The Mitochondrial Effects of Small Organic Ligands of BCL-2
2006; Elsevier BV; Volume: 281; Issue: 15 Linguagem: Inglês
10.1074/jbc.m513708200
ISSN1083-351X
AutoresEva Milanesi, Paola Costantini, Alberto Gambalunga, Raffaele Colonna, Valeria Petronilli, Anna Cabrelle, Gianpietro Semenzato, Andrea M. Cesura, Emmanuel Pinard, Paolo Bernardi,
Tópico(s)Antibiotics Pharmacokinetics and Efficacy
ResumoWe 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. 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. Antiapoptotic proteins of the BCL-2 family contribute to neoplastic cell expansion by suppressing physiological cell death mechanisms and by increasing the resistance to anticancer drugs. High levels of the BCL-2 protein can be found in cells selected for their resistance to chemotherapeutic agents (1Sartorius U.A. Krammer P.H. Int. J. Cancer. 2002; 97: 584-592Crossref PubMed Scopus (184) Google Scholar), and this makes BCL-2 an attractive target for cancer therapy (2Reed J.C. Nat. Rev. Drug Discov. 2002; 1: 111-121Crossref PubMed Scopus (603) Google Scholar, 3Letai A. Sorcinelli M.D. Beard C. Korsmeyer S.J. Cancer Cell. 2004; 6: 241-249Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Despite intense research on this topic, the mechanisms through which BCL-2 prevents apoptosis are not fully understood (4Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1646) Google Scholar). The protein dimerizes with other members of the family, which comprises both pro- and antiapoptotic members; and it is widely believed that the outcome for cell survival depends on the ratio of pro- to antiapoptotic BCL-2-like proteins (5Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). A computer screening based on the structure of the close BCL-2 relative, BCL-XL, has identified HA14-1, 6The abbreviations used are: HA14-1, ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate; B-CLL, B-chronic lymphocytic leukemia; BH3I-2′, 3-iodo-5-chloro-N-[2-chloro-5-((4-chlorophenyl)sulfonyl)phenyl]-2-hydroxybenzamide; Chelerythrine, 1,2-dimethoxy-12-methyl[1,3]benzodioxolo[5,6-c]phenantridinium; Cs, cyclosporin; EM20-25, 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; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA, human influenza hemagglutinin; Mops, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PT, permeability transition; PTP, permeability transition pore; siRNA, small interfering RNA; TMRM, tetramethylrhodamine methyl ester. a small organic ligand that is able to displace a peptide modeled on the BCL-2 binding region of BAK, a proapoptotic member of the family (6Wang J.-L. Liu D. Zhang Z.-J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Crossref PubMed Scopus (1178) Google Scholar). Remarkably, HA14-1 was able to cause cell death that was preceded by activation of caspase-9 and -3 and caused mitochondrial depolarization in situ (6Wang J.-L. Liu D. Zhang Z.-J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Crossref PubMed Scopus (1178) Google Scholar). Similar results have been reported for antimycin A, which displays a striking BCL-2 binding activity that is retained by a 2-methoxy derivative devoid of inhibitory effects on respiration (7Tzung S.P. Kim K.M. Basanez G. Giedt C.D. Simon J. Zimmerberg J. Zhang K.Y. Hockenbery D.M. Nat. Cell Biol. 2001; 3: 183-191Crossref PubMed Scopus (439) Google Scholar), and for BH3Is (8Degterev A. Lugovskoy A. Cardone M. Mulley B. Wagner G. Mitchison T. Yuan J. Nat. Cell Biol. 2001; 3: 173-182Crossref PubMed Scopus (543) Google Scholar) and Chelerythrine (9Chan S.-L. Lee M.C. Tan K.O. Yang L.K. Lee A.S.Y. Flotow H. Fu N.Y. Butler M.S. Soejarto D.D. Buss A.D. Yu V.C. J. Biol. Chem. 2003; 278: 20453-20456Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), which were identified by high throughput screening of chemical libraries. These findings point to mitochondria as the targets for the effects of BCL-2 ligands. Mitochondria are important players in the pathways to cell death through at least three mechanisms: (i) changes of ATP production, (ii) alteration of Ca2+ homeostasis, (iii) release of apoptogenic proteins like cytochrome c and Smac-Diablo that activate procaspase-9 and then downstream caspases (10Bernardi P. Petronilli V. Di Lisa F. Forte M. Trends Biochem. Sci. 2001; 26: 112-117Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). A mechanistic link between BCL-2, mitochondria, and cell death is provided by the reported inhibition of the release of cytochrome c and of other mitochondrial proteins by BCL-2 overexpression (11Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar, 12Yang J. Liu X. Bhalla K. Kim C.N. Ibrado A.M. Cai J. Peng T.I. Jones D.P. Wang X. