Anti-proliferative activities of finasteride in benign prostate epithelial cells require stromal fibroblasts and c-Jun gene

Abstract

This study aimed to identify the role of mouse fibroblast-mediated c-Jun and IGF-1 signaling in the therapeutic effect of finasteride on benign prostatic epithelial cells. BPH-1 cells, alone or with fibroblasts (c-Jun+/+ or c-Jun-/-), were implanted subcutaneously in male nude mice who were then treated with finasteride. The degrees of cell proliferation, apoptosis, and sizes of the xenografts were determined. BPH-1 cells were grown alone or co-cultured with mouse fibroblasts in the presence of finasteride and the level of IGF-1 secreted into the medium by the fibroblasts was determined. The proliferation-associated signaling pathway in BPH-1 cells was also evaluated. Fibroblasts and c-Jun promoted xenograft growth, stimulated Ki-67 expression, and inhibited BPH-1 apoptosis. Finasteride did not induce the shrinkage of xenografts in the combined-grafted groups despite repressing Ki-67 expression and inducing cell apoptosis. The addition of c-Jun-/- fibroblasts did not promote xenograft growth. In the absence of c-Jun and fibroblasts, finasteride did not alter xenograft growth, Ki-67 expression, or cell apoptosis. The in vitro results demonstrated that when BPH-1 cells were grown in monoculture, treatment with finasteride did not induce cell death and stimulated the expression of pro-proliferative signaling molecules, while in the presence of fibroblasts containing c-Jun, finasteride treatment repressed epithelial cell proliferation, the level of IGF-1 in the medium, and the activation of downstream pro-proliferative signaling pathways. Taken together, our results suggest that fibroblasts, c-Jun, and IGF-1 play key roles in mediating stromal-epithelial interactions that are required for the therapeutic effects of finasteride in benign prostate epithelial cells.

Fig 1. Expression of AR, 5-AR1 and 5-AR2 in mouse fibroblast and BPH-1 cells.

(A) mRNA transcription of AR and 5-AR in BPH-1 cells. Low level of AR mRNA, high level of 5-AR1 mRNA and negative 5-AR2 mRNA were detected in BPH-1 cells. (B) mRNA transcription of AR and 5-AR in mouse fibroblasts. A small quantity of AR mRNA and 5-AR2 mRNA, high level of 5-AR1 mRNA were found in mouse fibroblasts.

Fig 2. Cell proliferation and apoptosis in vitro and in xenografts of BPH-1-fibroblast-combined-grafted model.

(A) Cell proliferation of BPH-1 cells in mono-culture. High concentrations of finasteride did not inhibit cell proliferation in mono-culture. (B) Cell proliferation of BPH-1 cells co-cultured with wild-type fibroblasts or c-Jun^-/- fibroblasts. In the presence of fibroblasts, finasteride repressed cell proliferation by 30% (p = 0.003). (C&D) Growth curve of xenografts in the BPH-1-fibroblast-combined-grafted model. Compared with the BPH-1 mono-grafted group, wild-type fibroblasts (c-Jun^+/+) promoted xenograft growth whereas c-Jun^-/- fibroblasts did not stimulate xenograft growth. Finasteride did not have a significant impact on xenograft growth in the presence or absence of fibroblasts in limited 5-week experimental period.

Fig 3. Cell proliferation and apoptosis in xenograft tissues.

(A) Immunostaining of Ki-67, CK, vimentin and TUNEL in xenografted tissues. (B) Fibroblasts and c-Jun promoted the expression of Ki-67 in BPH-1 cells. Finasteride decreased the ratio of Ki-67-positive cells in epithelial cells in BPH-1-fibroblasts (c-Jun^+/+) combined-grafted group. (C) Fibroblasts and c-Jun repressed the ratio of apoptotic BPH-1 cells, whereas finasteride promoted cell apoptosis in the presence of fibroblasts.

Fig 4. The therapeutic effect of finasteride on fibroblasts.

(A) Finasteride induced cell death of fibroblasts. (B) Finasteride repressed the transcription of IGF-1 mRNA in fibroblasts in epithelia-fibroblasts co-culture system. (C) The level of IGF-1 protein in the medium of co-culture system was decreased in the presence of finasteride.

Fig 5. The effect of finasteride on proliferation-associated signaling pathway in BPH-1 in mono-culture or co-culture with fibroblasts.

(A) Finasteride stimulated the expression of p-AKT, p-ERK1/2, cyclin D1 and cyclin D3 in a concentration-dependent manner in BPH-1 mono-culture. (B) Finasteride repressed the expression of p-AKT, p-ERK1/2 and cyclin D1 in BPH-1 cells when co-cultured with fibroblasts. The inhibiting effect of finasteride on downstream proliferation-associated signaling pathway was compromised by c-Jun knockout in fibroblasts.