Methods: The HERG mRNA, protein and current in gastric cancer cells transfected with or without COX-2 antisense vector were measured by RT-PCR, Western blot and patch-clamp, respectively. Cyclic adenosine monophosphate (cAMP) concentration in gastric cancer cells transfected with or without COX-2 antisense vector was measured by ELISA. Construction of HERG mutant without cAMP-binding domain was completed
by PCR and the mutant was transfected into gastric cancer cells. The impact of COX-2 inhibitor and prostaglandin selleck screening library E2 (PGE2) on HERG current in gastric cancer cells transfected with or without HERG mutant was investigated by patch clamp. The effects of agonist and antagonist of cAMP and inhibitor of protein kinase A (PKA) on HERG current in gastric cancer cells transfected with or without HERG mutant were observed by patch clamp. Results: Transfection with COX-2 antisense vector did not alter the expression of HERG mRNA and protein, but it diminished the amplitude of HERG current
in gastric cancer cells (p < 0.05). The cAMP concentration in gastric cancer cells transfected selleck chemical with COX-2 antisense vector was lower than that in parental gastric cancer cells (p < 0.05). COX-2 inhibitor and PGE2 had influence on the HERG current in gastric cancer cells. COX-2 inhibitor reduced the amplitude of HERG current in gastric cancer cells and PGE2 enhanced the amplitude. However, in gastric cancer cells transfected with HERG mutant deleting cAMP-binding domain, both COX-2 inhibitor and PGE2 did not show significant effects on HERG current. cAMP agonist enhanced the amplitude of HERG current and cAMP antagonist reduced the amplitude in gastric cancer cells. Both agonist and antagonist of cAMP had no significant
effect on HERG current in gastric cancer cells transfected with HERG mutant deleting cAMP 上海皓元 binding domain. PKA inhibitor did not influence the HERG current whether in parental gastric cancer cells or in gastric cancer cells transfected with HERG mutant. Conclusion: COX-2 regulates HERG current through its catalytic product PGE2, which alters cAMP level in gastric cancer cells. cAMP interacts with HERG protein by binding with cAMP-binding domain of HERG protein and exerts impact on HERG current. PKA does not participate in this process. Key Word(s): 1. gastric cancer; 2. COX-2; 3. HERG; 4. potassium channel; Presenting Author: SHAO XIAO-DONG Additional Authors: ZHANG YONG-GUO, CHEN JIANG, LIN HAO, GUO XIAO-ZHONG Corresponding Author: GUO XIAO-ZHONG Affiliations: General Hospital of Shenyang Military Area Command Objective: To investigate the effect of HERG protein on the proliferation of gastric cancer cells. Methods: HERG-siRNA vector was constructed and transfected into gastric cancer cells, followed by screening and verifying.
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