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar, 13Kluck R.M. Bossy-Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 275: 1132-1136Crossref PubMed Scopus (4277) Google Scholar). Although it appears reasonable that the proapoptotic effects of HA14-1, BH3Is, and Chelerythrine are mediated by interactions with BCL-2 in mitochondria, the mechanistic basis for the mitochondrial effects of these drugs has not been established. Specifically, the depolarizing and/or cytochrome c-releasing effects of BCL-2 ligands could be due to opening of the PTP, an inner membrane channel that is reportedly inhibited by BCL-2 overexpression (11Susin S.A. Zamzami N. Castedo M. Hirsch T. Marchetti P. Macho A. Daugas E. Geuskens M. Kroemer G. J. Exp. Med. 1996; 184: 1331-1341Crossref PubMed Scopus (1028) Google Scholar), or to interference with energy coupling and/or respiration. Assessing whether BCL-2 ligands directly affect electron transfer appears particularly important in the light of the finding that antimycin A, the selective inhibitor of electron transfer at the bc1 complex, also binds to BCL-2 at the same site as HA14-1 (7Tzung S.P. Kim K.M. Basanez G. Giedt C.D. Simon J. Zimmerberg J. Zhang K.Y. Hockenbery D.M. Nat. Cell Biol. 2001; 3: 183-191Crossref PubMed Scopus (439) Google Scholar). In this paper we have investigated the mitochondrial and cellular effects of BH3I-2′, 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-2′ > Chelerythrine > 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. Measurements on Isolated Mitochondria—Liver mitochondria were isolated from albino Wistar rats weighing about 300 g by standard centrifugation techniques, as described previously (14Costantini P. Petronilli V. Colonna R. Bernardi P. Toxicology. 1995; 99: 77-88Crossref PubMed Scopus (129) Google Scholar). 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 × ml–1 penicillin, and 50 μg × ml–1 streptomycin. Cells were kept in a humidified atmosphere of 95% air and 5% CO2 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 × 106 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 μm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/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 μg) 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 anti-human BCL-2 antibody (clone 7, BD Biosciences) or a rabbit anti-human 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 (15Cerutti A. Trentin L. Zambello R. Sancetta R. Milani A. Tassinari C. Adami F. Agostini C. Semenzato G. J. Immunol. 1996; 157: 1854-1862PubMed Google Scholar). 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 μg/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+, CD16+, and CD56+ lymphocytes using high gradient magnetic separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany), as described previously (16Trentin L. Zambello R. Agostini C. Enthammer C. Cerutti A. Adami F. Zamboni S. Semenzato G. Blood. 1994; 84: 4249-4256Crossref PubMed Google Scholar). Briefly, 10 × 106 cells obtained as above were incubated for 30 min at 4 °C in 80 μl 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 μl of colloidal superparamagnetic microbeads conjugated with goat-anti-mouse-IgG antibodies were added. The CD3+, CD16+, and CD56+ 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+ and BCL-2+ 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 μm CsH or 1 μm CsA for 30 min at 37 °C prior to further additions. Fluorescence images were acquired with a 560-nm dichroic mirror using a 40×/1.3 oil immersion objective (Zeiss), with excitation at 546 ± 5 nm and emission at 580 ± 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 (17O'Brien J. Wilson I. Orton T. Pognan F. Eur. J. Biochem. 2000; 267: 5421-5426Crossref PubMed Scopus (2545) Google Scholar, 18Gugliucci A. Ranzato L. Scorrano L. Colonna R. Petronilli V. Cusan C. Prato M. Mancini M. Pagano F. Bernardi P. J. Biol. Chem. 2002; 277: 31789-31795Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, cells were grown in 96-well microtiter plates (20 × 103 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™ 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 × 106 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 μg 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 μg of pcDNA3 alone, using the Lipofectamine™ 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 Me2SO. 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 μg in a final volume of 1 ml) were incubated overnight at 4 °C on a rocker platform with 50 μl 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 –20 °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 μl 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 μl 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 5′-GCUGCACCUGACGCCCUUCtg-3′, 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™ 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 μg) 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 anti-mouse, 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-2′ 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. We tested the effects of BH3I-2′, 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-2′ (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 μm), followed by HA14-1 (half-maximal uncoupling at about 20 μm). We next investigated whether these compounds affect the PTP with a sensitive technique that we introduced in 1993 (19Petronilli V. Cola C. Massari S. Colonna R. Bernardi P. J. Biol. Chem. 1993; 268: 21939-21945Abstract Full Text PDF PubMed Google Scholar), which is illustrated for HA14-1 (Fig. 2A). In these protocols mitochondria are first loaded with a small Ca2+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 μm 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 μm 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 (Fig. 3) revealed that PC3 cells (lane 1) expressed high levels of BCL-XL but no detectable BCL-2, while HeLa Neo (lane 2) and HeLa BCL-2 cells (lane 3) expressed comparable levels of BCL-XL, BCL-2 being expectedly up-regulated in the latter cell type only. 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 (20Bernardi P. Scorrano L. Colonna R. Petronilli V. Di Lisa F. