ORIGINAL ARTICLE Effects of permeability transition inhibition and decrease in cytochrome c content on doxorubicin toxicity in K562 cells F De Oliveira 1 , C Chauvin 1 , X Ronot 2 , M Mousseau 3 , X Leverve 1 and E Fontaine 1 1 INSERM E-0221 Bioe´nerge´tique Fondamentale et Applique´e, Universite´Joseph Fourier, Grenoble, France; 2 Laboratoire de Dynamique Cellulaire, EPHE, UMR-CNRS, Institut Albert Bonniot, La Tronche, France; 3 De´partement de cance´rologie et d’he´matologie, Hoˆpital Albert Michallon, Grenoble, France As mitochondria play a key role in the commitment to cell death, we have investigated the mitochondrial conse- quences of resistance to doxorubicin (DOX) in K562 cells. We found that the permeability transition pore (PTP) inhibitor cyclosporine A (CsA) failed to inhibit PTP opening in the resistant clone. Moreover, the Ca 2 þ loading capacity in the resistant clone was identical to that observed in the parent cells in the presence of CsA, suggesting that the PTP was already inhibited in a CsA- like manner in the resistant cells. In agreement with this proposal, the mitochondrial target of CsA cyclophilin D (CyD) decreased by half in the resistant cells. The levels of adenine nucleotide translocator, voltage anion-dependent channel, Bax, Bcl-2, Bcl-x L , AIF and Smac/Diablo, were similar in both cell lines, whereas cytochrome c content was divided by three in the resistant cells. Since P-glycoprotein inhibition did not restore DOX toxicity in the resistant cells, while DOX-induced cell death in the parent cells was prevented by either PTP inhibition or siRNA-induced decrease in cytochrome c content, we conclude that the inhibition of PTP opening and the decrease in cytochrome c content participate in the mechanism that makes K562 cells resistant to DOX. Oncogene (2006) 25, 2646–2655. doi:10.1038/sj.onc.1209293; published online 12 December 2005 Keywords: chemoresistance; PTP; mitochondria; cyto- chrome c Introduction In addition to their known role in energy metabolism, mitochondria are now recognized to play a key role in the commitment to cell death (Desagher and Martinou, 2000; Cory and Adams, 2002; Ravagnan et al., 2002). Several intermembrane space proteins such as cyto- chrome c, AIF, endonuclease G, Smac/Diablo and Omi/ HtrA2, which have no proapoptotic activity when they remain inside mitochondria, prompt cell death once released into the cytosol. Evidence suggests that two nonmutually exclusive pathways make the outer mito- chondrial membrane permeable to these proapoptotic proteins (Green and Kroemer, 2004). One relies on outer membrane channel(s) involving Bcl-2 family proteins, while the other is due to the opening of an inner membrane channel: the permeability transition pore (PTP). The PTP is a multiprotein complex, whose molecular nature remains unelucidated (Green and Kroemer, 2004). The adenine nucleotide translocator (ANT) regulates PTP opening but is not mandatory for PTP formation since permeability transition persists in ANT- deficient cells (Kokoszka et al., 2004). The matrix chaperone protein cyclophilin D (CyD) is another nonmandatory component of the PTP. CyD-deficient animals have an inhibited PTP, and are resistant to cell death induced either by ischemia-reperfusion or oxida- tive stress (Baines et al., 2005; Basso et al., 2005; Nakagawa et al., 2005; Schinzel et al., 2005). Moreover, several unrelated drugs known to inhibit PTP opening have been shown to potently inhibit cell death in response to many cytotoxic insults (Chauvin et al., 2001). Finally, pharmacological agents used in anti- cancer therapy have been reported to target the PTP and to induce cell death via PTP opening (Green and Kroemer, 2004). Doxorubicin (DOX) is an anthracycline antibiotic used for the treatment of a variety of malignancies. Among the multiple mechanisms leading to resistance to DOX (Nielsen et al., 1996), the overexpression of P-glycoprotein (P-gp), a protein that pumps out of the cell unrelated agents including DOX, is believed to account for resistance to DOX in several cell lines. It must be noted, however, that P-gp inhibition did not systematically restore DOX cytotoxicity in P-gp over- expressing cells (success (Smyth et al., 1998; Fukushima et al., 2000); failure (Campone et al., 2001)). Moreover, cells overexpressing P-gp have also been reported to resist to non-P-gp substrates such as cytosine arabino- side, staurosporine, UV irradiation, Fas receptor liga- tion and serum starvation (Smyth et al., 1998; Trindade et al., 1999; Campone et al., 2001). Taken together, these Received 21 October 2004; revised 18 October 2005; accepted 4 November 2005; published online 12 December 2005 Correspondence: Professor E Fontaine, Laboratoire de Bioe´nerge´tique Fondamentale et Applique´e, INSERM E-0221, Universite´ Joseph Fourier – BP 53, F-38041 Grenoble Cedex, France. E-mail: eric.fontaine@ujf-grenoble.fr Oncogene (2006) 25, 2646–2655 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc