Effect Of IL-8 On Cell Proliferation And Migration In Hepatocellular Carcinoma Cells With Different Metastatic Potentials And The Links Between Chemokine And Epidermal Growth Factor Receptor Signaling Pathways

Background & AimsInterleukin-8 (IL-8), a member of the chemokine superfamily of structurally and functionally related inflammatory cytokines, was first purified on the basis of its neutrophil chemoattractant activity. The human IL-8 gene has a length of 5191 bp and contains four exons separated by three introns. It maps to human chromosome 4q12-21. IL-8 is generated as a precursor of 99 amino acids and is secreted after cleavage of a single sequence of 20 residues. N-terminal extracellular processing of the mature form yields several biologically active variants consisting of 79, 77, 72, 70 and 69 amino acids. The predominant variant consists of 72 amina acids and the molecular mass determined by SDS-PAGE was～8KDa. The promoter region of IL-8 contains bingding sites for AP-1, NF-κB, and NF-IL6. The IL-8 receptor (IL-8R), named CDw128, is a dimeric glycoprotein consisting of a 59 KDa and a 67 KDa subunit and belongs to the family of G-protein-coupled receptors (GPCR). IL-8 receptors are coupled to Bordetella pertussis toxin-sensitive GTP-bingding proteins. Signal transduction depends on the activation of a phospholipase C specific for phosphatidylinositol 4, 5-bisphosphate, which delivers two second messengers, IP3 and diacylglycerol. IP₃ induces a rise in cytosolic free calcium, and diacylglycerol activates protein kinase C. ADP-ribosylation of GTP-bingding proteins by pertussis toxin, depletion of mobilizable calcium, and exposure to protein kinase inhibitors or to wortmannin can be used to modulate signal transduction and to influence shape change, exocytosis and the respiratory burst. There are at least two different receptor types (CXCR1 and CXCR2). CXCR1 only binds IL-8, but CXCR2 also binds other CXC chemokines. Both IL-8R are encoded by a singe cope gene mapped to human chromosome 2q34-2q35 and they share 77% amino-acid identity.Tumor progression is closely linked to neovascularization. Since Koch et al found that IL-8 is also an angiogenic factor, the correlation between IL-8 expression and tumor progression has received considerable attention. The majority of studies demonstrated that IL-8 contributes to tumor progression by enhancing angiogenesis and metastasis via up-regulation of matrix metalloproteinase expression and collagenase activity in non-small cell lung cancer, breast cancer, prostate cancer, gall bladder and stomach cancer. In addition, it may act as an autocrine growth factor for malignant melanoma and colon carcinoma. But there is also contrary evidence that it may reduce tumorigenicity. Lee et al transfected human ovarian cell lines with an expression vector for human IL-8 and tested for their ability to form tumors in nude mice. They found that IL-8 expression by the transfected cells did not alter their growth properties in vitro. In contrast, tumor growth in vivo was significantly attenuated in animals receiving IL-8-expressing cells when compared with mice injected with control cells. Injection of neutralizing Ab to IL-8 reverted the phenotype and caused tumor growth in vivo. They analyzed that this effect may be mediated by the increased targeting of neutrophil and other mononuclear cells to the tumor injection site. Furthermore, the antitumor effect of Taxol has been attributed to an enhancement of tumor infiltration by neutrophils via up-regulation of tumor-associated IL-8. Expression of IL-8 has been reported in hepatocellular carcinoma, however, whether the IL-8 receptor also expressed in hepatocellular carcinoma and whether the expression of IL-8 is related to the metastatic potential remains unclear. The purpose of this study was to examine the expression of IL-8 and its receptors in hepatocellular carcinoma cells with different metastatic potentials and determine the effect of IL-8 on cell proliferation and migration.Epidermal growth factor receptor (EGFR) is one of the best characterized ligand -receptor systems and is frequently detected in a variety of tumor cell types of epithelial origin including carcinomas of breast and liver. Overexpression of EGFR by tumors has been associated with a poor clinical prognosis. The activation of EGFR signaling occurs via two distinct mechanisms: ligand-dependent and ligand-independent signaling pathways. The EGFR ligand family comprises several structurally related proteins such as EGF, transforming growth factor alpha, amphiregulin, heparin-binding EGF (HB-EGF) , betacellulin, epiregulin and