Rapid Jurkat Apoptosis by Clofibrate Figure 1. Kinetic of clofibrate-induced death in Jurkat cells. Morphological appearance (A: phase contrast microscopy, B: DAPI staining) of Jurkat cells exposed to clofibrate (CF) 1 mM for 45 min (Co: control). (C) percentages of cells with hypodiploid DNA content (apoptotic; see Penna et al., 2009) after exposure to CF (concentration on top of the bars, time on X axis). Data are expressed as mean 6 SD (n = 3). ** p,0,01 vs Co; *** p,0,001 vs Co. doi:10.1371/journal.pone.0045327.g001 by inhibition of HMG-CoA reductase (HMGR), a key enzyme in isoprenoid biosynthesis. Indeed, the mRNA level and enzymatic activity of HGMR as well as the cholesterol content in mitochondria are reduced in Yoshida AH-130 cells soon after CF treatment, while cell death can be attenuated by supplement- ing cells with mevalonate, the reaction product of HMGR [25]. Quite recently we also demonstrated [26] that the rapid apoptosis induced by CF in the AH130 hepatoma cells is amenable to a classic caspase-dependent intrinsic pathway. The possibility that CF, and fibrate derivatives in general, in addition to the hypolipidemic action, could be exploited also in view of their cytotoxic activity is an interesting one. Indeed, these drugs are currently used in the clinical practice and their side- effects are generally compatible with a good quality of life. In this regard, their possible use in combination with classical antineo- plastic treatments is intriguing. The data actually available in the literature are scanty and confusing, showing that CF may be cytotoxic for several tumor cell lines, with poor attention given to the underlying mechanisms. On this line, the present study tries to fill this gap, investigating CF-induced apoptosis in Jurkat cells, a human neoplastic line of hematopoietic origin. The results show that CF quickly activates the apoptotic machinery through a non conventional pathway that at least involves the initiator caspase 2, likely activated via ER stress, and the effector caspase 3, though not caspases 8 and 9. Materials and Methods All materials were supplied by Sigma (St. Louis, MO, USA), unless differently specified. Jurkat cells (from human leukemic transformed T lymphoma, clone E6-1) were purchased from ATCC (Manassas, VA, USA), seeded in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L- glutamine, and maintained at 37uC in a humidified atmosphere of 5% CO2 in air. CF, dissolved in DMSO, was added to the medium at the final concentration of 1 mM (or as specified). Controls were treated with the solvent alone. In some experiments Jurkat cells were pretreated with one of the following molecules: zVAD-fmk (zVAD, 1 h, 20 mM), DEVD-cho (DEVD, 1 h, 20 mM), VDVAD-cho (VDVAD, 1 h, 20 mM) LEHD-cho (LEHD, 1 h, 20 mM), IETD-cho (IETD, 1 h, 20 mM) from Alexis Biochemicals (Lausen, Switzerland), cyclosporine A (CsA, 15 min, 1 mM), calpeptin (1 h, 250 mM), bapta-AM (1h, 10 mM) from Biomol (Plymouth Meeting, PA, USA), SP600125 (1 h, 20 mM), PD150606 (1 h, 20 mM), form Calbiochem (La Jolla, Ca, USA) and EGTA (2mM, 30 min). Flow Cytometry DNA distribution analysis was performed as described else- where [26]. Briefly, cells were washed in PBS, fixed in ice-cold PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e45327