shown are presented after normalization with GAPDH expression and were analysed using 1-way ANOVA. *p < 0.01, ^#p < 0.05. e Light and scanning-electron microscopy observations showed tubular network and channel formation by OVCAR-3 cells in the 3D matrix after exposure to 5000 mU/ml hCG. Representative morphological changes are shown

Fig. 2

Inhibition of hCG expression using siRNA resulted in suppressed vascular marker and HIF-1α expression. a, b Expression of hCG in OVCAR-3 cells was inhibited by siRNA targeting hCG mRNA, but not by a negative control siRNA. Compared with untransfected OVCAR-3 cells and mock-transfected OVCAR-3 cells, the expression of CD31, VEGF, factor VIII, and HIF-1α decreased in OVCAR-3 cells transfected with hCG siRNA. a mRNA expression of the vascular cell marker, HIF-1α and hCG was analysed by RT-PCR. b Protein expression of the vascular cell marker, HIF-1α and hCG was analysed by western blotting. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown are presented after normalization with GAPDH bands and analysed by 1-way ANOVA. *p < 0.01

Fig. 3

Up-regulated expression of vascular markers and HIF-1α in OVCAR-3 cells transfected with the phCMV1 vector expressing β-hCG (phCMV1-hCGβ). a, b HCG expression increased significantly in transfected OVCAR-3 cells, as determined by RT-PCR and western blot analysis. Compared with untransfected and mock-transfected cells, both mRNA (a) and protein expression (b) of CD31, VEGF, Factor VIII, and HIF-1α increased significantly. c, d Band densities were quantified by densitometric analysis. Protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown were normalized to GAPDH bands and analysed by 1-way ANOVA. *p < 0.01

Fig. 4

Expression of the hCG receptor (hCG-R) in the ovarian cancer cell line OVACR-3. a Confocal image of OVCAR-3 cells following immunofluorescence staining with an hCG-R antibody. Green fluorescence was localized to the periphery of OVCAR-3 cells. Blue fluorescence (PI) was used to demonstrate the nucleus. b, d Different concentrations of hCG did not significantly affect hCG-R expression in OVCAR-3 cells. b Protein expression of hCG-R in OVCAR-3 cells treated with hCG (0, 50, 500, or 5000 mU/ml) for 7 days was detected by western blot analysis. d Expression of hCG-R mRNA in OVCAR-3 treated with hCG (50, 500, or 5000 mU/ml) was detected by RT-PCR. c, e Band densities were quantified by densitometric analysis. The protein and mRNA content measured in 3 independent replicates was quantified and the data are presented as the mean ± SD. The data shown were normalized to GAPDH bands and analysed using 1-way ANOVA. p > 0.05