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NSAIDs such as celecoxib induce apoptosis in cancer cells. Although this apoptotic effect is involved in the anti-tumor activity associated with such drugs, the mechanism by which this occurs is not fully understood. We report here that various NSAIDs, including celecoxib, up-regulate PUMA, a Bcl-2 family protein with potent apoptosis-inducing activity, in human gastric carcinoma cell line, accompanying the induction of apoptosis. Experiments using siRNA and an intracellular Ca(2+) chelator revealed that Ca(2+)-dependent up-regulation of ATF4 and CHOP is involved in this up-regulation of PUMA. The siRNA for PUMA inhibited the celecoxib-induced activation and translocation of Bax, release of cytochrome c into the cytosol and induction of apoptosis, suggesting that PUMA plays an important role in celecoxib-induced mitochondrial dysfunction and the resulting apoptosis.

In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.

Previous studies have shown that modification of activating transcription factor 6 (ATF6) protein is important for the endoplasmic reticulum (ER) stress response; ER stressors stimulate the degradation of ATF6 by Site-1 protease (S1P) and Site-2 protease (S2P) into p50-ATF6, which acts as a transcription factor. In the current study, we found that all of the ER stressors tested (such as thapsigargin) up-regulate ATF6 mRNA expression. As thapsigargin did not affect the stability of the ATF6 mRNA, it was concluded that this up-regulation is due to transcriptional activation of ATF6. An inhibitor of S1P suppressed this up-regulation of ATF6 mRNA expression and putative ATF6-binding elements in the promoter of ATF6 were identified, suggesting that p50-ATF6 positively regulates the gene expression of ATF6. Since cells over-expressing ATF6 showed an enhanced ER stress response, we propose that up-regulation of ATF6 mRNA expression is involved in enhancing the ER stress response.

The cytotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs) is involved in the formation of NSAID-induced gastric lesions. The mechanism(s) behind these cytotoxic effects, however, is not well understood. We found here that several NSAIDs tested caused hemolysis when employed at concentrations similar to those that result in cytotoxicity. Moreover, these same NSAIDs were found to directly permeabilize the membranes of calcein-loaded liposomes. Given the similarity in NSAID concentrations for cytotoxic and membrane permeabilization effects, the cytotoxic action of these NSAIDs may be mediated through the permeabilization of biological membranes. Increase in the intracellular Ca(2+) level can lead to cell death. We here found that all of NSAIDs tested increased the intracellular Ca(2+) level at concentrations similar to those that result in cytotoxicity. Based on these results, we consider a possibility that membrane permeabilization by NSAIDs induces cell death through increase in the intracellular Ca(2+) level.