Expression Of CCR8in Colon Cancer And Its Effects On Biological Behavior Of Colon Cancer Cells

Part I Expression of CCR8in colon cancer and its clinical significanceObjective:To investigate the expression of C-C chemokine receptor8(CCR8) in colon cancer tissues and colon cancer cell lines, and explore its clinical significance.Methods:The expression of CCR8in colon cancer tissues and adjacent normal tissues were detected in70colon cancer cases by immunohistochemistry, the expression in6colon cancer cell lines were detected by western blot and flowcytometry. The relationship between CCR8expression and pathological features in colon cancer was analyzed.Results:Thirty-three of70colon cancer tissues were positive for CCR8expression (47.1%), and the adjacent normal tissues barely express CCR8, chemokine CCL1was detected in mesenteric lymph nodes. The expression of CCR8in colon cancer is highly correlated with invasion depth, lymph node metastasis and TNM staging(p<0.01), but shows a negative correlation with gender, age, tumor size and differentiation (p>0.05). CCR8is expressed in all six colon cancer cell lines, and SW480cell line has the strongest expression.Conclusion:The expression of CCR8was detected in a portion of colon cancer cases, and highly correlates with the invasive and metastasis potential of colon cancer. Colon cancer cell line SW480is suitable for further investigation. Part II Effect of CCL1/CCR8axis on calcium mobilization, proliferation, apoptosis and migration of SW480cell lineObjective:To assess the role of CCL1/CCR8axis in SW480cell line calcium mobilization, proliferation and migration.Methods:Calcium indicator Fluo-3and flowcytometry were used to detect the intracellular calcium concentration changes induced by CCL1in SW480cells. MTT assay, Annexin V/PI apoptosis assay and Transwell migration assay were performed to assess the effects of CCLl on SW480cells proliferation, apoptosis and migration respectively.Results:CCL1can lead to a rapid increase in the intracellular calcium level in SW480cells, and its effect can be blocked by the CCR8selective antagonist MC148. After incubation with different concentrations of CCL1for24hours,48hours or72hours, no significant differences were observed in SW480cells proliferation. Neither CCL1nor MC148has effects on apoptosis of SW480cells induced by5-FU. CCL1concentration gradient stimulated SW480cells migration in a concentration dependent manner, which can be blocked by MC148.Conclusions:The CCR8expressed on SW480cells are functional. CCL1/CCR8axis can significantly increase SW480cells migration in a chemotactic manner, but has no effects on SW480cells proliferation and anti-apoptosis. Part III Effect of CCL1/CCR8axis on invasion and metastatic factors expression level of SW480cell lineObjective:To investigate the role of CCL1/CCR8axis in invasion and expression of Matrix Metalloproteinase2(MMP2), Matrix Metalloproteinase9(MMP9), E-cadherin and Vascular Endothelial Growth Factor (VEGF) of SW480cell line.Methods:Transwell invasion assay was used to assess the effect of CCL1on SW480cells invasion. Changes in expressions of MMP2, MMP9, E-cadherin and VEGF in SW480cells induced by CCL1were assessed by Real-Time PCR and western blot.Results:The Transwell invasion assay showed CCL1can promote SW480cells invasion, and this effect can be blocked by MC148. After12hours incubation with100ng/ml CCL1, MMP2mRNA and protein expression were significantly up-regulated in SW480cells, while E-cadherin mRNA and protein expression were significantly suppressed. No significant differences were observed in SW480cells MMP9and VEGF expression in both mRNA and protein level after incubation with100ng/ml CCL1or MC148for12hours.Conclusions:CCL1/CCR8axis can promote SW480cells invasion, up-regulate the expression of MMP2and down-regulate the expression of E-cadherin, but has no effect on MMP9and VEGF expression in SW480cell line.