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Celecoxib activates a novel mitochondrial apoptosis signaling pathway ¹

Department of Radiation Oncology, Experimental Radiotherapy Group, University of Tübingen, D-72076 Tübingen, Germany

²Correspondence: Department of Radiation Oncology, Experimental Radiotherapy Group, University of Tübingen, Hoppe Seyler Str. 3, D-72076 Tübingen, Germany. E-mail: claus.belka{at}uni-tuebingen.de

SPECIFIC AIMS

The COX-2 inhibitor Celecoxib inhibits cancer cell growth via proapoptotic effects independent of its capacity to block COX-2. To gain insight into the mechanisms of Celecoxib-induced apoptosis and to discriminate between death receptor and mitochondrial signaling pathways, the activation of caspases, and the induction of mitochondrial alterations were tested in Jurkat T cells, Jurkat cells lacking caspase-8 or FADD, Jurkat cells overexpressing Bcl-2 or a dominant negative caspase-9, BJAB cells, BJAB cells expressing a dominant negative FADD and embryonic fibroblasts from Apaf-1-expressing as well as Apaf-1 knock out mice upon treatment with Celecoxib.

PRINICIPAL FINDINGS

1. Celecoxib induces dose- and time-dependent apoptosis in Jurkat T cells
Treatment of Jurkat T cells with Celecoxib led to a time-and dose-dependent induction of apoptosis as determined by flow cytometry using light scatter characteristics. Doses of 75–100 µM were sufficient to induce significant apoptotic changes. Analysis of the nuclear morphology by fluorescence microscopy upon staining with Hoechst 33342 revealed all the typical characteristics of apoptotic cells, including nuclear condensation and segmentation. Activation of caspases-9, -8, and -3 could be detected as early as 3 h after treatment with 75 µM Celecoxib. That caspases are activated by Celecoxib treatment was further substantiated by detection of the characteristic PARP processing pattern and the decrease in full-length ICAD, both substrates of caspase-3.

In parallel to the activation of caspases, characteristic hallmarks of mitochondrial involvement in Celecoxib-induced apoptosis were detected.

2. Celecoxib-induced apoptosis is independent of death receptor pathways
Celecoxib induced apoptosis in Jurkat and BJAB cells irrespective of the expression of functional FADD whereas lack of FADD abrogated death receptor-induced apoptosis in FADD-negative cells. Since caspase-8 was activated in response to Celecoxib, we tested in caspase-8-negative Jurkat cells and the respective A3 control cells to what extent caspase-8 is required for induction or execution of apoptosis in response to Celecoxib. No significant differences in the apoptosis induction and cleavage pattern of the caspase-3 substrate PARP were detected. These findings indicate that Celecoxib-induced apoptosis occurs independent of caspase-8 and without a requirement for FADD.

3. Caspase-9 and Apaf-1 are essential for Celecoxib-induced apoptosis
In general, death receptor-independent pathways are mediated via the mitochondria involving caspase-9- and Apaf-1-dependent activation of effector caspases downstream of the mitochondria. To test the assumption that caspase-9 is involved in Celecoxib-induced apoptosis, Jurkat cells were transfected with a dominant negative mutant of caspase-9. The dominant negative mutant significantly reduced Celecoxib-induced apoptosis and caspase activation. To elucidate the putative role of caspase-9 as the initiator caspase for the induction of apoptosis in response to Celecoxib, cells were treated with 40 µM of LEHD-fmk (caspase-9 inhibitor), IETD-fmk (caspase-8 inhibitor) and zVAD-fmk (pan caspase inhibitor) 10 min prior to treatment with Celecoxib. Characteristic morphological alterations induced by Celecoxib such as chromatin condensation and segmentation were abrogated by the pan-caspase inhibitor z-VAD and the caspase-9 inhibitor LEHD; in accordance with results using caspase-8-negative cells, the caspase-8 inhibitor IETD had no relevant influence on Celecoxib-induced nuclear changes. Specificity of the LEHD action was demonstrated by the lack of an influence on TRAIL-induced caspase activation and apoptosis. Thus, caspase-9 functions as the initiator caspase during Celecoxib-induced apoptosis.

