Regulatory Mechanism Of CXCL5and IL-8Production Induced By Epithelial Growth Factor In Human Hepatocellular Carcinoma Cells

Background and AimsHeptaocellular carcinoma (HCC) is one of most common maliganacies with a high rate of metastasis, responsible for the main cause of death, the morbidity and mortality of which ranked sixth and third in the world respectively. In China, about140000people die of liver cancer every year, accounting for about50%of global death toll of HCC. HCC slowly unfolds on a background of chronic inflammation triggered by exposure to infectious agents, mainly HBV infection. Chronic liver inflammation was thought to be the main risk factor prompting the carcinogenesis, tumor growth and progression, which may relevant to the continuous, non-specific and inefficient immune system activation. HCC inflammatory micro-environment consists of immune cells and inflammatory cytokines. On one hand, tumor cells secret inflammatory cytokines, thus induce migration of epithelial cells and immune cells in circulation and immune response, involving the process of angiogenesis, tumor growth and metastasis; on the other hand, immune cells recruited also produce inflammatory cytokines regulating the tumor progression. EGF and EGFR are found over-expressed in liver cancer, related with poor prognosis. EGF and EGFR signaling pathway play vital roles in HCC inflammatory micro-environment.The present studies aimed at exploring the pattern and potential mechanism of IL-8and CXCL5production from HCC, the involvement of their intracellular signaling pathways induced by EGF, and the extensive cross-talk between IL-8and CXCL5.MethodsThe inflammatory cytokines and receptors PCR array was used to measured the expression of inflammatory cytokines in HCC cells with different metastasis. The concentration of IL-8and CXCL5from HCC cells were measured by ELISA. The phosphorylations of ERK and PI3K/Akt were analyzed by western blot. Cell proliferation and apoptosis were measure by CCK-8and flow cytometry. Cell bio-behaviors including cell numbers, cell movements and cell morphology, were measured by Cell IQ Platform.Results1. The HCC cell lines (HepG2, HCCLM3) can produce IL-8and CXCL5; The production of IL-8and CXCL5in HCC cells with high metastatic potential (IICCLM3) is higher, compared with the cells with low metastatic potential (HepG2).2. The IL-8and CXCL5production from HCC cell lines stimulated with EGF significantly increased in a time-dependent and dose-dependent manners, as compared with those without EGF challenge.3. Erlotinib (EGFR inhibitor), U0126(ERK pathway inhibitor), BEZ235and SHBM1009(PI3K pathway inhibitor) could inhibit the IL-8and CXCL5production induced by EGF.4. EGF prompts the cell proliferation and cell movement in HepG2cell lines, while acted differently in HCCLM3cell lines. Meanwhile, U0126, BEZ235and SHBM1009inhibited HCC cell proliferation and cell movements.5. EGF up-regulated the phosphorylation levels of p38MAPK, ERK and Akt in HCC cells. The pathway inhibitor down-regulated the the phosphorylation levels of p38MAPK, ERK and Akt in HCC cells.6. CXCL5could act as a role of negative feeback to regulate the production of IL-8in HCC cell lines in time-dependent and dose-dependent way.7. CXCR2inhibitor down-regulated the production of IL-8induced by EGF.ConclusionThe expression of IL-8and CXCL5in HepG2and HCCLM3cells is significantly different. Compared with HepG2, the production of IL-8and CXCL5in HCCLM3cells is significantly higher. EGF could increase the production of IL-8and CXCL5in HCCLM3and HepG2cells probably through p38MAPK, ERK and Akt signaling pathways. ERK and PI3K/Akt signaling pathways involved in the regulation of HCC cell proliferation, apoptosis and movements. EGF prompts the HepG2cell proliferation and movements, and acted reversely in HCCLM3cells. Our results also indicated that CXCL5could act as a role of negative feeback to regulate the production of IL-8in HCC cell lines in time-dependent and dose-dependent way. EGF induced IL-8production through a potential cross-talk between CXCR2and EGFR signaling pathway. Thus, our data suggest that inflammatory cytokines such as IL-8and CXCL5play critical roles in the tumor growth and metastasis, as the potential therapeutic target for HCC.