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CXCL-8, a chemokine secreted by melanoma and stromal cells, serves as a growth and angiogenic factor for melanoma progression. This study evaluated how modulation of CXCL-8 levels in melanoma cell lines with different tumorigenic and metastatic potentials affected multiple tumor phenotypes. A375P cells (CXCL-8 low expressor) were stably transfected with a CXCL-8 mammalian expression vector to overexpress CXCL-8, whereas A375SM cells (CXCL-8 high expressor) were transfected with a CXCL-8 antisense expression vector to suppress CXCL-8 expression. Subsequent cell proliferation, migration, invasion, and soft-agar colony formation were analyzed, and in vivo tumor growth and metastasis were evaluated using mouse xenograft models. Our data demonstrate that overexpression of CXCL-8 significantly enhanced primary tumor growth and lung metastasis, accompanied by increased microvessel density in vivo, as compared with vector control-transfected cells. We also observed increased clonogenic ability, growth, and invasive potential of CXCL-8 overexpressing cells in vitro. Knockdown of CXCL-8 using an antisense vector resulted in increased cell death and reduced tumor growth relative to control. Taken together, these data confirm that CXCL-8 expression plays a critical role in regulating multiple cellular phenotypes associated with melanoma growth and metastasis. more...

We examined the expression of CXCL8 (interleukin-8), its receptors, CXCR1 and CXCR2, and vessel density in human melanoma by immunohistochemical analysis of tumors from different Clark levels, depths, and thicknesses. Expression of CXCL8 and CXCR2 was lower in Clark level I and II specimens than in level III through V specimens and metastases. CXCR1 expression was observed ubiquitously in the majority of human melanoma tumor specimens irrespective of disease state, with the highest intensity in Clark level III specimens. We observed a significant difference in CXCL8 and CXCR2 expression between thin (0.75 mm) melanomas and between thin and metastatic lesions. Positive correlations were observed between Clark level and CXCL8 or CXCR2 and between thickness and CXCR2 expression. We found no correlation between vessel density and Clark level or thickness. Our data suggest that expression of CXCL8 and CXCR2 contributes to aggressive growth and metastasis in human malignant melanoma. Consistent with the transition from radial to vertical growth phase melanoma, expression of CXCL8 and its receptor, CXCR2, may be key in the switch to an aggressive, more metastatic phenotype. more...

Interleukin-8 (IL-8/CXCL8), a paracrine angiogenic factor, modulates multiple biologic functions in CXCR1 and CXCR2 expressing endothelial cells. Several reports suggest that inflammation, infection, cellular stress and tumor presence regulate IL-8 production in endothelial cells. In the present study, we test the hypothesis that IL-8 regulates multiple biological effects in endothelial cells in an autocrine manner. We examined the autocrine role of IL-8 in regulating angiogenesis by using a neutralizing antibody to IL-8, CXCR1 or CXCR2 in human vein umbilical endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMEC). Neutralizing antibody to IL-8, CXCR1 or CXCR2 inhibited endothelial cell proliferation, and MMP-2 production as compared to cells cultured with medium alone or control antibody. In addition, we observed that the number of apoptotic cells was significantly higher in anti-IL-8, anti-CXCR1 and anti-CXCR2 treated endothelial cells, which coincided with decreased survival-associated gene expression. We observed reduced migration of endothelial cells treated with anti-IL-8 and anti-CXCR2 antibody, but not anti-CXCR1 antibody as compared to controls. Further, we observed an inhibition of capillary tube formation and neovascularization following treatment with anti-IL-8, anti-CXCR1 and anti-CXCR2 antibodies. Together these data suggest that IL-8 functions as an important autocrine growth and angiogenic factor in regulating multiple biological activities in endothelial cells. more...

IL-8, a member of the chemokine family, has been shown to play an important role in tumor growth, angiogenesis, and metastasis. The objective of this study was to determine the mechanism of IL-8-mediated angiogenesis. We examined the direct role of IL-8 in angiogenesis by examining IL-8 receptor expression on endothelial cells and their proliferation, survival, and matrix metalloproteinases (MMPs) production. We demonstrate that HUVEC and human dermal microvascular endothelial cells constitutively express CXCR1 and CXCR2 mRNA and protein. Recombinant human IL-8 induced endothelial cell proliferation and capillary tube organization while neutralization of IL-8 by anti-IL-8 Ab blocks IL-8-mediated capillary tube organization. Incubation of endothelial cells with IL-8 inhibited endothelial cell apoptosis and enhanced antiapoptotic gene expression. Endothelial cells incubated with IL-8 had higher levels of Bcl-x(L):Bcl-x(S) and Bcl-2:Bax ratios. Furthermore, incubation of endothelial cells with IL-8 up-regulated MMP-2 and MMP-9 production and mRNA expression. Our data suggest that IL-8 directly enhanced endothelial cell proliferation, survival, and MMP expression in CXCR1- and CXCR2-expressing endothelial cells and regulated angiogenesis. more...