To test for an involvement of Apaf-1 in Celecoxib-induced apoptosis, we performed experiments using embryonic fibroblasts from Apaf-1-expressing and Apaf-1 knockout mice. Since ionizing radiation is known to rely on intact Apaf-1 signaling, radiation experiments were performed as a control. Those experiments clearly showed that significant apoptosis upon Celecoxib treatment and radiation could only be detected in Apaf-1-expressing fibroblasts, pointing to an important role of the apoptosome in Celecoxib-induced apoptosis.

4. Mitochondrial damage and Celecoxib-induced apoptosis occur in the presence of Bcl-2
The data indicated that activation of caspase-9 may be a key step during Celecoxib-induced apoptosis whereas death receptor pathways were not involved. Based on previous observations that Bcl-2 interferes with mitochondrial damage and subsequent activation of caspase-9, we speculated that Celecoxib-induced cell death might be abrogated by overexpression of antiapoptotic members of the Bcl-2 family, Bcl-2 or Bcl-x_L. Surprisingly, our data revealed that neither Bcl-2 nor Bcl-x_L significantly interfered with Celecoxib-induced apoptosis. To further substantiate our finding that Celecoxib-induced apoptosis is mediated by a Bcl-2-independent process, the activation of caspases upon Celecoxib treatment was determined. As shown in Fig. 1 A, caspase-9 was activated in the Bcl-2-overexpressing cells albeit with a little delay in the time kinetic. A similar pattern was detectable when the processing of caspase-8, caspase-3, and PARP was analyzed (Fig. 1A ). Thus, Celecoxib-induced activation of caspases-9, -3, and -8 occurred irrespective of a strong Bcl-2 overexpression.

View larger version (33K): [in this window] [in a new window]   Figure 1. Overexpression of Bcl-2 does not abrogate Celecoxib-induced caspase activation and mitochondrial alterations. A) Jurkat vector cells or Bcl-2-overexpressing Jurkat cells were treated for 0, 3, or 6 h with 75 µM Celecoxib. Caspase activation was then determined by Western blot analysis of cytosolic extracts with specific antibodies against caspase-8 and the cleavage products of caspase-9 and -3. Caspase-3 activation was verified by detection of the cleavage of the caspase-3 substrate PARP. B) Bcl-2-overexpressing Jurkat cells were treated with various concentrations of Celecoxib. The mitochondrial membrane potential was then determined by FACS analysis of TMRE-stained cells. Depolarization of the cells with the proton ionophore CCCP was used as a positive control. Data show 1 representative of 3 independent experiments. C) Celecoxib treatment does not induce Bcl-2 cleavage or phosphorylation. Jurkat vector cells or Bcl-2-overexpressing Jurkat cells were treated with various concentrations of Celecoxib. Post-translational modification of Bcl-2 was analyzed by Western blot using an antibody against Bcl-2.

Bcl-2 controlled release of cytochrome c and the breakdown of the mitochondrial membrane potential are paradigmatic for the activation caspase-9 via complex formation between cytochrome c, dATP, Apaf-1, and procaspase-9. Therefore, we tested to what extent the Celecoxib-induced breakdown of the mitochondrial membrane potential, and release of cytochrome c were influenced by Bcl-2 overexpression. Bcl-2 abrogated neither depolarization of the mitochondrial membrane potential (Fig. 1B ) nor Celecoxib-induced release of cytochrome c (not shown). The lack of inhibitory Bcl-2 effects was not caused by Celecoxib-induced cleavage of Bcl-2 or an alteration of its phosphorylation state (Fig. 1C ).

CONCLUSIONS AND SIGNIFICANCE

Taken together, our data show that programmed cell death induced by Celecoxib was mediated by caspases and had all the hallmarks of apoptosis. Celecoxib-induced apoptosis was dependent on activation of caspase-9 and the expression of Apaf-1. However, Celecoxib-induced apoptosis did not require the presence of intact death receptor pathways nor was it abrogated by overexpression of antiapoptotic Bcl-2 family members.

