| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
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 Besançon, France [J. W.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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, 8, 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 |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Quantification of Apoptosis.
DiOC6(3), a cationic lipophilic fluorochrome (Molecular Probes, Eugene, OR), was used to measure the m. Briefly, cells were incubated at 37°C for 15 min in the presence of DiOC6(3) (40 nM), followed by immediate analysis of fluorochrome incorporation in an Epics Profile II cytofluorometer (Coulter, Miami, FL). Hydroethydine (2 µM; 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.
 | (1) |
Immunofluoresence Detection of Apoptosis-regulatory Proteins on Fixed Permeabilized Cells.
SHEP-control cells were cultured on coverglasses for 24 h in the presence or absence of RO5-4864 (100 µM) and/or anti-CD95 antibody (1 µg/ml), washed once with PBS, and fixed with paraformaldehyde (4% w/v) and picric acid fixative solution. A Mab specific for cytochrome c (Mab 6H2.B4; BD PharMingen; dilution, 1:250) was used and revealed by a goat antimouse IgG ALEXA 488 conjugate (Molecular Probes). Cells were also stained for the detection of Smac/DIABLO (Mab 1067; Calbiochem; dilution, 1:50) and cleaved caspase 9 and cleaved caspase 3 (Cell Signaling Technology; dilution, 1:50), all revealed by appropriate secondary antibodies conjugated to ALEXA 568 and counterstained with the nuclear dye Hoescht 33342 (2 µM; Molecular Probes). Quantification of mitochondrial cytochrome c and Smac/DIABLO release and of caspase-9 and caspase-3 induction was determined as a percentage of total cells.
Evaluation of FasR and mPBR mRNA and CD95 Expression.
mRNA expressions of CD95 and mPBR were measured by a reverse transcription-PCR-based method with total RNA isolated from living cells using Trizol reagent (Life Technologies, Inc., Cergy Pontoise, France) according to the manufacturer’s instructions, as described previously (16)
. Briefly, cDNA was prepared in an oligodeoxythymidylic acid-primed reverse transcription reaction consisting of 2 µg of total RNA; a total volume of 20 µl of cDNA was then used for PCR amplification. The final PCR reaction volume was 50 µl:1 µl of the cDNA solution, and the following mixture containing 5 µl of 10x Taq buffer (Appligene-Oncor, Illkirch, France), 1 µl of deoxynucleotide triphosphate (final concentration, 0.25 mM each), 1 µl of each of the 5' and 3' primers (100 ng/µl), 40.5 µl of water, and 0.5 µl (2.5 units) of Taq polymerase (Appligene-Oncor) was added. Primers used were as follows: (a) for CD95, 5'-CAA-GTC-ACT-GAC-ATC-AAC-TTC-3' (sense) and 5'-CCT-TTG-TTT-TCC-TTT-CTG-TGC-3' (antisense); and (b) for mPBR, 5'-GTG-AAT-GCT-TGG-GAA-GTG-GTA-3' (sense) and 5'-TGG-TTC-ATA-CGT-GAC-CTG-ACA-3' (antisense).
Thermal cycling was as follows: (a) denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, 25 cycles for CD95 PCR; and (b) denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, 35 and 40 cycles for mPBR PCR. PCR products (7 µl) were separated by electrophoresis in a 1.8% agarose gel and visualized by ethidium bromide (0.5 µg/ml) staining under UV exposure for 6–30 s. The image was then processed by software, analyzed using the NIH version 1.60 Image program, and photographed.
To determine cell surface CD95 expression, cells were incubated with a murine antihuman IgG1 anti-Fas Mab (dilution, 1:800; Immunotech) for 30 min on ice in 1% FCS-PBS, washed twice, and then incubated for 30 min on ice with FITC-conjugated goat antimouse IgG1 Mab (Coulter, Coultronics, Marseille, France). After additional washing with PBS, cells were analyzed using an Epics Profile II cytofluorometer. Fluorescence data were collected in 104 viable cells, as determined by forward light scatter intensity. Negative control was performed using isotype control IgG followed by FITC-conjugated anti-IgG secondary antibody (PharMingen).
In Vivo Enhancement of Chemotherapy-induced Apoptosis by mPBR Ligands.
