The Mitochondrial Effects of Small Organic Ligands of BCL-2

We have investigated the mitochondrial effects of BH3I-2′, Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2′ (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 μm) > HA14-1 (half-maximal uncoupling at about 20 μm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

In this paper we have investigated the mitochondrial and cellular effects of BH3I-2Ј, Chelerythrine, and HA14-1. All three compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2Ј Ͼ Chelerythrine Ͼ HA14-1, and they all sensitized the PTP to opening at concentrations lower than required for uncoupling both in isolated mitochondria and intact cells. BCL-2 overexpression did not sensitize but rather protected cells from the cytotoxic effects of the BCL-2 ligands. We show that the BCL-2 binding and PTP-inducing effects can be separated from the potentially toxic effects on respiration through the identification of EM20-25, a molecule devoid of effects on mitochondrial respiration that is able to induce PTP opening in isolated mitochondria and intact cells, to disrupt the BCL-2/BAX interactions in situ with activation of caspase-9 and to sensitize leukemic cells to the killing effects of staurosporine, chlorambucil, and fludarabine.

EXPERIMENTAL PROCEDURES
Measurements on Isolated Mitochondria-Liver mitochondria were isolated from albino Wistar rats weighing about 300 g by standard centrifugation techniques, as described previously (14). Oxygen consumption was measured polarographically with a Clark oxygen electrode in a closed 2-ml vessel equipped with magnetic stirring and thermostated at 25°C. Mitochondrial swelling was followed as the change of light scattering of the mitochondrial suspension at 545 nm with a PerkinElmer Life Sciences 650-40 fluorescence spectrophotometer equipped with magnetic stirring and thermostatic control.
Cell Cultures-PC3 human prostate cancer cells were grown in RPMI 1640 medium supplemented with 2 mM glutamine. HeLa Neo and HeLa BCL-2 cells (a generous gift of Dr. Naoufal Zamzami, Institut Gustave Roussy, Villejuif, France) were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine. The media were all supplemented with 10% fetal calf serum, 50 units ϫ ml Ϫ1 penicillin, and 50 g ϫ ml Ϫ1 streptomycin. Cells were kept in a humidified atmosphere of 95% air and 5% CO 2 at 37°C in a Forma tissue culture water jacketed incubator.
Analysis of BCL-2, BCL-XL, and GAPDH Expression in Different Cell Lines-One day before the experiment, 1 ϫ 10 6 PC3, HeLa Neo, and HeLa BCL-2 cells were plated onto 100-mm diameter tissue culture dishes in the appropriate growth medium and incubated at 37°C. Cells were then harvested, sedimented, washed once with ice-cold PBS, resuspended in 1 ml of ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 100 M phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/ml pepstatin), incubated 30 min on ice, and finally Dounce-homogenized. The homogenates were sedimented at full speed in a microcentrifuge for 10 min at 4°C to remove cell debris and nuclei. The supernatants, corresponding to the soluble cellular extracts, were transferred to clean tubes, and the protein concentration was determined by the Bradford assay. Equal protein amounts (100 g) were solubilized in Laemmli gel sample buffer containing 5% 2-mercaptoethanol, separated electrophoretically by SDS-PAGE, and subjected to Western blotting analysis using a mouse antihuman BCL-2 antibody (clone 7, BD Biosciences) or a rabbit antihuman BCL-XL antibody (clone 54HD, Cell Signaling Technology), as described below. The same membranes were then washed, stripped as described below, and probed with a mouse monoclonal antibody against rabbit skeletal muscle GAPDH (clone 6C5, Chemicon International, Inc.).