In the present study, we examined the autocrine/paracrine role of IL-8 in melanoma growth and metastasis by analyzing the expression and functional significance of IL-8 receptors, CXCR1 and CXCR2 in human malignant melanoma cells with different metastatic potential. CXCR1 and CXCR2 mRNA and protein levels were analyzed by reverse trannscriptase-based polymerase chain reaction, immunohistochemistry, immunoprecipitation, flow cytometry and ligand binding assay in melanoma cells in vitro and xenografted in nude mice. Melanoma cells constitutively expressed CXCR1 and CXCR2 mRNA and protein. Highly metastatic A375SM cells expressed higher levels of CXCR1 and CXCR2 mRNA and protein in vitro and in vivo as compared to low metastatic A375P and non-metastatic SBC-2 melanoma cells. Treatment of SBC-2 and A375P cells with exogenously added recombinant IL-8 significantly enhanced their proliferation and invasive potential. Further neutralizing antibodies to CXCR1 and CXCR2 inhibited proliferation and invasive potential of unstimulated and IL-8-stimulated A375P cells. In summary, the data suggest that constitutive expression of CXCR1 and CXCR2 play an important role regulating the IL-8-mediated metastatic phenotype in human malignant melanoma cells. more...

Tumor-associated macrophages (TAM) have been shown to play an important role in tumor angiogenesis. The purpose of this study was to determine whether monocyte recruitment, activation and differentiation mediated by monocyte chemotactic protein-1 (MCP-1) and macrophage colony stimulating factor (M-CSF) modulate the expression of the angiogenic factor, Interleukin (IL)-8. Isolated human peripheral blood monocytes secreted low basal levels of IL-8. Incubation of monocytes with M-CSF or MCP-1 resulted in an up-regulation of IL-8 mRNA and protein expression. The differential expression of IL-8 by monocytes following MCP-1 and M-CSF treatments involved activation of the NFkB transcription factor. Further activation with lipopolysaccharide (LPS) caused an increase in IL-8 secretion in monocytes but not in monocyte-derived macrophages (MDM). MDM-conditioned media significantly up-regulated IL-8 expression in human malignant melanoma cells in vitro. In summary, we demonstrated that MCP-1 and M-CSF, critical for monocyte recruitment, activation and differentiation, differentially regulate IL-8 expression and may play an important role in monocyte/macrophage-mediated tumor angiogenesis. more...

In the present study, we demonstrate that upregulation of interleukin-1beta(IL-1beta)-mediated and tumor necrosis factor-alpha (TNF-alpha)-mediated IL-8 expression in human malignant melanoma cells is modulated by the activation of nuclear factor-kappaB (NF-kappaB). Addition of capsaicin (8-methyl-N-vanillyl-6-nonenamide), a known inhibitor of NF-kappaB, resulted in the inhibition of constitutive as well as IL-1beta-induced and TNF-alpha-induced IL-8 expression in melanoma cells. The inhibition of IL-8 expression was dependent on the concentration of capsaicin and duration of treatment. Further, electrophoretic mobility shift assay (EMSA) of nuclear extracts from melanoma cells showed a constitutive activation of NF-kappaB and activated protein 1 (AP-1), which was upregulated following treatment with IL-1beta. Treatment of melanoma cells with capsaicin inhibited activation of constitutive and IL-1beta-induced NF-kappaB, but not AP-1, leading to inhibition of IL-8 expression. Further, downregulation of IL-8 expression in capsaicin-treated melanoma cells resulted in inhibition of in vitro cell proliferation. These results demonstrate that constitutive and induced NF-kappaB activation regulates IL-8 expression in melanoma cells. Downregulation of constitutive and induced NF-kappaB activation in malignant melanoma cells leads to inhibition of IL-8 production and in vitro cell proliferation. more...

Purpose: In the present study, we examined the expression of a multifunctional cytokine, interleukin 8 (IL-8), and its receptors, CXCR1 and CXCR2, in human colon carcinoma cells with different metastatic potentials and determined their role in modulating phenotypes associated with metastasis., Experimental Design: IL-8, CXCR1, and CXCR2 protein and mRNA expression were examined using ELISA, immunocytochemistry, and reverse transcription-PCR in human colon carcinoma cells with different metastatic potentials. IL-8-mediated proliferation, migration, and tumor-endothelial cell interaction were analyzed., Results: IL-8 mRNA and protein expression was very low in Caco2 cells but elevated in KM12C cells and very high in KM12L4 cells, suggesting an association between the IL-8 production and metastatic potential. Similarly, CXCR1 and CXCR2 expression was lower in Caco2 cells than in low and high metastatic KM12C and KM12L4 cells. The recombinant human IL-8 enhanced the proliferation of colon carcinoma cells. Furthermore, proliferation of low and high metastatic cells expressing different levels of IL-8 was inhibited by neutralizing antibodies to IL-8, CXCR1, and CXCR2. We observed significant differences in the invasive potential of colon carcinoma cells expressing different levels of IL-8. In addition, we observed that IL-8 modulates adhesion of tumor cells to endothelial cells in an autocrine and paracrine manner., Conclusion: Our present data suggest an association between constitutive expression of IL-8 and aggressiveness in human colon carcinoma cells and the possible role of IL-8 in modulating different metastatic phenotypes associated with progression and metastasis. more...

