Human amniotic epithelial cells inhibit growth of epithelial ovarian cancer cells via TGF‑β1-mediated cell cycle arrest

Abstract

It is reported that human amniotic epithelial cells (hAECs) endow intrinsic antitumor effects on certain kinds of cancer. This research was designed to evaluate whether hAECs endowed potential anticancer properties on epithelial ovarian cancer (EOC) cells in vivo and in vitro, which has not been reported before. In this study, we established a xenografted BALB/c nude mouse model by subcutaneously co-injecting ovarian cancer cell line, SK-OV-3, and hAECs for 28 days. In ex vivo experiments, CCK‑8 cell viability assay, real-time PCR, cell counting assay, cell cycle analysis and immunohistochemistry (IHC) assay were used to detect the effects of hAEC‑secreted factors on the proliferation and cell cycle progression of EOC cells. A cytokine array was conducted to detect anticancer-related cytokines released from hAECs. Human recombinant TGF‑β1 and TGF‑β1 antibody were used to treat EOC cells and analyzed whether TGF‑β1 contributed to the cell cycle arrest. Results from in vivo and ex vivo experiments showed that hAEC-secreted factors and rhTGF‑β1 decreased proliferation of EOC cells and induced G0/G1 cell cycle arrest in cancer cells, which could be partially reversed by excess TGF‑β1 antibody. These data indicate that hAECs endow potential anticancer properties on epithelial ovarian cancer in vivo and in vitro which is partially mediated by hAEC‑secreted TGF‑β1-induced cell cycle arrest. This study suggests a potential application of hAEC‑based therapy against epithelial ovarian cancer.

Figures

Figure 1

hAECs inhibit proliferation of EOC cells in vitro in a paracrine manner. (A) hAECs was negative for mesenchymal marker vimentin (green) and positive for epithelial marker CK-7 (red) by using immunofluorescence. DAPI (blue) staining showed the nuclei (scale bar, 100 µm). (B) CCK-8 cell viability assay used to test the effects of hAEC-CM in different concentrations on the viability of EOC cells at 48 h (n=6; performed in triplicate). Ordinary ANOVA was used for statistical analysis. (C) Real-time PCR were used to test the effects of hAECs on the expression levels of PCNA and Ki-67 in EOC cells cultured in Transwell system for 48 h (n=3; performed in triplicate). Data are represented as means ± SEM. *p<0.05 and ***p<0.001.

Figure 2

hAECs inhibit growth of SK-OV-3 cells in vivo. (A) Gross observation of subcutaneous xenografts obtained from SK-OV-3 injected group and SK-OV-3/hAECs co-injected group (scale bar, l cm). (B) The average tumor weight of SK-OV-3 injected group was significantly higher than that of SK-OV-3/hAECs co-injected group at day 28 (n=10). (C) The average tumor volume of SK-OV-3 injected group was significantly bigger than that of SK-OV-3/hAECs co-injected group at day 14 and day 28 (n=10). (D) (a) Representative image of GFP-transfected hAECs (scale bar, 200 µm). (b) We did not find positive signal of hAECsGFP+ in the negative control (scale bar, 100 µm). (c) The positive signals were found in the xenografted tumor tissues obtained from SK-OV-3/hAECsGFP+ injected group (scale bar, 100 µm). (E and F) Proliferation of cancer cells were tested by IHC using antibodies against PCNA and Ki-67 in SK-OV-3 and SK-OV-3/hAECs tumor tissues (scale bar, 100 µm; n=5). Data were analyzed by Mann-Whitney U test and were represented as means ± SEM. *p<0.05, **p<0.01 and ***p<0.001.

Figure 3

hAECs induce G0/G1 cell cycle arrest in EOC cells in a paracrine manner. (A) The effects of hAEC-CM on EOC cell division were tested by cell counting assay (n=6; performed in triplicate). (B) Cell cycle analysis revealed that hAEC-secreted factors induced G0/G1 cell cycle arrest in EOC cells in Transwell system (n=3; performed in triplicate). Data are represented as means ± SEM. *p<0.05, **p<0.01 and ***p<0.001.

Figure 4

hAECs regulate the expression of proteins related to G0/G1 cell cycle arrest determined by IHC. hAECs upregulated p16INK4A, p21 and phospho-JNK expression and downregulated phospho-pRB (Ser807) expression in EOC cells (n=5). Scale bar of (A), (B) and (C) were 100 µm and that of (D) was 50 µm. H-score system was used for semi-quantification of p16INK4A, p21 and phospho-JNK between two groups. Data were analyzed by Mann-Whitney U test and presented as means ± SEM. **p<0.01.

Figure 5

TGF-β1 was enriched in hAEC-released cell cycle-regulatory cytokines and induced G0/G1 cell cycle arrest in EOC cells. (A) (a) Selective map of the human antibody array 1000. hAECs released multiple cell cycle-regulatory cytokines, including TGF-β1, GM-CSF, IL-8, IFN-γ, IL-6, IL-1, TNF-α, TGF-β2 and Smad 7. (b) The intensities of the signals were quantified by densitometry and the expression of Smad 7 was regarded as control. (B) CCK-8 cell viability assay used to test the effects of rhTGF-β1 in different concentrations on the viability of EOC cells at 48 h (n=6; performed in triplicate). Data were analyzed by Mann-Whitney U test. (C) After being treated with rhTGF-β1, cell cycle of EOC cells was analyzed by flow cytometry. (D) ELISA was used to test the efficiency of TGF-β1 antibody added to neutralize the function of hAEC-secreted TGF-β1. Data were analyzed by Kruskal-Wallis test. (E) After being treated with TGF-β1 antibody in the Transwell system, cell cycle of EOC cells was analyzed by flow cytometry. Data are presented as means ± SEM. *p<0.05, **p<0.01 and ***p<0.001.