Swiss mice (nu/nu; female, 6–8 weeks old) weighing 30 g were bred in the animal facilities (Institut Curie, Paris, France), maintained under specific pathogen-free conditions with artificial lighting (12-h light/12-h dark cycle), and fed a regular diet and water ad libitum. The care, housing, and handling of the mice were performed in accordance with the recommendations of the French Ethics Committee and under the supervision of authorized investigators. For therapeutic trials, the tumor-bearing mice were randomly divided into equivalent groups of four to eight animals, and mice were treated as soon as the xenografted tumors reached a 5-mm diameter (or a tumor volume of approximately 60 mm3).
Two different human tumors, small cell lung cancers SCLC6 and SCLC61, were used and treated by a combination of etoposide (Pierre Fabre, Boulogne, France) and ifosfamide (Asta Medica, Mérignac, France) or etoposide alone, respectively, with or without RO5-4864. RO5-4864 was diluted in 100 µl of excipient solution containing ethanol and Tween 80 and was injected s.c. Etoposide and ifosfamide were diluted in 200 µl of 0.9% sodium chloride and injected i.p. The control group received injections according to the same schedule as experimentally treated mice.
All mice were weighed once weekly. Tumor growth was monitored by measuring two perpendicular diameters with calipers. Mice were sacrificed when the tumor volume reached 2500 mm3 in the control group.
Statistical Analysis.
We used Student’s t test to assess the in vivo enhancement of chemotherapy-induced apoptosis by RO5-4864 on growth of xenografted tumors in nude mice. The t values were two-sided and were considered significant when they were less than or equal to 0.05.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. RO5-4864, PK11195, or diazepam used at concentrations that were not cytotoxic enhanced the incidence of nuclear apoptosis (measured as subdiploidy) induced by the anti-CD95 antibody (Fig. 1A)
. The induction of apoptosis depended on the concentrations of the mPBR ligands, RO5-4864, PK11195, or diazepam. A cooperative apoptosis-inducing effect between CD95 and PBR ligands was confirmed when, instead of DNA loss, other features of apoptosis, namely, the drop in m and production of anion superoxide, were assessed (Fig. 1B)
.
|
, 3A
, and 4A
). In contrast, PK11195 and diazepam sensitized only SHEP-control cells (data not shown), but not 143N2 (Fig. 3A)
or SNB79 cells, to CD95-induced apoptosis (data not shown). RO5-4864, but not PK11195 or diazepam, sensitized SHEP-Bcl-2 (Fig. 2)
but not SHEP.Bcl-XL cells (data not shown). The RO5-4864-mediated sensitization to anti-CD95 was observed over a wide range of anti-CD95 antibody concentrations (0.25–2 µg/ml; Fig. 3B
). When cells were cultured for 24 h in the presence of different mPBR ligands before exposure to anti-CD95, no apoptogenic activity was detected in any of the cell lines tested (Fig. 4B)
. To test the dose dependency of apoptosis enhancement, human osteosarcoma (143N2) and neuroblastoma (SHEP-control or SHEP-Bcl-2) cell lines were cultured for 24 h concomitantly with anti-CD95 and low doses of RO5-4864 (1–100 nM); no potentiation of the cytotoxic effect of the anti-CD95 Mab was observed, showing that doses of up to 30 µM of the various mPBR ligands were necessary to induce apoptosis (data not shown).
|
|
|
, green fluorescence) and Smac/DIABLO (Fig. 5B
, red fluorescence) revealed a mitochondrial punctuate staining pattern for untreated SHEP cells as well as SHEP cells cultured in the presence of anti-CD95 antibody alone and RO5-4864 alone. In contrast, a significant portion of cells cultured in the presence of both anti-CD95 antibody and RO5-4864 exhibited a diffuse staining pattern of cytochrome c and Smac/DIABLO. Because both cytochrome c and Smac/DIABLO participate in the activation of caspases (in particular, caspases 9 and 3), we determined caspase activation using antibodies that recognized cleaved processed caspases. Cleaved caspase 9 (Fig. 5C)
and 3 (Fig. 5D)
were detected in SHEP-control cells cultured in the simultaneous presence of anti-CD95 antibody and RO5-4864, but not in cells cultured in the presence of CH-11 alone or RO5-4864 alone. Taken together, these data are compatible with the notion that RO5-4864 sensitizes cells to CH-11-induced mitochondrial release of caspase activators, thereby resulting in enhanced caspase activation and apoptosis.