Purification of B-CLL Cells-B-CLL was diagnosed according to standard clinical and laboratory criteria, and patients who had not yet received treatment were studied. Mononuclear cells were recovered following centrifugation on Ficoll-Hypaque gradient (15). Cell samples were washed three times with PBS and resuspended in endotoxin free RPMI 1640 medium (Sigma) supplemented with 20 mM HEPES and L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum (ICN Flow, Costa Mesa, CA). T cells were removed from the entire cell suspension by rosetting with neuroaminidase (Sigma)treated sheep red blood cells. Additional enrichment of B cells was obtained by removing residual CD3 ϩ , CD16 ϩ , and CD56 ϩ lymphocytes using high gradient magnetic separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany), as described previously (16). Briefly, 10 ϫ 10 6 cells obtained as above were incubated for 30 min at 4°C in 80 l of PBS with purified azide-free CD3 (OKT3, Ortho Pharmaceuticals, Raritan, NJ), CD16 (Leu-11c, BD Biosciences) and CD56 (Leu-19) monoclonal antibodies. After two washes with PBS supplemented with 0.5% bovine serum albumin, 20 l of colloidal superparamagnetic microbeads conjugated with goat-anti-mouse-IgG antibodies were added. The CD3 ϩ , CD16 ϩ , and CD56 ϩ cells rosetting with microbeads were then isolated and removed applying a magnetic system to the outer wall of the columns. Following this multistep negative selection procedure, more than 98% of the resulting cell population was CD19 ϩ and BCL-2 ϩ with high density expression of BCL-2 as defined by mean fluorescence intensities, which were comparable in all cells. The expression of BCL-2 was detected using flow cytometric analysis with fluorescein isothiocyanate-labeled mouse anti-hBcl-2 monoclonal antibody (Clone 124, Dako, Glostrup, Denmark). Cells were fixed and permeabilized using Fix and Perm kit (Caltag) for 15 min at room temperature and then stained with anti-BCL-2 antibody for 30 min. A fluorescein isothiocyanate-labeled mouse IgG1 monoclonal antibody was used as a negative control.
Fluorescence Microscopy-One-hundred thousand cells were seeded onto 24-mm diameter round glass coverslips in 6-well plates and grown for 1 day. The coverslips were then transferred onto the stage of a Zeiss Axiovert 100TV inverted microscope equipped with a HBO mercury lamp (100 watts), and epifluorescence was detected with a 12-bit digital cooled CCD camera (Micromax, Princeton Instruments). Cells were incubated in Hanks' balanced salt solution without bicarbonate and phenol red and allowed to equilibrate with 20 nM TMRM in the presence of 1.6 M CsH or 1 M CsA for 30 min at 37°C prior to further additions. Fluorescence images were acquired with a 560-nm dichroic mirror using a 40ϫ/1.3 oil immersion objective (Zeiss), with excitation at 546 Ϯ 5 nm and emission at 580 Ϯ 15 nm. Exposure time was 80 ms, and data were acquired and analyzed with the MetaMorph Metafluour Imaging Software. Clusters of several mitochondria were identified as regions of interest, whereas background was taken from fields not containing cells. Sequential digital images were acquired every 2 min for 60 min, and the average fluorescence intensity of all the regions of interest and of the background was recorded and stored for subsequent analysis. Mitochondrial fluorescence intensities minus background were normalized to the initial fluorescence for comparative purposes.
Cell Viability-The number of viable cells was assessed based on the Resazurin assay as described (17,18). Briefly, cells were grown in 96-well microtiter plates (20 ϫ 10 3 cells/well) in their medium (0.2 ml/well) for 1 day. Cells grown on each well were then treated for 16 h as described in the legend to Fig. 5 in the Forma incubator. After treatment the medium was replaced with fresh medium supplemented with 10% (v/v) Resazurin for 3 h. The ratio of oxidized to reduced Resazurin (which reflects the metabolic activity of viable cells) was detected at 540/620 nm with a microplate reader (Spectracount TM Packard). We verified that the ratio increased linearly with the number of cells in the range used in the experiments.
