Expression Of Cyclooxygenase-2and CCR7and Gastric Cancer Transfer

Chapter one Correlation between the expression of COX-2and CCR7in gastric cancer and tumor metastasisObjective This study was designed to evaluate the coexpression of COX-2and CCR7in human gastric cancer and determine their relationships and correlations with lymph node metastasis, tumor differentiation, invasion depth, lymphatic metastasis and other clinicopathologic parameters.Methods Tissue samples of primary tumors from100gastric cancer patients and normal gastric tissue samples from40gastric cancer patients, undergoing paraffin imbedding slices were immunohistochemically examined for COX-2and CCR7expressions.Results COX-2and CCR7were highly expressed in gastric cancer tissues, the positive rate of COX-2and CCR7in the primary tumor was72.5%and67.5%, respectively. A significant correlation was found between the expression scores of COX-2and CCR7(P<0.05), and both also were correlated with several clinicopathologic parameters, including invasion depth, lymph node metastasis, TNM stage and primary tumor size, but both were not related to patients’age, sex and other factors (P>0.05). In addition, only CCR7expression was associated with histology and tumor site (P<0.05).Conclusions High expression of COX-2and CCR7in human gastric cancer was a significantly correlated. COX-2and CCR7were significantly associated with clinicopathologic parameters, including invasion depth, lymph node metastasis, TNM stage and primary tumor size. This indicate that detection of COX-2and CCR7will be helpful to predicting the metastasis of gastric cancer in clinic. Chapter2Regulation of CCR7expression in tumor cells by COX-2Objective To study the regulation of CCR7expression by COX-2and its mechanism.Methods Detected the expression of CCR7in mRNA and protein levels by RT-PCR, immune-histochemical and Western blot in SCG-7901and MGC80-3cells, which incubated with NS-398, PGE2, Con A, EP receptor agonists or antagonist, then investigated the effect of the influx of [ca2+]i, chemotactic and invasive responses after the treated tumor cells stimulated by CCL21, through Fura-2/AM and Chemotactic chamber.Results COX-2was over expressed in SCG-7901and MGC80-3cells, CCR7was expressed in these cells at different levels, EP1, EP2, EP4receptors were expressioned in SCG-7901and MGC80-3cells, but only can be detected the expression of EP3in SCG-7901cells. In both cells could be found the expressed of CCR7, and the calcium mobilization, chemotactic and invasive responses activated by CCL21were significantly inhibited when treated with NS-398(10μmol/L) and AH-23848(10μmol/L). The negative results could be detected when treated with PGE2(10μmol/L) and Con A (1mg/ml). There were not significant differences when treated with SC-19220,17-phenyl trinor PGE2, AH-6809. These showed that NS-398, AH-23848could reduce the functional expression of CCR7in SCG-7901,MGC80-3cells, and promote tumor metastasis, but PGE2, Con A were the negative.Conclusions In the SCG-7901and MGC80-3cells, the inhibitors of COX-2and EP4receptor can down-regulate the functional expression of CCR7, will be helpful to further studying the mechanism of COX-2and CCR7in promoting tumour metastasis, and indicate the synergic effect of COX-2and CCR7in tumour metastasis and the potential role in cancer therapy. Chapter3Silencing of CCR7for inhibition of tumor metastasisObjective To study the effect of siRNA-CCR7in tumor metastasis and its relationship with VEGF-C, VEGF-D, VEGF-A in vitro.Methods The varied expression of CCR7, VEGF-C, VEGF-D and VEGF-A in mRNA and/or protein levels in SCG-7901and MGC80-3cells stable transfected into siRNAs vectors targeted CCR7was detected, then the effect of the influx of [ca2+]I, chemotactic and invasive responses after the stable transfected cells stimulated by CCL21, through Ca2+Mobilization and Matrigel invasion assay were assessed.Results Inhibition of CCR7expression at the mRNA and protein levels could be detected in SCG-7901and MGC80-3cells stable transfected siRNA1, siRNA2vectors, and the inhibition of influx of [ca2+]i, chemotactic and invasive responses stimulated by CCL21also could be found in these cells. In SCG-7901cells, siRNA1, siRNA2could inhibit the expression of CCR7, VEGF-C, but not affected the expression of VEGF-D, VEGF-A in both cells. The expression of VEGF-C in MGC80-3cells was not detected.Conclusions Inhibition of CCR7expression by a siRNA impairs chemotactic, invasion and VEGF-C expression of cancer cell lines. It will lay a solid basis for us to further study the role of CCR7in tumour metastasis and CCR7specific inhibitor.