|
. CD95 expression was low for the SNB19 cell line and high for all other cell lines. CD95 expression was not affected by a 12-h exposure to anti-CD95 (1 µg/ml) and mPBR ligands (60 µM), as shown in 143N2 and SHEP-control cell lines (Fig. 6B)
.
|
). Similar results were obtained when mice bearing SCLC6 xenografts received a combination of etoposide (12 mg/kg) and ifosfamide (90 mg/kg) with or without concomitant administration of 40 mg/kg RO5-4864. RO5-4864 significantly enhanced the growth-inhibitory effect of chemotherapy (P < 0.05; Fig. 7B
) but had no effect on tumor growth alone.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Enhancement of anti-CD95 antibody-induced apoptosis by RO5-4864 was characterized by numerous mitochondrial alterations, namely, the drop of the m and the production of superoxide anion. We also observed enhancement of the mitochondrial release of cytochrome c and Smac/DIABLO regulatory proteins, as well as activation of caspases 9 and 3. Note that quantification of mitochondrial cytochrome c and Smac/DIABLO release and caspase 9 and 3 induction was performed on adherent cells, leading to an underestimation of apoptosis resulting in loss of cellular adhesion. These observations suggest that activation of caspase 3 results from a mitochondrial megachannel disrupter responsible for cytochrome c and Smac/DIABLO release and caspase 9 activation (17
, 18)
and argue in favor of a mitochondrial-dependent mechanism of anti-CD95 antibody-induced apoptosis by RO5-4864.
Mitochondrial benzodiazepine receptors have already been reported as therapeutic targets using a PBR ligand-melphalan conjugate (19)
or porphyrins in photodynamic tumor therapy (20)
. Similarly, we have previously reported potentiation of lonidamine-induced cytotoxicity by concomitant exposure to diazepam in human glioblastoma tumor xenografted into nude mice (21)
. Bono et al. (22)
showed that PK11195 enhanced the sensitivity of cells to tumor necrosis factor 
and abolished the apoptosis-inhibitory effect of Bcl-2 via a direct effect on mitochondria. Nevertheless, the same authors reported that at concentrations between 10 and 100 nM, RO5-4864 reduced the proapoptotic effect of tumor necrosis factor . This observation is not in agreement with our own data showing that at concentrations <1 µM, RO5-4864, PK11195, and diazepam failed to stimulate CD95-induced apoptosis. This apparent discrepancy could be explained by the fact that we used different cell lines. An alternative explanation could be that PK11195 (an isoquinoline carboxamide) and RO5-4864 (a benzodiazepine) exert different conformational and/or physicochemical changes on their mitochondrial binding partners. Indeed, differential effects observed with RO5-4864 and PK11195 supported the assumption that RO5-4864 acts as an agonist and PK11195 as an antagonist of the mPBR (12)
and that they interact with two different conformations or domains of the mPBR (23)
. It could be speculated that the binding of the mPBR ligand to its receptor induces a peculiar conformation of the mitochondrial PT pore, which sensitizes the cell to an apoptotic message. This would emphasize the importance of mPBR-associated proteins modulating the cell death process, such as Bcl-2, Bcl-XL, or Bax (24)
. All these hypotheses must be explored in additional studies to explain the enhancement of apoptosis by mPBR ligands.
In conclusion, the present data indicate that RO5-4864 mPBR ligand can sensitize a panel of different human tumor cells to apoptosis induction, both in vitro and in vivo. Irrespective of the exact molecular mechanisms accounting for their chemosensitizing effect, mPBR ligands therefore appear to be potential antineoplastic agents.
|
FOOTNOTES |
|---|
1 Supported by a special grant from the Ligue Nationale contre le cancer and the European Commission (QL61-1999-739). 
2 To whom requests for reprints should be addressed, at Service d’Hématologie, Institut Curie, 26 rue d’Ulm, 75248 Paris cedex 05, France. Phone: 33-1-44-32-46-90; Fax: 33-1-44-32-46-93; E-mail: didier.decaudin{at}curie.net.
3 The abbreviations used are: PT, permeability transition; mPBR, mitochondrial peripheral benzodiazepine receptor; PBR, peripheral benzodiazepine receptor; Mab, monoclonal antibody; m, inner mitochondrial transmembrane potential.