Immunoprecipitation-The day before the experiment, 1 ϫ 10 6 HeLa BCL-2 cells were plated onto 100-mm diameter tissue culture dishes in the appropriate growth medium and incubated at 37°C until a confluence of about 80 -90% was reached. Cells were then transiently transfected with 4 g of the eukaryotic expression vector pcDNA3 containing the BAX cDNA sequence fused to a HA tag sequence (a generous gift of Atan Gross, Weizmann Institute of Science, Rehovot, Israel) or with 4 g of pcDNA3 alone, using the Lipofectamine TM reagent (Invitrogen) and following the protocol described in the product technical sheet. Exactly 46 h after the start of transfection, the medium was removed, and the cells were incubated for 6 h with fresh medium containing EM20-25 at the concentrations indicated in the legend to Fig. 8 or the same volume of Me 2 SO. Cells were then harvested, sedimented, washed once with ice-cold PBS, resuspended in 1 ml of ice-cold lysis buffer, incubated 30 min on ice, and finally Dounce-homogenized. The homogenate was sedimented at full speed in a microcentrifuge for 10 min at 4°C to remove cell debris and nuclei. The supernatant, corresponding to the soluble cellular extract, was transferred to a clean tube and the protein concentration determined by the Bradford assay (Bio-Rad). Equal protein amounts (450 g in a final volume of 1 ml) were incubated overnight at 4°C on a rocker platform with 50 l of an anti-HA affinity matrix (Roche Applied Science) previously equilibrated with lysis buffer. The matrix was then sedimented at full speed in a microcentrifuge for 10 s and the supernatant (corresponding to the flow-through) carefully removed and stored at Ϫ20°C. The matrix was washed with (i) 1 ml of ice-cold lysis buffer, (ii) 1 ml of ice-cold buffer containing 500 mM NaCl and 0.1% Nonidet P-40 and otherwise identical to the lysis buffer, and (iii) 1 ml of ice-cold buffer containing 0.1% Nonidet P-40 and otherwise identical to the lysis buffer except that it did not contain NaCl. The matrix was carefully pelleted and the supernatant removed at each wash step. The matrix was finally resuspended in 40 l of Laemmli gel sample buffer containing 5% 2-mercaptoethanol, boiled for 5 min, and pelleted again. The supernatants were transferred to clean tubes and subjected to Western blotting analysis using a mouse anti-human BCL-2 antibody, as described below. The same membrane was then washed, stripped as described below, and probed with a rat monoclonal anti-HA antibody (clone 3F10, Roche Applied Science).
Detection of Caspase-9 Cleavage-The flow-through fractions from the anti-HA affinity matrix incubation were precipitated with 4 volumes of ice-cold acetone for 10 min followed by centrifugation at full speed in a microcentrifuge for 15 min at 4°C. The acetone was carefully removed and the pellets were air-dried at room temperature, resuspended in 40 l of Laemmli gel sample buffer containing 5% 2-mercaptoethanol, and boiled for 5 min. SDS-PAGE and Western blotting were performed as described below. Both full-length and cleaved caspase-9 were detected by using a rabbit polyclonal anti caspase-9 antibody (Santa Cruz Biotechnology, Inc.).
