¹Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan

Correspondence: Professor T Mizushima, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University 5-1, Oe-honmachi, Kumamoto 862-0973, Japan. E-mail: mizu@gpo.kumamoto-u.ac.jp

Received 7 April 2005; Revised 23 August 2005; Accepted 23 August 2005; Published online 3 October 2005.

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2 (eIF2) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca²⁺ concentration, while 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, an intracellular Ca²⁺ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca²⁺-dependent activation of the PERK-eIF2-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.