Interleukin-6 is an autocrine growth factor in human prostate cancer

Abstract

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. We have found that interleukin (IL)-6 protein concentrations are increased approximately 18-fold in clinically localized prostate cancers when compared to normal prostate tissue. Normal and neoplastic prostatic epithelial cells in culture, with the exception of LNCaP cells, secrete IL-6. Addition of exogenous IL-6 to primary epithelial cells in culture or the LNCaP prostate cancer cell line leads to phosphorylation of Stat-3 and increases in net cell proliferation. The concentration of IL-6 receptor is increased eightfold in the prostate cancer tissues and is increased in the cancer cells by immunohistochemistry. The increased expression of IL-6 receptor is correlated with increased proliferation of prostate cancer cells in vivo as assessed by Ki67 immunohistochemistry. These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo for primary, androgen-dependent prostate cancers.

Figure 1.

IL-6 content of benign and neoplastic prostate tissues. Protein extracts were prepared from prostate tissue samples from radical prostatectomy specimens that contained only benign prostate (BN) or 30 to 80% carcinoma (CA) by frozen section analysis. IL-6 content was then determined by ELISA and is expressed as ng per gram wet weight of tissue.

Figure 2.

Phosphorylation of Stat-3 after treatment of LNCaP prostate cancer cells with IL-6. LNCaP prostate cancer cells were treated with recombinant IL-6 for 0 to 60 minutes and cells were separated into nuclear and cytoplasmic fractions at the indicated times and lysates prepared. Equal quantities of protein were then analyzed by Western blot using antibodies specific for Stat-3 protein (Stat-3) or phosphorylated Stat-3 (pStat-3).

Figure 3.

Proliferation of prostate epithelial cells and LNCaP prostate cancer cells in response to exogenous IL-6. A: Primary epithelial cultures were plated at 1 × 10 cells per 35-mm dish and the next day refed with MCDB 153 containing insulin as the only growth factor. Control plates were incubated in this medium and experimental plates were treated with 1 or 10 ng/ml of recombinant IL-6. Cells were harvested by trypsinization after 1 to 3 days and cell number determined by counting using a Coulter counter. Standard deviations of triplicate determinations are shown. B: LNCaP cells were plated at 5 × 10 cells per 35-mm dish and the next day refed with RPMI 1640 containing insulin as the only growth factor. Control plates were incubated in this medium and experimental plates were treated with 10 or 100 ng/ml of recombinant IL-6. Cells were harvested by trypsinization after 1 to 3 days and cell number determined by counting using a Coulter counter. Standard deviations of triplicate determinations are shown.

Figure 4.

Cell cycle analysis of LNCaP prostate cancer cells treated with exogenous IL-6. LNCaP cells were plated at 2 × 10 cells per 100-mm dish and the next day refed with RPMI 1640 containing insulin as the only growth factor. Control plates were incubated in this medium and experimental plates were treated with 100 ng/ml of recombinant IL-6. Cells were harvested after 1 to 3 days and the number of dead cell fragments and the distribution within the cell cycle of viable cells determined by flow cytometry after staining with propidium iodide. A: Percentage of total events that are dead cell fragments at 24, 48, and 72 hours with or without 100 ng/ml IL-6. B: Percentage of viable cells in the S phase of the cell cycle after 24, 48, and 72 hours with or without 100 ng/ml IL-6.

Figure 5.

Immunohistochemistry with anti-phospho-Stat-3 antibody. Frozen sections of prostate cancer tissues were analyzed by immunohistochemistry using an anti-phospho-Stat-3 antibody. Variable staining of prostate cancer nuclei was seen in 10 of 12 cases. A: Anti-phospho-Stat-3 antibody. B: Control. Original magnifications, ×200.

Figure 6.

Correlation of tissue IL-6 receptor levels and proliferation. IL-6 receptor concentration was determined by ELISA of protein extracts of 12 human prostate cancer tissues and proliferation assessed by Ki67 immunostaining in the same cancers using frozen sections obtained before protein extraction. Total Ki67-positive nuclei were counted in 20 high-power (×400) fields of tumor. The cancers were divided into three groups of four cases each based on the IL-6 receptor protein concentration: low (12.5 ng/g wet weight), intermediate (38.0 ng/g), and high (82.5 ng/g). The standard deviation of the number of Ki67-positive nuclei per 20 high-power fields for each group is indicated.