Small Interfering RNA (siRNA) Transfection-The human Bcl-2 siRNA sequence was designed as 5Ј-GCUGCACCUGACGCCCU-UCtg-3Ј, and the corresponding annealed oligonucleotide was purchased from Ambion, Inc. (Austin, TX). A validated, non-targeting siRNA (negative control comprised of a 19-bp scrambled sequence with no significant homology to any known human gene sequences) was also purchased from Ambion, Inc. Transient transfection of siRNA was carried out using Oligofectamine TM reagent (Invitrogen) according to the manufacturer's protocol. One day before transfection with siRNA, HeLa BCL-2 cells were plated on 12-well plates and grown in the appropriate medium supplemented with 10% serum and without antibiotics until a confluence of about 30 -50% was reached. Cells were transfected with 100 pmol of Bcl-2 siRNA or 25 pmol of negative control RNA per well in serum-free medium for 4 h and then cultured in the presence of serum (10%) and without antibiotics at 37°C until they were ready to be assayed for cell viability or Western blotting analysis (40 h after RNA addition). The number of viable cells after RNA addition was assessed based on the Resazurin assay, as described previously. For Western blotting analysis, cells were harvested, sedimented, washed once with ice-cold PBS, resuspended in 1 ml of ice-cold lysis buffer, incubated 30 min on ice, and finally Dounce-homogenized. The homogenates were sedimented at full speed in a microcentrifuge for 10 min at 4°C to remove cell debris and nuclei. The supernatants, corresponding to the soluble cellular extracts, were transferred to clean tubes and the protein concentration was determined by the Bradford assay. Equal protein amounts (100 g) were solubilized in Laemmli gel sample buffer containing 5% 2-mercaptoethanol, separated electrophoretically by SDS-PAGE, and subjected to Western blotting analysis, as described below, using a mouse anti-human BCL-2 antibody. The same membrane was then washed, stripped as described in the following paragraph, and probed with a mouse monoclonal antibody against rabbit skeletal muscle GAPDH (clone 6C5, Chemicon International. Inc.).

SDS-PAGE and Western
Blotting-The proteins from each solubilized sample, obtained as described in the preceding paragraphs, were separated electrophoretically in SDS-polyacrylamide 1.5-mm-thick minigels (12% acrylamide-0.4% bisacrylamide) and electroblotted onto nitrocellulose membranes. For immunoblotting analysis, the membrane was blocked in PBS containing 0.05% Tween 20 and 5% nonfat milk (blocking buffer) and incubated with the proper antibody for 2 h (anti-BCL-2 and anti-HA antibodies) or overnight (anti-BCL-XL, anti-GAPDH, and anti-caspase-9 antibodies). The membrane was then washed with PBS containing 0.05% Tween 20 and incubated with blocking buffer containing horseradish peroxidase-conjugated goat antimouse, anti-rat, or anti-rabbit IgG (1:5,000 dilution) for 1 additional hour. After further washing in PBS containing 0.05% Tween 20, labeled proteins were visualized with an ECL Western blotting detection kit (Bio-Rad). Membrane stripping for sequential blotting was carried out using the Re-Blot Plus Western blot recycling kit (Chemicon International, Inc.) according to the manufacturer's protocol.
Reagents-TMRM was purchased from Molecular Probes (Eugene, OR); CsA was purchased from Fluka Riedel-de Haen. BH3I-2Ј and Chelerythrine were from Calbiochem and Sigma, respectively, while HA14-1 and EM20-25 were supplied from Hoffmann-La Roche (Basel, Switzerland). The antibody against caspase-9 was from Santa Cruz Biotechnology, Inc., and it recognized both the uncleaved and cleaved forms of caspase; the secondary peroxidase-conjugated antibodies were from Southern Biotechnology, and the peroxidase detection kit was from Pierce. All other chemicals and tissue culture reagents were purchased from Sigma and were of the highest available grade.

RESULTS
We tested the effects of BH3I-2Ј, Chelerythrine, and HA14-1 on the respiration of isolated rat liver mitochondria. All compounds displayed a biphasic effect, with uncoupling at lower concentrations and respiratory inhibition as the concentration was raised further (Fig. 1). The most effective was BH3I-2Ј (half-maximal activity at about 80 nM, which is equivalent to the uncoupling activity of the most potent protonophore, FCCP). Chelerythrine had an intermediate potency (half-maximal uncoupling at about 2 M), followed by HA14-1 (half-maximal uncoupling at about 20 M).
