Caspase 2 Activation and ER Stress Drive Rapid Jurkat Cell Apoptosis by Clofibrate Fabio Penna, Fabrizio Pin, Domiziana Costamagna, Patrizia Reffo, Francesco Maria Baccino, Gabriella Bonelli, Paola Costelli* Department of Experimental Medicine and Oncology, University of Torino, Torino, Italy Abstract Differently from the antiapoptotic action most commonly assigned to peroxisome proliferators (PPs), we demonstrated that some of them, clofibrate (CF) in particular, display clearcut apoptogenic properties on rat hepatoma cell lines. We and others could confirm that CF as well as various other PPs can induce apoptosis in a variety of cells, including human liver, breast and lung cancer cell lines. The present study was aimed at investigating the cytotoxic action of CF on a neoplastic line of different origin, the human T leukemia Jurkat cells. We observed that CF rapidly triggers an extensive and morphologically typical apoptotic process on Jurkat cells, though not in primary T cells, which is completely prevented by the polycaspase inhibitor zVADfmk. Gene silencing studies demonstrated that CF-induced apoptosis in Jurkat cells is partially dependent on activation of caspase 2. Looking for a possible trigger of caspase 2 activation, we observed increased levels of phosphorylated eIF2a and JNK in CF-treated cells. Moreover, intracellular Ca2+ homeostasis was perturbed. Together, these findings are suggestive for the occurrence of ER stress, an event that is known to have the potential to activate caspase 2. The present observations demonstrate that CF induces in Jurkat cells a very fast and extensive apoptosis, that involves induction of ER stress and activation of caspases 2 and 3. Since apoptosis in Jurkat cells occurs at pharmacologically relevant concentrations of CF, the present findings encourage further in depth analysis in order to work out the potential implications of CF cytotoxcity on leukemic cells. Citation: Penna F, Pin F, Costamagna D, Reffo P, Baccino FM, et al. (2012) Caspase 2 Activation and ER Stress Drive Rapid Jurkat Cell Apoptosis by Clofibrate. PLoS ONE 7(9): e45327. doi:10.1371/journal.pone.0045327 Editor: Marc Tjwa, University of Frankfurt - University Hospital Frankfurt, Germany Received November 28, 2010; Accepted August 20, 2012; Published September 18, 2012 Copyright: 2012 Penna et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work supported by Ministero per l’Universita` e la Ricerca Scientifica (MIUR, Roma; PRIN projects 2001 and 2003), University of Torino (ex-60% funds), Regione Piemonte, Compagnia di San Paolo, Torino, and Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano, MFAG6211, IG9153). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: paola.costelli@unito.it Introduction Clofibrate (CF) and other fibrate derivatives have long been used as hypolipidemic drugs [1]. These compounds are part of a largely heterogeneous class of chemicals known as peroxisome proliferators (PPs). Their mechanism of action typically requires binding to heterodimeric nuclear receptors in which a monomer of RXR combines with a monomer of PP-activated receptor (PPAR). Three different PPAR subfamilies (a, b/d, and c) have been described [2], PPARa being particularly involved in fibrate- activated signal transduction. PPs have been shown to behave as hepatocarcinogens in rodents [3]. Indeed, when administered to rats and mice they induce peroxisome proliferation, hepatomegaly, and hepatocarcinogen- esis [4,5]. By contrast, these effects cannot be observed in monkeys, pigs and humans [6,7,8]. PPs are considered non- genotoxic carcinogens, their oncogenicity apparently deriving from both the oxidative response consequent to peroxisome proliferation and their ability to interfere with the regulation of cell proliferation and death [8,9]. PPARa appears mainly in charge of these activities. Indeed, long term PPs administration does not result in hepatocarcinogenesis in PPARa-null mice [10]. However, several side effects such as rhabdomyolysis, liver and heart toxicity, anemia and leukopenia as well as rodent liver carcinogenesis are likely due to PPAR-independent mechanisms (rewieved in [11]). In addition, despite observations that various PPARa ligands exert a prosurvival action that was suggested to contribute to their carcinogenic potential [12], some of them have been demonstrated to induce apoptosis in different hepatoma cell lines. An initial report from our laboratory [13] showed, quite unexpectedly at the time, that treatment with CF promptly induces massive and typical apoptosis in hepatoma cells of both rat (Yoshida AH-130) and human (HepG2) origin, with no correlation with the species-specificity of hepatocarcinogenesis. Subsequently, similar observations were made on various cell lines exposed to CF or other PPARa ligands such as nafenopin, perfluorooctanoic acid, and BR931 [14,15,16]. Noterworthily, PPARa ligand cytotoxicity is not restricted to cells of the hepatocytic lineage, but it has also been observed in breast or lung cancer cell lines [17,18] as well as in human keratinocytes and lymphoblasts [19,20]. Furthermore, ligands of the other two PPAR isotypes, i.e. b/d, and c, have been shown to induce cell death as well [21,22,23], and CF itself can bind to all three PPAR subfamilies [24]. Of particular interest are several reports that suggest the potential use of PPARa ligands as antineoplastic drugs. In this connection, a good insight into cell death mechanisms triggered by these ligands becomes especially important. Previous results obtained in our laboratory suggested that a role may be played PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e45327