levels close to those produced by A23187, but quite higher than those caused by CF per se. These findings can be likely accounted for by the notion [35] that in Jurkat cells CF $0.5 mM, not only stimulates a transient [Ca2+]i increase, but also inhibits the external Ca2+ influx. They also seem to support the view that a [Ca2+]i rise neither plays a crucial role in CF- induced apoptosis of Jurkat cells nor is adequate to support calpain activation and fodrin clevage, as shown above. Discussion In recent years, we have been investigating what initially appeared as an unexpected property of CF, namely, its ability to elicit a fast and extensive apoptotic process in cell lines of neoplastic origin, particularly hepatocarcinomas [13,19,25,26], among which the rat Yoshida ascites hepatoma AH-130 provided the main paradigm. The observation that an important lethal action is exerted by CF as well as other PPs on cells of both rodent and human origin prompted us to evaluate carefully these agents for their possible use as antineoplastic drugs. We noticed that human T-lymphoma Jurkat cells are as susceptible as rat hepatoma AH-130 cells to CF-induced apoptosis. This finding suggests that CF could be useful in the treatment of lymphoid malignancies. In this regard, to resort therapies less aggressive in terms of systemic toxicity and side effects would represent a major advance. Mechanisms of CF-induced Apoptosis Among PPAR ligands, only few studies have investigated the cytotoxic mechanisms of those targeting PPARa. Among the latter, some have been reported to activate apoptotic death in various cell lines (reviewed by [36]). In our experience, PPARa activation is unlikely to play any significant role in CF-induced apoptosis of Jurkat (this report) or hepatoma cells, particularly in view of its very rapid time-course [25]. All the potentially relevant parameters herewith evaluated in Jurkat cells are already altered within 5 min exposure to CF, and [Ca2+]i within 1 min. Therefore, this fast apoptotic death should be likely categorized as an extrareceptor activity, not differently from other effects of PPARa ligands, such as ER or oxidative stress [12]. In both human Jurkat T-leukemia cells and rat AH-130 hepatoma cells [13,19,25,26] CF-induced apoptosis is caspase- dependent, yet the two death processes follow different courses. As previously found, caspases 3, 8, and 9 are rapidly activated in AH- 130 cells exposed to CF and relevant caspase inhibitors afford a significant protection from apoptosis [26]. At variance, we presently found that caspases 8 and 9 activation in Jurkat cells is delayed, particularly with respect to caspase 3 and, in less striking manner, to caspase 2. Moreover, while the mitochondrial pathway appears to play a significant role in CF-treated AH-130 cells [26], in Jurkat cells the pattern is quite different. Briefly, the mitochondrial membrane potential rapidly falls and cytochrome c is released in both cell lines, once exposed to CF, but Smac- Diablo is released from mitochondria in AH-130 cells only (unpublished data). Moreover, caspase-9 inhibition or treatment with cyclosporine A are completely ineffective in protecting Jurkat cells from death. Altogether, these observations suggest that the mithocondrial pathway only marginally contributes to CF-induced apoptosis of Jurkat cells. The occurrence of ER stress in Jurkat cells exposed to CF is documented by the early and marked increased phosphorylation of two typical targets, eIF2a and JNK. Moreover, a selective JNK inhibitor (SP600125) affords a partial protection from CF-induced apoptosis, suggesting a role for JNK activation in this death. In last Rapid Jurkat Apoptosis by Clofibrate years, ER stress has been proposed to trigger apoptosis through mechanisms involving either caspase 12 [37] or 4 or 2, apart from other initiator (8, 9) and effector (3, 7) caspases [38]. Since we ruled out an involvement of both caspases 12 and 4 (see Results), our attention was focused onto caspase 2, which is regarded as an initiator caspase [39]. Dimerization and autocleavage of the p51 caspase 2 precursor lead to formation of an active p37 complex that is subsequently cleaved by an unknown caspase into an inactive 19 kDa fragment [39]. In the present study, the p51 precursor is already decreased in Jurkat cells after 5 min on CF, indicating early activation of caspase 2 that subsequently further increase through the whole experimental time. Of interest, the inactive p19 fragment becomes detectable after 30 min on CF (data not shown), suggesting that a late inhibitory cleavage might maintain constant levels of active caspase 2. Interestingly, CF has been reported to cause retrograde movement of Golgi constituents to the ER and, independently from PPARa activation, to disrupt the morphological and functional integrity of the Golgi complex in a manner similar to brefeldin A [40,41]. In conclusion, these observations are compatible with the hypothesis that activation of Golgi- or ER- associated caspase 2 are involved in the activation of caspase 3 in CF-treated cells. Role of Intracellular [Ca2+] in CF Cytotoxicity Both ER stress and mitochondrial depolarization may result in increases of cytosolic [Ca2+] i, which should be expected to activate calpains. The latter in turn can be involved in the early phases of apoptosis, leading to cleavage of the effector caspase 3 [42,43,44]. As an example, coincubation of Jurkat cells with Entamoeba histolytica results in calpain-dependent caspase 3 activa- tion and apoptosis, that can be prevented by calpeptin [45]. However, the involvement of calpain activation in CF-induced cell death appears unlikely, in view of two different observations: (i) even if calpeptin partially protects Jurkat cells from CF, this effect probably relies on non-specific caspase inhibition, since calpeptin only barely attenuates the intracellular fodrin cleavage if compared to the polycaspase inhibitor zVAD; (ii) no significant protection from CF is afforded by PD150606, another calpain inhibitor. A [Ca2+]i rise can result either in pro- or antiapoptotic effects [46]. In the present work, a [Ca2+]i elevation occurs after CF, yet treatments aimed at inhibiting it by means of extra- or intracellular chelators confer no protection against CF-induced cell death. By contrast, Ca2+ influx elicited by A23187 unexpectedly exerts a marked antiapoptotic action in CF-treated Jurkat cells, this protection being abrogated by adding EGTA into the medium. Therefore, protection by A23187 appears to depend on its ability to increase the Ca2+ influx in CF-treated cells, where it also inhibits both cytochrome c release and caspase 3 activation. The present data match a previous report of an antiapoptotic action of Ca2+ influx in puromycin-treated U937 cells [46]. The negligible toxicity of A23187 itself on Jurkat cells remains to be clarified. Noteworthily, T-cells tolerate high [Ca2+]i as required for activation, proliferation, and cytokine synthesis (reviewed by [47]), nor death occurs in B-lymphoma cell cultures exposed to anoxia/reoxygenation, in spite of a sustained [Ca2+]i rise [48]. Conclusion The present study demonstrates that exposing Jurkat cells to CF triggers a caspase-dependent apoptotic process not conforming to a canonical pathway, wherein caspases 2, likely activated by ER stress, and caspase 3 are involved. Finally, both apoptosis and caspase 3 activation are prevented by Ca2+ influx, suggesting that mobilization of Ca2+ stores from the ER combined with PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e45327