Esophageal Epithelial Cells, Cox-2 Expression, Regulation And Cell Immortalization, And Mitomycin C Resistance

11.2.1 Aims and methodsEsophageal carcinoma (EC) is one of the six most common malignancies in the world. Linzhou, formerly Linxian, and nearby county Huixian in Henan Province, China, is one of the highest incidence areas for EC. Etiology for EC is not clear. Second prevention is one of the very important strategies in reducing the incidence for EC, however, the progress in this area is not very promising. Recent studies have suggested that nonsteroid anti-inflammatory drugs could reduce the risk of gastric and colon cancer, presumably by inhibiting the COX enzyme. Cyclooxygenase includes two isoforms,known as cyclooxygenase-1 and cyclooxygenase-2 (COX-2),whichhave different functions. Aberrant expression of COX-2 has been identified to increase cell proliferation and carcinogenesis. It has been documented that the expression of COX-2 is up-regulated in several epithelial tumor, but little is known about COX-2 for its expression in EC tissues in Henan high incidence area, the regulation factors, the effect on the esophageal epithelial ccarcinogenesis and the possible application in tumor chemotherapy. This study was designed to characterize COX-2 expression in EC tissues by immunohistochemical method and to correlate the alterations of COX-2 expression with EC cell differentiation and lymph node metastasis, to analysis the effect of serum concentrations on COX-2 expression in EC cell lines, to explore the function of TGF- β receptor and the interaction between TGF- β₁ and fibroblast in regulation of COX-2 expression through primary cell culture, Western Blotting and RT-PCR technology, to establish immortalization cell models by HPV-16- E6E7 and hTERT transfection and to determine the function of COX-2 expression in this process, and to investigate the function of COX-2 expression in EC cells after Mitomycin C treatment with flow cytometry.II.2.2 Key results and conclusions11.2.2.1 Increased expression of COX-2 protein in human ECThe positive immunostaining rate for COX-2 in 31 EC patients was 51.6% (16/31). No immunoreactivity was observed in control group. The differentiation progression of EC was inversely related to the expression of COX-2 (r=-0.564; P=0.001), but there was no correlation between metastasis characteristics of EC and intensity of COX-2 protein expression (Z=0.830;P=0.496). This study indicates that COX-2 protein over-expression may be one of the key molecular events correlated to the differentiation of EC, and it may shed new light on selecting molecular targets for EC prevention and treatment.11.2.2.2 Effect of serum concentrations on COX-2 expression and cell growth in two EC cell lines.Both the expressions of TGF- β RI and II were detected in primary culturednormal esophageal epithelial cells and EC-18 cells, while only trace expressions of TGF- β receptor I and no TGF- β receptor II expression was found in ECa-109 cells. After 48hrs culture in gradient serum concentrations, including 10%, 5%, 2.5%, 1.25%, 0.625% and 0%, both the cell growth rates and COX-2 expression increased. ECa-109 cells, in which the functions of TGF- β receptor were lost, showed serum depended expression of COX-2 mRNA and protein, while EC-18 cells, in which the expression of TGF- β receptor I and II were active, showed peak COX-2 mRNA and protein expression in 10% and 2.5% PCS serum concentration groups (p<0.05). On the other hand, serum depended growth tendency was found in the two EC cell lines. These data implied that TGF- β₁ should be at least one of the inhibitors for COX-2 expression in EC cells in vitro, while COX-2 may not be the key factor in cell proliferation control. Different serum concentrations had various effects on the expression of COX-2 in EC cells with different TGF- β₁ expression subtypes. The effect of serum concentration should not be ignored in the studies focusing on COX-2 expression and esophageal epithelial carcinogenesis in vitro.II.2.2.3 Expression of COX-2 in EC cells was affected by the interaction between TGF- β₁ a...