[CANCER RESEARCH 62, 1388 –1393, March 1, 2002]
Peripheral Benzodiazepine Receptor Ligands Reverse Apoptosis Resistance of Cancer Cells in Vitro and in Vivo1 Didier Decaudin,2 Maria Castedo, Fariba Nemati, Arnaud Beurdeley-Thomas, Gonzague De Pinieux, Antoine Caron, Pierre Pouillart, John Wijdenes, Dany Rouillard, Guido Kroemer, and Marie-France Poupon Departments of Hematology [D. D.], Medical Oncology [P. P.], and Cytopathology [D. R.] and UMR 147 CNRS, Section de Recherche [D. D., F. N., A. B-T., G. D. P., A. C., M-F. P.], Institut Curie, 75005 Paris, France; UMR 1599 CNRS, Institut Gustave Roussy, 94800 Villejuif, France [M. C., G. K.]; and Diaclone, 25020 Besanc¸on, France [J. W.]
ABSTRACT The mitochondrial peripheral benzodiazepine receptor (mPBR) is involved in a functional structure designated as the permeability transition pore, which controls apoptosis. Binding of Fas/APO-1/CD95 triggers a prototypic apoptosis-inducing pathway. Using four different human tumor cell lines (T-cell Jurkat, neuroblastoma SHEP, osteosarcoma 143N2, and glioblastoma SNB79 cell lines), all of which express CD95 and mPBR, we investigated the potential role of mPBR ligands in CD95-induced apoptosis. We show that, in vitro, the three mPBR ligands tested (RO54864, PK11195, and diazepam) enhanced apoptosis induced by anti-CD95 antibody in Jurkat cells, as demonstrated by mitochondrial transmembrane potential drop and DNA fragmentation. In contrast, RO5-4864, but not PK11195 or diazepam, enhanced anti-CD95 apoptosis in all other cell lines. These effects were obtained in Bcl-2-overexpressing SHEP cell lines, but not in Bcl-XL SHEP cell lines. Enhancement of anti-CD95 antibodyinduced apoptosis by RO5-4864 was characterized by an increased mitochondrial release of cytochrome c and Smac/DIABLO proteins and an enhanced activation of caspases 9 and 3, suggesting a mitochondriondependent mechanism. Preincubation of cells with the different mPBR ligands or anti-CD95 did not affect the levels of expression of either mPBR or CD95. In vivo, we found that the RO5-4864 mPBR ligand significantly increased the growth inhibition induced by two chemotherapeutic agents, etoposide and ifosfamide, using two human small cell lung cancers xenografted into nude mice. Peripheral benzodiazepine receptor ligands may therefore act as chemosensitizing agents for the treatment of human neoplasms.
INTRODUCTION Failure of cell death induction leads to resistance of cancer cells to antitumor therapies. Reversion of this apoptosis resistance and the consequent potentiation of cytotoxic effects constitute a challenge for the improvement of chemotherapy. The apoptotic process is marked by a series of morphological and molecular alterations, including disruption of mitochondrial membrane integrity, caspase activation, and DNA fragmentation (1). Over recent years, it has been widely accepted that apoptosis is under the control of mitochondria and that the PT3 pore plays a key role in this regulation (2). The PT pore is a multiprotein complex located at the contact site between the mitochondrial inner and outer membranes. Several proteins contribute to PT pore formation, including mitochondrial hexokinase, the PBR, and the voltage-dependent anion channel in the outer membrane, creatine kinase in the intermembrane space, adenine nucleotide translocator in the inner membrane, and cyclophilin D in the matrix (1, 3). Opening of the PT pore induces apoptosis, whereas pharmacological inhibition
of this pore prevents cell death. Concurrently, the oncoprotein Bcl-2, which is particularly abundant at the location of the PT pore (4), prevents apoptosis induction (5). The mPBR belongs to the PT pore complex (6) and is up-regulated in several tumors such as glioblastoma and ovarian and hepatocellular carcinomas (7–9). We reported recently that PK11195, a ligand of the mPBR, enhanced apoptosis initiated by a number of different agents, such as etoposide, doxorubicin, dexamethasone, ␥-irradiation (10), lonidamine (11), and the proapoptotic second messenger ceramide (10). When added to isolated mitochondria, PK11195 facilitated dissipation of the ⌬m and release of apoptogenic factors such as cytochrome c and apoptosis-inducing factor. Moreover, PK11195 abolished apoptosis inhibition by Bcl-2 via a direct effect on mitochondria (10). Altogether, these findings suggest that mPBR ligands act on the PT pore to facilitate apoptosis induction and that they appear to be pharmacological targets for apoptosis modulation. Surprisingly, apoptosis enhancement by PK11195 is only observed at a concentration 1000-fold higher than that required for its specific binding to the high-affinity mPBR, thus questioning the relationship between the apoptosis-sensitizing effect of PK11195 and its mitochondrial binding. To confirm the therapeutic potential of mPBR ligands and to elucidate the mechanisms by which they enhance apoptosis, we studied the in vitro and in vivo antitumor activities of several apoptotic inducers in combination with various mPBR ligands, namely, RO5-4864, PK11195, and diazepam (6, 12). We focused our investigations on the Fas/APO-1/CD95 pathway, using the human anti-CD95 Mab CH-11, and looked for enhancement of the in vitro cytotoxic effect of CH-11 by several mPBR ligands. Finally, we showed in vivo enhancement of chemotherapy-induced apoptosis by RO5-4864 in two human small cell lung cancers xenografted into nude mice. These results support the hypothesis that mPBR ligands could be used for chemosensitization of solid cancers. MATERIALS AND METHODS