We next investigated whether these compounds affect the PTP with a sensitive technique that we introduced in 1993 (19), which is illustrated for HA14-1 ( Fig. 2A). In these protocols mitochondria are first loaded with a small Ca 2ϩ pulse that is not sufficient to open the PTP per se. Under these conditions, the addition of a low concentration of FCCP (40 nM in this experiment) was not sufficient to trigger opening of the voltage-dependent PTP because the threshold potential for PTP opening was not reached (Fig. 2, trace a). Yet, increasing concentrations of HA14-1 in the range between 1 and 10 M caused a marked effect on PTP opening by 40 nM FCCP, which now occurred in increasing fractions of the mitochondria (traces b-f). The effect was due to PTP opening because it was fully inhibited by CsA (trace g). It should be stressed that 10 M HA14-1 did not depolarize mitochondria nor did it potentiate the depolarizing effects of concentrations of FCCP between 10 and 150 nM (results not shown). Of note, all BCL-2 ligands displayed a prominent PTP-sensitizing effect at concentrations that did not affect mitochondrial respiration (Fig. 2B).
To test whether these complex effects of BCL-2 ligands could also be observed in intact cells, we preliminarly studied the pattern of expression of BCL-XL and BCL-2 in three cell lines. Western blot analysis ( We then incubated human prostate cancer PC3 cells with TMRM, which accumulates inside energized mitochondria. Mitochondrial depolarization causes probe release, which can be detected as the decrease of mitochondrial fluorescence with sensitive imaging techniques (20). The experiments of Fig. 4  We then tested the effects of BCL-2 ligands on the survival of PC3, HeLa Neo, and HeLa BCL-2 cells (Fig. 5). BCL-2-overexpressing cells (open squares in all panels) were significantly protected from the killing effects of staurosporine (A) as compared with Neo-transfected cells (closed squares in all panels). In striking contrast, HeLa BCL-2 cells, as well as PC3 cells, were as sensitive as HeLa Neo cells to the toxic effects of BH3I-2Ј (B), Chelerythrine (C), and HA14-1 (D). These results suggest that the cytotoxicity of BCL-2 ligands may be largely independent of expression of BCL-2 and BCL-XL and that it may rather be mediated by the effects of these compounds on respiration.
To assess whether the toxic mitochondrial effects were inevitably linked to the BCL-2 binding activity, we screened a series of HA14-1 analogs from the Hoffmann-La Roche chemical library. Fig. 6 reports the structure of one such compound, whose synthesis has been reported in the literature (21) and which we named EM20-25, together with that of HA14-1. EM20-25 did not cause uncoupling, and it only slightly inhibited uncoupled respiration at concentrations above 30 M (Fig.  7A), but it did cause sensitization of the PTP at concentrations higher than 2 M (Fig. 7B).
To address the key issue of whether EM20-25 interacts with BCL-2, HeLa BCL-2 cells were transiently transfected with HA-BAX or empty vector, and transfected cells were then treated with 1 mM EM20-25 or vehicle (Me 2 SO) for 6 h. After detergent extraction, equal amounts of    APRIL 14, 2006 • VOLUME 281 • NUMBER 15 protein were immunoprecipitated using an anti-HA affinity matrix, separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antibody against BCL-2. One millimolar EM20-25 displaced BCL-2 (Fig. 8 It has been shown that BH3I-2Ј is a substrate of the multidrug resistance P-glycoprotein (22). To assess whether EM20-25 was transported by the same system, we tested the effects of CsH, which inhibits the pump but not the PTP (23). CsH did not modify the cytotoxic concen-tration of EM20-25 in PC3 cells (Fig. 9A), while it considerably decreased the cytotoxic concentration of EM20-25 both in HeLa Neo and HeLa BCL-2 cells (Fig. 9, B and C, respectively).

The Mitochondrial Effects of BCL-2 Ligands
We next assessed whether the cytotoxic effects of EM20-25 involved the PTP by studying the mitochondrial membrane potential in situ (Fig.  10). Cells were loaded with TMRM and analyzed with the same technique described in Fig. 4. Depending on the cell type, EM20-25 depolarized mitochondria at concentrations between 0.4 and 3 mM (open symbols), and this effect was sensitive to pretreatment with CsA (closed symbols). It should be mentioned that despite the clear PTP-inducing ability of EM20-25 cell death could not be prevented by CsA (results not shown), a finding that will be further addressed under "Discussion." In an attempt to resolve the question of whether EM20-25 had cytotoxic effects independent of binding to BCL-2, we successfully suppressed BCL-2 expression in HeLa BCL-2 cells by siRNA interference. Treatment with BCL-2 siRNA was able to kill all cells surviving toxicity by the transfection technique itself (about 40% of the total), a finding that prevented further investigation of this problem (results not shown).
