The Effect Of Cyclooxygenase-2 Gene Inhibiting On Arachidonic Acid Metabolism Pathway Of Esophageal Squamous Cell Carcinoma Cells

Background: Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) are two of the key enzymes of arachidonic acid metabolism pathway. Our previously studies have found that selected COX-2 inhibitor alone may induce esophageal squamous cell carcinoma(ESCC) cell proliferation in several specific concentrations, which may be due to the activation of 5-LOX shunt. However, whether the COX-2 mRNA inhibiting may result in the same outcome is still unknown.AIMS: To investigate the effect of COX-2 gene silencing on the proliferation of ESCC cells by small interfering RNA (siRNA), the following alteration of COX-2, 5-LOX, and their downsteam products, and the alteration of .apoptosis related genes expression. To explore the potential utility and the mechanism of COX-2 inhibition for the prevention and treatment of ESCC.METHODS: Three sequences of COX-2 siRNA were selected and synthesised. ESCC cell lines, including TE-1 and Eca109 were used for this study. Cells were divided into 4 groups, including blank control, liposome transfection control, random sequence siRNA and COX-2 siRNA groups. Cell proliferation was assessed by Cell Counting Kit-8 assay. Protein and mRNA expression of COX-2 and 5-LOX were determined by Western blot and RT-PCR, respectively. The concentrations of prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) were measured by ELISA Kit. Flow cytometer was used for the cell cycle measurement.RESULTS: One of the three COX-2 siRNA sequences, which with the highest COX-2 expression inhibiting rates of TE-1(79%) and Eca109 (73%) cells was selected. In TE-1 and Eca109 cells, after COX-2 siRNA transfection: 1. cell proliferation inhibition was achieved, while the inhibiton rates were 45.86% (P<0.05) and 48.99% (P<0.05), respectively; 2. the expression of COX-2 was down-regualated significantly (P<0.05), while the expression of 5-LOX was stayed in the same level (P>0.05). 3. the alteration of PGE₂ and LTB₄ levels were highly according to the alteration of COX-2 and 5-LOX, respectively; 4. the percentages of cells in G1 stage were 63.16 % and 68.15 %, respectively, while those in blank control groups were 58.93% and 33.02%, respectively; 5.the expression of Bcl-2 were found decreased, while the expression of Caspase-9 and Bax were found increased (P<0.05).CONCLUSIONS: The COX-2 expression of ESCC cells was inhibited by highly effective sequence selected siRNA. G1 arrested and apoptosis were induced in ESCC cells after COX-2 siRNA treatment. However, the expression of 5-LOX showed no significient difference before and after COX-2 siRNA treatment, which implied that highly effective inhibition of COX-2 expression may avoid the 5-LOX shunt of arachidonic acid metabolism pathway and the following up-regulation expression of LTB₄, a cell proliferation factor. On the other hand, the result also suggested that a successfully anti-cancer effect of ESCC may be achieved by only high dose selected COX-2 inhibitor with a significant inhibiting effect of COX-2.