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The non-steroidal anti-inflammatory drug Celecoxib is a specific inhibitor of cyclooxygenase-2. Apart from its inhibitor function, Celecoxib induces apoptosis through the intrinsic pathway which is controlled by the Bcl-2 family members. In Jurkat T lymphoma cells, treatment with Celecoxib results in a rapid decline of the anti-apoptotic Bcl-2-related protein Mcl-1. The depletion of Mcl-1 is sufficient for apoptosis induction and can be blocked by overexpression of Bcl-xL but not by the close homologue Bcl-2. The present investigation analyzed the mechanism by which Bcl-xL prevents apoptosis induction whereas Bcl-2 failed to. Our data show that the involvement of the orphan nuclear receptor Nur77/TR3 specifically targeting Bcl-2 but not Bcl-xL was not involved in Celecoxib-induced apoptosis. Surprisingly, BH3-only proteins Bid, Bim, and Puma of the Bcl-2 family were not needed either. However, unlike Bcl-2, Mcl-1, and Bcl-xL sequestered Bak preventing it from activation through a direct interaction. Thus, when abundantly expressed, Bcl-xL can substitute for the loss of Mcl-1 whereas Bcl-2, incapable of forming a high affinity complex with Bak, could not., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The cyclooxygenase-2 inhibitor Celecoxib is a potent inducer of apoptosis in tumor cells. In most cellular systems Celecoxib induces apoptosis via an intrinsic, mitochondrial apoptosis pathway. We recently showed that in Bax-negative Jurkat cells expression of pro-apoptotic Bak is essential for Celecoxib-induced mitochondrial damage and apoptosis induction. Aim of the present study was to identify specific pro- and anti-apoptotic members of the Bcl-2 family involved in the regulation of Bak activation, and subsequent apoptosis upon treatment with Celecoxib in the Jurkat cell model. Our results show that apoptosis in response to Celecoxib required the presence of Noxa and downregulation of the anti-apoptotic protein Mcl-1. Celecoxib-induced Bak activation and subsequent apoptosis could be inhibited by overexpression of Bcl-xL but not by the very similar Bcl-2. In Bcl-xL-overexpressing cells neutralization of both, Mcl-1 and Bcl-xL, was prerequisite for an efficient induction of apoptosis. Our data reveal an important role of the Mcl-1/Noxa axis for Celecoxib-induced apoptosis and suggest that Celecoxib may be of value for treatment of tumors addicted to Mcl-1 and for combined treatment approaches targeting anti-apoptotic Bcl-2 family members.

The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib constitutes the prototype of pro-apoptotic agents acting through the intrinsic death pathway in a Bcl-2 independent manner. To gain further insight into celecoxib-mediated apoptosis regulation at the level of the mitochondria we tested in how far the crucial pro-apoptotic Bcl-2 proteins Bak and Bax were involved using clones of the Bax-deficient Jurkat T-lymphoma cell model either expressing Bak (Jurkat Bak positive) or being negative for Bak (Jurkat Bak negative), or overexpressing Bcl-2 (Jurkat Bcl-2). Celecoxib induced substantial apoptosis in Jurkat Bak-positive cells. Overexpression of Bcl-2 had only limited protective effects. However, loss of Bak-expression conferred almost complete resistance of Jurkat cells to celecoxib-induced apoptosis. Neither enhanced celecoxib-concentrations nor prolonged incubation times were sufficient to normalize apoptotic rates upon celecoxib-treatment in these Bak/Bax-negative cells. In line with that observation, siRNA-mediated silencing of Bak in the Bak-positive Jurkat cells largely reduced the extent of celecoxib-induced cell death. Interestingly, in celecoxib-sensitive Bak-positive cells, celecoxib-treatment resulted in down-regulation of the anti-apoptotic Bcl-2 protein Mcl-1 which may contribute to celecoxib-mediated activation of Bak-dependent apoptosis. Taken together our data clearly show for the first time the functional relevance of Bak for celecoxib-induced apoptosis in Bax-deficient Jurkat T-lymphoma cells.