We finally tested whether EM20-25 affected the response of BCL-2expressing leukemic B-CLL cells from untreated patients to staurosporine, chlorambucil, and fludarabine. A measurable cytotoxic effect of EM20-25 could be observed at concentrations between 20 and 40 M (Fig. 11). Most remarkably, these concentrations of EM20-25 potentiated the killing effects of staurosporine (A), chlorambucil (B), and fludarabine (C).

FIGURE 7. Effects of EM20-25 on respiration and volume in isolated rat liver mitochondria.
A, the experimental conditions were exactly as described for Fig. 1. One min after the addition of mitochondria the indicated concentrations of EM20-25 were added, followed by 0.2 M FCCP after a further 3 min. Values on the ordinate refer to the rate of respiration before (closed symbols) or after (open symbols) the addition of FCCP. B, the incubation medium was the same as in Fig. 2, except that CsA was omitted. The final volume was 2 ml at pH 7.4 and 25°C. The experiments were started by the addition of 0.5 mg ϫ ml Ϫ1 of mitochondria followed by 30 M Ca 2ϩ . One min later, the indicated concentrations of EM20-25 were added, followed by 0.5 mM EGTA-Tris and 40 nM FCCP after one further min. Values on the ordinate refer to the fraction (⌽) of mitochondria that had undergone the permeability transition as measured from the absorbance changes at 540 nm.

DISCUSSION
In this manuscript we have characterized the complex mitochondrial effects of BH3I-2Ј, Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. We have shown that these compounds also share a series of mitochondrial effects that can be summarized as follows: (i) sensitization of the PTP to opening at concentrations that do not interfere with energy coupling and (ii) uncoupling of mitochondrial respiration, which is then superceded by inhibition as the drug concentration is increased further. Analysis of structural analogs of HA14-1 from the Hoffmann-La Roche chemical library has then allowed to identify EM20-25 as a molecule devoid of toxic side effects on respiration but possessing the ability (i) to sensitize PTP opening in isolated mitochondria and intact cells, (ii) to displace BAX from BCL-2 in HeLa cells, with activation of caspase-9, (iii) to sensitize BCL-2 overexpressing cells to treatment with staurosporine, and of B-CLL cells from patients to treatment with staurosporine, fludarabine, and chlorambucil. Our results on BH3I-2Ј, Chelerythrine, and HA14-1 provide a novel reading frame for previous studies where the cytotoxic effects of these drugs have been addressed.
Independent studies from two laboratories have shown that treatment with HA14-1 and/or BH3I-2Ј depolarized mitochondria in situ (22,24,25) and increased mitochondrial respiration, an effect that sensitized to cell death by tumor necrosis factor-related apoptosis-inducing ligand the human T lymphoblastic leukemia cell CEM (25). A similar effect was observed with the protonophore carbonyl cyanide m-chlorophenyl hydrazone, which by itself was devoid of apoptosis-inducing properties (25). The results of An et al. (24) are entirely consistent with our finding that at low concentrations HA14-1 sensitizes the mitochondrial proapoptotic pathway, while at higher concentrations the direct effects on mitochondria predominate. Indeed, low concentrations of HA14-1 caused activation of caspase-9 and -3 and DNA fragmentation, while higher concentrations of the drug caused caspase-independent cell death. In the study of Hao et al. (25) caspases were apparently not involved in sensitization to tumor necrosis factor-related apoptosisinducing ligand by HA14-1 and BH3I-2Ј, consistent with a direct effect of these drugs on mitochondrial energy coupling in that model system.