In the present study, we analyzed the expression of a multifunctional cytokine, interleukin-8 (IL-8), in metastatic endometrial carcinoma cells. Our data demonstrate that human serous papillary endometrial adenocarcinoma (SPEC) and human endometrial adenocarcinoma (HEC) cells expressed steady-state IL-8-specific mRNA transcript and secreted IL-8 protein. The levels of IL-8 mRNA in SPEC-2 cells established from stage IV serous papillary adenocarcinoma were three-fold higher as compared to endometrial adenocarcinoma cells, HEC-1 A, established from stage IA endometrial cancer. Further, we observed higher levels of IL-8 mRNA and protein expression in the metastatic variants of SPEC-2 and HEC-1A cells as compared to the parent cell lines, demonstrating that IL-8 expression was associated with metastatic potential. Further, the treatment of endometrial carcinoma cells with inflammatory cytokines, IL-1beta and tumor necrosis factor-alpha (TNF-alpha), demonstrated that IL-1beta and TNF-alpha induced IL-8 expression in endometrial cancer cells. IL-1beta was a more potent inducer of IL-8 expression than TNF-alpha in our studies. These data demonstrate that constitutive and induced IL-8 expression in endometrial carcinoma cells might be an important regulatory mechanism of tumor growth and metastasis. more...

This review article has described briefly studies supporting the concept that IL-8 expression and its regulation by inflammatory cytokines like IL-1 may play an important role in controlling the phenotypes associated with melanoma progression and metastasis. It is clear from the experiments presented here that IL-8 is an important autocrine multifunctional cytokine that modulates melanoma/cell proliferation, migration by induction of extracellular matrix degradation enzymes and induces neovascularization, all of which are critical for melanoma growth and metastasis. In addition, their expression in melanoma tumor specimens suggests an association between IL-8 expression and tumor aggressiveness. Further, inflammatory cytokines produced by either tumor cells or stromal cells may regulate IL-8 expression, which can control melanoma growth and enhance our current knowledge regarding melanoma progression and metastasis. Understanding these events and their significance will allow us to design novel therapeutic approaches for treatment of melanoma. more...

It was recently demonstrated that interleukin-8 (IL-8) is produced by cultured melanoma cells and acts as an essential autocrine growth factor. Earlier studies from our laboratory demonstrated a direct correlation between IL-8 expression in human variant cell lines and their metastatic potential in nude mice. In the present study we examined the expression of IL-8 in human malignant melanomas using immunohistochemistry to correlate IL-8 levels with disease stage. None of the radial growth phase (RGP) tumours (melanoma in situ) expressed IL-8. In contrast, 50% of the vertical growth phase (VGP) tumours (invasive melanoma), which have a high risk of metastasis, showed IL-8 immunoreactivity. Further, all the metastatic lesions analysed showed intense staining for IL-8; the levels were higher than those observed in the primary skin tumours. In summary, the data suggest an association between the expression of IL-8 and the metastatic phenotype. more...

Here, we report the molecular regulation of interleukin (IL)-8 expression in human melanoma cells. The inflammatory cytokines IL-1beta and tumor necrosis factor-alpha (TNF-alpha) up-regulated IL-8 expression, in a time- and concentration-dependent manner, in three metastatic melanoma variants, SBC-2 (nonmetastatic), A375P (low metastatic), and A375SM (high metastatic), by increased transcription of the IL-8 gene, leading to increased levels of IL-8 mRNA and protein production. Furthermore, we report that IFN-alpha and IFN-beta did not inhibit steady-state IL-8 production. However, IFN-alpha and IFN-beta inhibited IL-1beta or TNF-alpha-mediated up-regulation of IL-8 mRNA. In addition, IFN-beta demonstrated a more potent inhibitory effect at a lower concentration than did IFN-alpha. Both pretreatment and simultaneous treatment of melanoma cells with IFN-alpha or IFN-beta inhibited the IL-1beta and TNF-alpha up-regulation of IL-8 mRNA levels. This inhibition was at the transcriptional levels and was unaffected by a protein synthesis inhibitor, suggesting that this did not require de novo protein synthesis. Further, modulation of IL-8 levels by IL-1beta, alone or in combination with IFN-beta, affected the proliferation of melanoma cells. In summary, our data suggest that the up-regulation of IL-8 expression in melanoma cells is regulated at the transcriptional level and is rapidly and specifically inhibited by IFN-alpha or IFN-beta, independent of de novo protein synthesis, perhaps due to a transient modification of a preexisting factor(s). more...