Several observations suggested that an up-regulation of death ligands and subsequent autocrine death receptor stimulation may be involved in stress- and drug-induced apoptosis. Alternatively, it was suggested that death receptor pathways may be activated by a ligand-independent engagement of FADD. Similarly, it has been suggested that the NSAID sulindac may induce apoptosis via direct activation of FADD. Our observations make the involvement of such mechanisms for Celecoxib-induced apoptosis highly unlikely. Using FADD-negative Jurkat cells, it was evident that Celecoxib-induced apoptosis was completely independent of FADD. Thus, apoptosis induction via up-regulation of CD95-L or TRAIL and subsequent autocrine stimulation of the respective death receptor is not involved in Celecoxib-induced cell death. Our data also exclude the necessity of another FADD-dependent mechanism for Celecoxib-induced apoptosis. The interpretation that death receptor signaling pathways are not required for Celecoxib-induced apoptosis was further substantiated by the observation that both the lack of caspase-8 and pretreatment with a caspase-8-specific peptide inhibitor had no effect on Celecoxib-induced apoptosis.

Since the results discussed above clearly indicated that death receptor pathways are not involved in Celecoxib-induced apoptosis, we speculated that Celecoxib may act via mitochondrial apoptosis pathways. Indeed, Celecoxib induced several cellular events, including caspase-9 activation, release of cytochrome c, and breakdown of the mitochondrial membrane potential, all of which are indicative of an involvement of mitochondrial apoptosis pathways. The interpretation that Celecoxib-induced apoptosis is mediated via cytochrome c release and subsequent caspase-9 activation was substantiated by the finding that a dominant negative caspase-9 mutant and a caspase-9-specific peptide inhibitor were able to block Celecoxib-induced cell death.

Bcl-2 had been shown to interfere with apoptosis induction in response to many cellular stressors acting via mitochondrial signaling pathways. In contrast, Bcl-2 does not generally interfere with receptor-induced cell death, which is mediated in most cases by a direct caspase-8 and caspase-3 activation. Thus, we speculated that Bcl-2 would interfere with Celecoxib-induced apoptosis. However, our data clearly show that Bcl-2 did not block Celecoxib-induced apoptosis, caspase activation, or mitochondrial alterations. Bcl-2 itself was not modified by Celecoxib treatment, since neither the size nor abundance of the nonphosphorylated 26 kDa Bcl-2 was altered, and no significant phosphorylation of Bcl-2 could be detected that would be characterized by the appearance of a 28 kDa band. The finding that in addition to Bcl-2, Bcl-x_L also lacked significant inhibitory effects on Celecoxib-induced apoptosis allows a more generalized conclusion that Celecoxib activates apoptosis via a mitochondrial pathway that is not inhibitable by anti-apoptotic members of the Bcl-2 family (Fig. 2 ).

View larger version (18K): [in this window] [in a new window]   Figure 2. Diagram of the apoptotic signaling pathway induced by Celecoxib. Celecoxib induces mitochondrial signaling including loss of m, release of cytochrome c, activation of caspase-9, caspase-3, caspase-8, and nuclear condensation. In contrast to its influence on radiation-induced mitochondrial alterations, Bcl-2 did not abrogate Celecoxib-induced breakdown of m, release of cytochrome c, and subsequent activation of caspase-9. Independence of FADD and caspase-8 proves that Celecoxib-induced apoptosis does not involve death receptor signaling pathways.

In parallel, our findings make an involvement of Bcl-2 like proapoptotic molecules in Celecoxib-induced apoptosis highly unlikely since Bcl-2 would counteract their function. Only very few models of cytochrome c-dependent but Bcl-2-independent apoptosis have been found. It has been shown that treatment of BAF3 cells with the potassium ionophore valinomycin induced breakdown of the mitochondrial membrane potential, release of cytochrome c, and activation of caspases. However, Bcl-2 overexpression did not influence the induction of apoptosis by valinomycin. Thus, a direct action on the mitochondrial ion homeostasis may also be operative in Celecoxib-induced apoptosis. Nevertheless, the exact mechanism of Celecoxib-mediated cytochrome c release remains to be defined.

In conclusion, our findings indicate that Celecoxib-induced apoptosis is mediated via a novel caspase-9- and Apaf-1-dependent but Bcl-2/Bcl-x_L-independent mitochondrial pathway. Since Bcl-2 overexpression is associated with carcinogenesis and possibly with a reduced efficacy of conventional cytostatic drugs and ionizing radiation, the observation that Celecoxib acts independent of anti-apoptotic Bcl-2 family members is of high interest for its clinical use, either alone or in combination with other treatment modalities.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0947fje; doi: 10.1096/fj.02-0947fje

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