Insulin-like growth factor binding protein-6 inhibits prostate cancer cell proliferation: implication for anticancer effect of diethylstilbestrol in hormone refractory prostate cancer

Abstract

Diethylstilbestrol (DES) is a synthetic oestrogen, and its anticancer effects are exerted in androgen-dependent prostate cancer. The administration of DES decreases serum testosterone to castration levels. However, in androgen-independent prostate cancer patients, who are already orchiectomised, the administration of DES improves symptoms and decreases prostate-specific antigen (PSA). The mechanisms responsible for these direct inhibitory effects have been explained as biological actions not mediated by oestrogen receptors. We assessed the gene expression profiles of prostate cancer cells treated with DES, and investigated direct inhibitory effects of DES. DES inhibited the proliferation of LNCaP and PC-3 cells. cDNA microarray analysis showed that expression of many genes was downregulated by DES. However, insulin-like growth factor binding protein 6 (IGFBP-6) gene expression levels were upregulated in PC-3 cells. IGFBP-6 gene expression and protein levels significantly increased after DES treatment. Recombinant IGFBP-6 inhibited cell proliferation, and the inhibitory effect of DES was neutralised by anti-IGFBP-6 antibody. From the immunohistochemical analysis of IGFBP-6 using biopsy samples from androgen-independent prostate cancer, we found IGFBP-6 expression in androgen independent prostate cancer, and that DES treatment increased the IGFBP-6 staining intensity of the cancer cells in one sample. These findings suggested that DES induces IGFBP-6, which inhibits cell proliferation in an androgen-independent prostate cancer cell line, PC-3. IGFBP-6 therefore might be involved in the direct effects of DES in androgen-independent prostate cancer.

Figure 1

Inhibition of the proliferation of human prostate cancer cell lines LNCaP and PC-3 by diethylstilbestrol (DES). Cells were incubated for 24 h, and thereafter CM was aspirated away and the cells were incubated with CM containing various concentrations of DES. After 72 h, the number of viable cells was measured by MTT assay. Optical densities (OD) of cell lysates were measured at a wavelength of 540 nm. The values are expressed as means+s.d. (n=3), and the P-values were <0.05 (^*) and <0.01 (†).

Figure 2

Gel electrophoresis of DNA of LNCaP and PC-3 treated with diethylstilbestrol (DES). Cells treated with various concentrations of DES for 72 h were harvested, and DNA was extracted. DNA was electrophoresed in a 1.0% agarose gel. DNA fragmentation was observed after DES treatment in both LNCaP (A) and PC-3 cells (B).

Figure 3

Insulin-like growth factor binding protein-6 (IGFBP-6) gene expression levels in PC-3 cells. Transcript levels were measured by quantitative real-time PCR after treatment with various concentrations of DES. 18s ribosomal RNA transcript levels were used for the internal control, and gene expression levels were expressed as fold changes relative to those of the controls. IGFBP-6 gene expression was significantly increased after DES treatment in a dose-dependent manner in PC-3 cells. The values are expressed as means+s.d. (n=3), and the P-values were <0.05 (^*) and <0.01 (†).

Figure 4

Insulin-like growth factor binding protein-6 (IGFBP-6) protein levels in PC-3 cells. Protein levels were measured by Western blot analysis after treatment with various concentrations of DES. Proteins levels were expressed as fold changes. IGFBP-6 protein expression was significantly increased after DES treatment in a dose-dependent manner in PC-3 cells. The values were expressed as means+s.d. (n=3), and the P-values were <0.05 (^*) and <0.01 (†).

Figure 5

Inhibition of proliferation of human prostate cancer PC-3 cells by recombinant IGFBP-6. Cells were incubated for 24 h, and thereafter CM was aspirated away and the cells were incubated with CM containing various concentrations of IGFBP-6. After 48 h, the viable cell number was measured by MTT assay. The optical density (OD) of the cell lysates is expressed as the fold change. Recombinant IGFBP-6 inhibited the proliferation of PC-3 cells in a dose-dependent manner. The values are expressed as means+s.d. (n=3), and the P-values were <0.05 (^*).

Figure 6

Effects of IGFBP-6 and anti-IGFBP-6 antibody on PC-3 cell proliferation. Cells were incubated for 24 h, and thereafter CM was aspirated away. After 48 h of incubation of PC-3 cells with 800 ng ml⁻¹ recombinant IGFBP-6 and various concentrations of anti-IGFBP-6 or normal goat IgG, the viable cell number was measured by MTT assay. The optical density (OD) of the cell lysates was expressed as the fold change. In total, 20–30 μg ml⁻¹ of anti-IGFBP-6 antibody significantly neutralised the inhibitory effects of cell proliferation. The values are expressed as means+s.d. (n=3), and the P-values were <0.05 (^*) and <0.1 (#).

Figure 7

Effect of neutralising anti-IGFBP-6 antibody on DES-treated PC-3 cells. Cells were incubated for 24 h, and thereafter CM was aspirated away. After 48 h incubation of PC-3 cells with 6.25 μM DES and various concentrations of anti-IGFBP-6 antibody or normal goat IgG, the viable cell number was measured by MTT assay. The optical density (OD) of the cell lysates was expressed as the fold change. Cell proliferation was significantly upregulated by incubation with high-dose antibody (30 μg ml⁻¹). Anti-IGFBP-6 antibody inhibited DES-induced cell growth suppression. The values are expressed as means+s.d. (n=3), and the P-values were <0.01 (†).

Figure 8

Immunohistochemical staining of IGFBP-6. IGFBP-6 was visible in the cytoplasm of cancer cells in the organ (A). For one patient, DESdP treatment increased the IGFBP-6 staining intensity in cancer cells (B). The immunostained section in (B) is from the same patient as that shown in (A). Cells that stained positively for IGFBP-6 are brown in colour.