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, 3 (1), 33-42

Expression of interleukin-8 Correlates With Angiogenesis, Tumorigenicity, and Metastasis of Human Prostate Cancer Cells Implanted Orthotopically in Nude Mice

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Expression of interleukin-8 Correlates With Angiogenesis, Tumorigenicity, and Metastasis of Human Prostate Cancer Cells Implanted Orthotopically in Nude Mice

S J Kim et al. Neoplasia.

Abstract

We determined whether the expression of interleukin-8 (IL-8) by human prostate cancer cells correlates with induction of angiogenesis, tumorigenicity, and production of metastasis. Low and high IL-8-producing clones were isolated from the heterogeneous PC-3 human prostate cancer cell line. The secretion of IL-8 protein correlated with transcriptional activity and levels of IL-8 mRNA. All PC-3 cells expressed both IL-8 receptors, CXCR1 and CXCR2. The low and high IL-8-producing clones were injected into the prostate of nude mice. Titration studies indicated that PC-3 cells expressing high levels of IL-8 were highly tumorigenic, producing rapidly growing, highly vascularized prostate tumors with and a 100% incidence of lymph node metastasis. Low IL-8-expressing PC-3 cells were less tumorigenic, producing slower growing and less vascularized primary tumors and a significantly lower incidence of metastasis. In situ hybridization (ISH) analysis of the tumors for expression of genes that regulate angiogenesis and metastasis showed that the expression level of IL-8, matrix metalloproteinases, vascular endothelial growth factor (VEGF), and E-cadherin corresponded with microvascular density and biological behavior of the prostate cancers in nude mice. Collectively, the data show that the expression level of IL-8 in human prostate cancer cells is associated with angiogenesis, tumorigenicity, and metastasis.

Figures

Figure 1
Figure 1
Production of IL-8 protein by clones isolated from the PC-3 parental line. Clones were isolated by a limited dilution technique and expanded in culture. Cells were plated into wells and culture supernatants were harvested 48 hours later. Level of IL-8 was determined by ELISA. Arrows identify clones producing low or high levels of IL-8 protein.
Figure 2
Figure 2
IL-8 expression in PC-3 lines. (A) Northern blot analysis. PC-3 IL-8 high cells had 15.3-fold higher expression of IL-8 mRNA than PC-3 IL-8 low cells. (B) Nuclear run-on assays. IL-8 transcriptional activity in the PC-3 IL-8 high cells was 4.4-fold higher than in the PC-3 IL-8 low cells.
Figure 3
Figure 3
Expression of IL-8 receptors in PC-3 cells. The expression of the IL-8 receptors, CXCR1 (265 bp) and CXCR2 (361 bp), was determined by RT-PCR carried out in the absence of reverse transcriptase as negative control to rule out DNA contamination. Note that all PC-3 cells expressed both receptors for IL-8 receptors. P, PC-3P; M, PC-3 MM2; H, PC-3 IL-8 high; L, PC-3 IL-8 low; N, negative control.
Figure 4
Figure 4
Tumorigenic and metastatic potential. (A) PC-3 parental and PC-3 MM2 and (B) PC-3 IL-8 low and PC-3 IL-8 high cells were injected into the prostate of nude mice. At 3, 4, or 5 weeks after tumor cell injection, groups of mice (n=5) were killed and necropsied. All mice with large palpable prostate (>1.5 g) were killed and necropsied, regardless of the time of injection. Mean tumor weight ±SD is shown in the bar graph. Percent regional lymph node metastasis is shown over the bars.
Figure 5
Figure 5
ISH analysis for angiogenesis- and metastasis-regulating genes in prostate tumors. Hybridization with poly d(T)20 probe confirmed mRNA integrity. The numbers indicate expression intensities as compared with poly d(T)20, which was assigned the value of 100. All analyses were carried out at the periphery of the neoplasms.
Figure 6
Figure 6
Relative MVD in prostate tumors. Prostate tumors produced by PC-3 IL-8 high and PC-3 IL-8 low cells were harvested and prepared for immunostaining with anti-CD31 antibodies. The mean number of blood vessels was 587±66 in tumors produced by PC-3 IL-8 high and 247±57 in tumors produced by PC-3 IL-8 low cells (P <.001).

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