Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells

Abstract

Prostate cancer is the second leading cause of malignancy-related mortality in males in the United States. As a solid tumor, clinically significant tumor growth and metastasis are dependent on nutrients and oxygen supplied by tumor-associated neovasculature. As such, there is a selective tumorigenic advantage for those neoplasms that can produce angiogenic mediators. We show here that human prostate cancer cell lines can constitutively produce angiogenic CXC chemokines. Tumorigenesis of PC-3 prostate cancer cells was shown to be attributable, in part, to the production of the angiogenic CXC chemokine, interleukin (IL)-8. Neutralizing antisera to IL-8 inhibits PC-3 tumor growth in a human prostate cancer/SCID mouse model. Furthermore, angiogenic activity in PC-3 tumor homogenates was attributable to IL-8. In contrast, the Du145 prostate cancer cell line uses a different angiogenic CXC chemokine, GRO-alpha, to mediate tumorigenicity. Neutralizing antisera to GRO-alpha but not IL-8 reduced tumor growth in vivo and reduced the angiogenic activity in tumor homogenates. Thus, prostate cancer cell lines can use distinct CXC chemokines to mediate their tumorigenicity.

Figure 1.

PC-3 and Du145 cells constitutively express significant levels of angiogenic CXC chemokines. ELISA measurements of 96-hour conditioned media from PC-3, Du145, and LNCaP cell lines.

Figure 2.

PC-3 and Du145 cells grow progressively in SCID mice. Male SCID mice were injected bilaterally in the rear flank with 1 × 10 PC-3, Du145, or LNCaP cells. Tumors were measured weekly with digital engineer’s calipers.

Figure 3.

PC-3 tumor growth correlates with the in vivo production of IL-8 and ENA-78. A cohort of male SCID mice were injected with 1 × 10 PC-3 cells in the rear flanks bilaterally at day 0. Mice were measured weekly for tumor size. Each week, six mice were sacrificed and tumor biopsies were analyzed by ELISA for specific angiogenic CXC chemokines. Graphs show comparisons between weekly tumor growth and in vivo production of specific angiogenic CXC chemokines.

Figure 4.

Du145 Tumor growth correlates with the in vivo production of IL-8 and GRO-α. A cohort of male SCID mice were injected with 1 × 10 Du145 cells in the rear flanks bilaterally at day 0. Mice were measured weekly for tumor size. Each week, six mice were sacrificed, and tumor biopsies were analyzed by ELISA for specific angiogenic CXC chemokines. Graphs show comparisons between weekly tumor growth and in vivo production of specific angiogenic CXC chemokines.

Figure 5.

IL-8 is a tumorigenic factor for PC-3 cells but not for Du145 cells. Male SCID mice were injected with 1 × 10 PC-3 (A) or Du145 (B) cells bilaterally in the rear flanks on day 0. Starting on day 0, animals received i.p. injections of 0.5 ml of goat anti-human IL-8 neutralizing antisera or 0.5 ml of NGS as a control. Antibody injections were given i.p. every 48 hours for the duration of the study. Results presented represent weekly tumor measurements.

Figure 6.

Tumor homogenates from PC-3 tumor-bearing animals treated with anti-IL-8 show less angiogenic activity than NGS-treated tumor homogenates. PC-3 tumor-bearing mice that were treated with either goat anti-human IL-8 or NGS as a control were sacrificed, and tumor homogenates were analyzed in the rat corneal micropocket assay. A demonstrates the neovascular response seen in six of six eyes tested from the NGS-treated mice. B represents the neovascular response seen when the anti-IL-8 treated tumor homogenates were analyzed. In the anti-IL-8 treated mice, two of six eyes tested were negative, three of six showed only an occasional loop as seen in B, and one of six eyes tested was positive.

Figure 7.

GRO-α is a tumorigenic factor for Du145 cells. Male SCID mice were injected bilaterally in the rear flanks with 1 × 10 Du145 tumor cells on day 0. Starting on day 0 animals were given 0.5-ml i.p. injections of either rabbit anti-human GRO-α or NRS as a control. Graph represents weekly tumor measurements.

Figure 8.

Du145 tumor homogenates from anti-GRO-α treated animals have less angiogenic activity than NRS-treated animals. Tumors were excised from antibody-treated Du145 tumor-bearing mice, homogenized, and analyzed in the corneal micropocket assay. A represents the angiogenic response seen in three of five eyes tested from the NRS-treated animals. B represents the negative response seen in four of five eyes tested from the anti-GRO-α treated animals.