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Induction of Claudin-4 by Nonsteroidal Anti-inflammatory Drugs and Its Contribution to Their Chemopreventive Effect

¹ Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan; ² Faculty of Pharmaceutical Sciences and ³ Graduate School of Medicine and Dentistry, Okayama University and ⁴ Department of Clinical Pharmacy, Shujitsu University School of Pharmacy, Okayama, Japan

Requests for reprints: Tohru Mizushima, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japan. Phone: 81-96-371-4323; Fax: 81-96-371-4323. E-mail: mizu{at}gpo.kumamoto-u.ac.jp.

Nonsteroidal anti-inflammatory drugs (NSAID) have shown chemopreventive effects in both preclinical and clinical studies; however, the precise molecular mechanism governing this response remains unclear. We used DNA microarray techniques to search for genes whose expression is induced by the NSAID indomethacin in human gastric carcinoma (AGS) cells. Among identified genes, we focused on those related to tight junction function (claudin-4, claudin-1, and occludin), particularly claudin-4. Induction of claudin-4 by indomethacin was confirmed at both mRNA and protein levels. NSAIDs, other than indomethacin (diclofenac and celecoxib), also induced claudin-4. All of the tested NSAIDs increased the intracellular Ca²⁺ concentration. Other drugs that increased the intracellular Ca²⁺ concentration (thapsigargin and ionomycin) also induced claudin-4. Furthermore, an intracellular Ca²⁺ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] inhibited the indomethacin-dependent induction of claudin-4. These results strongly suggest that induction of claudin-4 by indomethacin is mediated through an increase in the intracellular Ca²⁺ concentration. Overexpression of claudin-4 in AGS cells did not affect cell growth or the induction of apoptosis by indomethacin. On the other hand, addition of indomethacin or overexpression of claudin-4 inhibited cell migration. Colony formation in soft agar was also inhibited. Suppression of claudin-4 expression by small interfering RNA restored the migration activity of AGS cells in the presence of indomethacin. Based on these results, we consider that the induction of claudin-4 and other tight junction–related genes by NSAIDs may be involved in the chemopreventive effect of NSAIDs through the suppression of anchorage-independent growth and cell migration.

Key Words: NSAIDs • tight junction • claudin-4 • calcium • cancer

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