Received 6/29/01. Accepted 1/ 2/02.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Fafalios, A. Akhavan, A. V. Parwani, R. R. Bies, K. J. McHugh, and B. R. Pflug Translocator Protein Blockade Reduces Prostate Tumor Growth Clin. Cancer Res., October 1, 2009; 15(19): 6177 - 6184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Kim, C.-W. Shu, W. Xu, C.-W. Shiau, D. Grant, S. Vasile, N. D. P. Cosford, and J. C. Reed Chemical Biology Investigation of Cell Death Pathways Activated by Endoplasmic Reticulum Stress Reveals Cytoprotective Modulators of ASK1 J. Biol. Chem., January 16, 2009; 284(3): 1593 - 1603. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. F. Santidrian, A. M. Cosialls, L. Coll-Mulet, D. Iglesias-Serret, M. de Frias, D. M. Gonzalez-Girones, C. Campas, A. Domingo, G. Pons, and J. Gil The potential anticancer agent PK11195 induces apoptosis irrespective of p53 and ATM status in chronic lymphocytic leukemia cells Haematologica, December 1, 2007; 92(12): 1631 - 1638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. N. Obame, R. Zini, R. Souktani, A. Berdeaux, and D. Morin Peripheral Benzodiazepine Receptor-Induced Myocardial Protection is Mediated by Inhibition of Mitochondrial Membrane Permeabilization J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 336 - 345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kroemer, L. Galluzzi, and C. Brenner Mitochondrial Membrane Permeabilization in Cell Death Physiol Rev, January 1, 2007; 87(1): 99 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Costa, A. Salvetti, L. Rossi, F. Spinetti, A. Lena, B. Chelli, M. Rechichi, E. Da Pozzo, V. Gremigni, and C. Martini Peripheral Benzodiazepine Receptor: Characterization in Human T-Lymphoma Jurkat Cells Mol. Pharmacol., January 1, 2006; 69(1): 37 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-C. Wu, M.-L. Chan, W.-Y. Chen, C.-Y. Tsai, F.-R. Chang, and Y.-C. Wu Pristimerin induces caspase-dependent apoptosis in MDA-MB-231 cells via direct effects on mitochondria Mol. Cancer Ther., August 1, 2005; 4(8): 1277 - 1285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Fischer and K. Schulze-Osthoff New Approaches and Therapeutics Targeting Apoptosis in Disease Pharmacol. Rev., June 1, 2005; 57(2): 187 - 215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. G. Stavrovskaya, M. V. Narayanan, W. Zhang, B. F. Krasnikov, J. Heemskerk, S. S. Young, J. P. Blass, A. M. Brown, M. F. Beal, R. M. Friedlander, et al. Clinically Approved Heterocyclics Act on a Mitochondrial Target and Reduce Stroke-induced Pathology J. Exp. Med., July 19, 2004; 200(2): 211 - 222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Ostuni, K. Marazova, G. Peranzi, B. Vidic, V. Papadopoulos, R. Ducroc, and J.-J. Lacapere Functional characterization and expression of PBR in rat gastric mucosa: stimulation of chloride secretion by PBR ligands Am J Physiol Gastrointest Liver Physiol, June 1, 2004; 286(6): G1069 - G1080. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. B. Walter, B. W. Raden, M. R. Cronk, I. D. Bernstein, F. R. Appelbaum, and D. E. Banker The peripheral benzodiazepine receptor ligand PK11195 overcomes different resistance mechanisms to sensitize AML cells to gemtuzumab ozogamicin Blood, June 1, 2004; 103(11): 4276 - 4284. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Muscarella, K. A. O'Brien, A. T. Lemley, and S. E. Bloom Reversal of Bcl-2-Mediated Resistance of the EW36 Human B-Cell Lymphoma Cell Line to Arsenite- and Pesticide-Induced Apoptosis by PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor Toxicol. Sci., July 1, 2003; 74(1): 66 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Castedo, J.-L. Perfettini, and G. Kroemer Mitochondrial Apoptosis and the Peripheral Benzodiazepine Receptor: A Novel Target for Viral and Pharmacological Manipulation J. Exp. Med., November 4, 2002; 196(9): 1121 - 1126. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or without (
) 1 µg/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 
) were assessed.
, 0.5 µg/ml; 
, 1 µg/ml; 
, 2 µg/ml). Determination of cell viability was performed using a methylene blue assay.
) or RO5-4864 excipient and etoposide (
). B, etoposide and ifosfamide were administered with (