epigen. Each of these specific ligands binds to the ectodomain of the EGFR and provokes its activation through a mechanism that involves dimerization, activation of the receptor tyrosine kinase cytosolic domain, and autophosphorylation of the receptor. Recently it has become apparent that the EGFR is also part of signaling networks activated by stimuli that do not directly interact with this receptor. These stimuli include agonists that specifically bind to other membrane receptors, membrane depolarization agents, and environmental stressors. The data not only show ligand-independent tyrosine phosphorylation of the EGFR, but also provide experimental evidence that the EGFR participates in the signaling events and cellular responses initiated by these various stimuli. Agonists for a diverse group of GPCR, such as purogenic, muscarnic acetylcholine, angiotensin, lysophosphatidic acid (LPA), thrombin, endothelin, adrenergic and bombesin, have been demonstrated to bring about increased levels of phosphotyrosine on the EGFR. These findings suggest important "cross-talk" between GPCR and EGFR signaling pathways. It is of interest whether IL-8, as one of the agonists for GPCR, may induce the transactivation of EGFR. Schraufstatter et al found that EGFR was phosphorylated following stimulation with IL-8 in microvascular endothelial cells, and which is mediated by the CXCR2 and involves cathepsin B. Studies about IL-8-induced transactivation of EGFR in gastric and colon carcinoma cells show that IL-8 induces shedding of HB-EGF because of an ADAM-dependent pathway. Venkatakrishnan et al found that transactivation of EGFR stimulated with IL-8 in ovarian cancer cells was associated with the adaptor molecules Shc and Grb2 , and was dependent on intracellular Ca²⁺ mobilization. The main point of this study is to examine whether there is cross-talk between IL-8-mediated GPCR pathway and EGFR signaling pathway in breast and hepatocellular carcinaoma cells.MethodsIL-8 protein and its recptors CXCR1, CXCR2 were measured by ELISA and immunocytochemistry in HepG2 cell line, low and high metastatic MHCC97-L and MHCC97-H cell lines. Effect of rhIL-8 and neutralizing antibody on cell proliferation and invasion were analyzed using MTT and matrigel invasion assay. IL-8 secretion responded to treatment with rhEGF and anti-EGFR in MDA-231 and HepG2 cell lines were measured by ELISA .EGFR transactivation stimulated with rhIL-8 and neutralizing antibody were assessed by western blot using anti-phosphotyrosine antibody in MDA-231 and HepG2 cell lines.Results1. The release of IL-8 were detected in all of the three cell lines, but no correlations were found between the level of IL-8 released and metastatic potential. CXCR1 and CXCR2 both expressed on cell membrane in three cell lines.2. Exogenous IL-8 and its neutralizing antibody did not significantly influence the proliferation of ihree cell lines, but rhIL-8 stimulated invasive activites in three cell lines and its neutralizing antibody inhibited the invasive activites (p < 0.05).3. Both EGF and anti-EGFR inhibited the secretion of IL-8 in breast carcinoma cell and IL-8 have no effect on EGFR phosphorylation, but anti-IL-8 induced transactivation of EGFR after 24h.4. EGF promoted the secretion of IL-8 and anti-EGFR inhibited the secretion of IL-8 in hepatic cell carcinoma HepG2 cell line, but both exogenous IL-8 and anti-IL-8 has no effect on the expression of EGFR.Conclusions1. The release of IL-8 were detected in all of the three cell lines with different metastatic potential in hepatocelllular carcinoma, and both CXCR1 and CXCR2 expressed on cell membrane in three cell lines, but no correlations were found between the level of IL-8 released and metastatic potential. IL-8 contributes to tumor progression in hepatocelllular carcinoma through its enhancement of invasive activites but not act as an autocrine growth factor. 2. Exogenous IL-8 has no effect on transactivation of EGFR in breast carcinoma. On the contrary, its neutralizing antibody induced tyrosine phosphorylation of EGFR. These observations, together with the finding that both EGF and anti-EGFR inhibited the secretion of IL-8 in breast carcinoma, suggests the correlation of competitive inhibition, rather than cross-talk, between GPCR-mediated IL-8 signaling pathway and EGFR pathway in breast carcinoma.3. Combined administration of two human antibodies using growth factor blockade in conjunction with chemokine blockade may provide a more effective approach for treatment of hepatocelllular carcinoma because the monoclonal antibody against the human EGFR obviously inhibited the release of IL-8.