Cell Lines, Culture Conditions, and Apoptosis Induction. The human Fas-resistant T-cell line Jurkat (kindly provided by J. Wijdenes; Diaclone, Besanc¸on, France); the human neuroblastoma cell line SHEP stably transfected with either Bcl-2 (SHEP-Bcl-2), Bcl-XL (SHEP-Bcl-XL), or control vector (SHEP-control; a gift from Klaus-Michael Debatin, University Hospital, Ulm, Germany); the human osteosarcoma cell line 143N2 (UMR 147 CNRS); and the human glioblastoma cell line SNB79 (UMR 147 CNRS) were cultured in DMEM or RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS (Dutscher, Brumath, France), penicillin G (102 IU/ml) ⫹ streptomycin (50 g/ml; Sigma Chemical Co.), and L-glutamine (2 mM; Sigma Chemical Co.). Cells were cultured in the presence of the CD95 cross-linking Received 6/29/01; accepted 1/2/02. IgM Mab CH-11 (1 g/ml; Immunotech, Marseille, France), during or after The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with exposure to RO5-4864 (Sigma Chemical Co.), PK11195 (Sigma Chemical 18 U.S.C. Section 1734 solely to indicate this fact. Co.), or diazepam (Roche, Neuilly/Seine, France) at different concentrations, 1 Supported by a special grant from the Ligue Nationale contre le cancer and the as indicated in the figures. After the indicated intervals, cells were studied for European Commission (QL61-1999-739). 2 To whom requests for reprints should be addressed, at Service d’He´matologie, apoptosis quantification or viability. Institut Curie, 26 rue d’Ulm, 75248 Paris cedex 05, France. Phone: 33-1-44-32-46-90; Quantification of Apoptosis. DiOC6(3), a cationic lipophilic fluoroFax: 33-1-44-32-46-93; E-mail: [email protected]
chrome (Molecular Probes, Eugene, OR), was used to measure the ⌬m. 3 The abbreviations used are: PT, permeability transition; mPBR, mitochondrial peBriefly, cells were incubated at 37°C for 15 min in the presence of DiOC6(3) ripheral benzodiazepine receptor; PBR, peripheral benzodiazepine receptor; Mab, mono(40 nM), followed by immediate analysis of fluorochrome incorporation in an clonal antibody; ⌬m, inner mitochondrial transmembrane potential. 1388
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PBR LIGANDS REVERSE APOPTOSIS RESISTANCE
Fig. 1. Enhancement of anti-CD95 Mab (CH-11)-induced apoptosis of T-cell Jurkat cells by various mPBR ligands. Cells were concomitantly cultured for 9 h with (f) or without (u) 1 g/ml CH-11, alone or in combination with the indicated concentration of RO5-4864, PK11195, or diazepam. In A, the percentage of subdiploid cells was determined by propidium iodide staining and fluorescence-activated cell-sorting analysis, as described in “Materials and Methods.” In B, after culture with CH-11 (Mab), with or without (0) mPBR ligands, dissipation of the ⌬m (f) or enhancement of superoxide anion generation (u) and subdiploidy (䡺) were assessed.
Epics Profile II cytofluorometer (Coulter, Miami, FL). Hydroethydine (2 M; 15 min at 37°C; Molecular Probes) was used to measure superoxide anion generation, as described previously (13). DiOC6(3) and hydroethydine fluorescences were recorded in FL1 and FL3, respectively. The frequency of subdiploid cells (cells that have lost part of their chromosomal DNA) was determined by propidium iodide staining of ethanol-permeabilized cells, as described previously (14). Determination of Cell Viability. The cytotoxic effect of CH-11, tested alone or in combination with other agents, was assessed after several exposure times in 96-well incubation plates using methylene blue, as described previously (15). Briefly, at the end of each experiment, culture medium was aspirated, surviving cells adhering to the well bottom were fixed with methanol and stained by methylene blue (1% in borate buffer), and cell-fixed dye was eluted with 0.1 N HCl. Absorbance (A) was measured in each well in an automatic scanning photometer at a wavelength of 630 nm. Each experimental point was determined in triplicate or sextuplicate. The percentage of cell survival was calculated as shown below.
P ⫽ 共 A in treated cells/A in control cells兲 ⫻ 100
reagent (Life Technologies, Inc., Cergy Pontoise, France) according to the manufacturer’s instructions, as descri...