It must be stressed that the effective concentrations cannot be readily compared in different studies and in different cell lines because the drugs are substrates of the multidrug resistance P-glycoprotein, as shown for BH3I-2Ј (22) and EM20-25 in HeLa cells (Fig. 9). These obser-    vations probably also account for our observation that B-CLL cells from untreated leukemic patients were much more sensitive to the proapoptotic effects of EM20-25 than HeLa cells. The study of Feng et al. (22) also demonstrated that the proapoptotic effects of BH3I-2Ј vary widely depending on the cell type, yet mitochondrial depolarization could always be observed, and this event could be counteracted by both BCL-2 and BCL-XL. These findings easily explain the depolarizing effects of EM20-25 in mitochondria of PC3 cells, which express large amounts of BCL-XL (Fig. 3).
A new entry among the ligands of BCL-2 family members is Chelerythrine, a natural product of plants that has been identified through high throughput screening of disruptors of the interaction of BCL-XL with a fluoresceinated peptide modeled on the BCL-2 homology 3 domain of BAK (9). Remarkably, a dual effect of Chelerythrine can be easily deduced from the results of Chan et al. (9). Indeed, overexpression of BCL-XL protected from the cytotoxic effects of low concentrations of Chelerythrine; and the protective effects could only be overcome by high drug concentrations (9) that according to our studies are more likely to affect mitochondrial energy conservation directly.
It is tempting to speculate that the effects of BH3I-2Ј, Chelerythrine, HA14-1, and EM20-25 on the PTP are related to BCL-2 binding. BCL-2 overexpression does desensitize the PTP to a variety of stimuli when assessed in isolated mitochondria (11); and we observed onset of PTP desensitization with the same time course of mitochondrial BCL-2 upregulation during hepatocarcinogenesis by 2-acetylaminofluorene, a condition that conferred resistance to hepatocyte apoptosis in vivo (26). On the other hand, the properties of the permeability transition are indistinguishable in mitochondria isolated from isogenic human colon cancer bax Ϫ/Ϫ and bax ϩ/ϩ HCT116 cell lines (27). On balance, we think that establishing whether the interactions of BH3I-2Ј, Chelerythrine, HA14-1, and EM20-25 with BCL-2 play a mechanistic role in PTP regulation will require further work and will probably require characterization of the PTP at the molecular level.
Our results do not necessarily imply that apoptosis induction by EM20-25 can be exclusively explained by its PTP-inducing effects on mitochondria. Indeed, CsA did not prevent cell killing by EM20-25, suggesting that the latter compound may also influence BCL-2-and BCL-XL-dependent events at different cellular locations (e.g. the endoplasmic reticulum) or affect cell survival through additional mechanisms. However, it should also be noted that CsA is not a blocker of the PTP but rather a desensitizer whose inhibitory effects can be overcome by proper stimuli such as an increased Ca 2ϩ concentration (28). Thus, the failure of CsA at inhibiting cell death is not in contrast with a role of PTP opening in triggering the process that will eventually lead to cell demise.
Irrespective of this issue, a clear point emerges from the present results, i.e. that the potentially toxic effects of BCL-2 ligands on respiration and energy coupling can be dissociated from the PTP-inducing effects. Indeed, EM20-25 possesses the ability to activate the mitochondrial proapoptotic pathway through the PTP without affecting energy coupling directly. It is also remarkable that EM20-25 displayed the unique property of sensitizing BCL-2-overexpressing cells to staurosporine and B-CLL cells from leukemic patients to staurosporine, chlorambucil, and fludarabine. These results represent a proof of principle that BCL-2 ligands with a selective effect on BCL-2-overexpressing cells can be developed and used for the selective killing of apoptosisresistant cells. Consistent with this prediction, after this manuscript was submitted a novel inhibitor of BCL-2 family proteins has been described, which induces regression of solid tumors in mice (29).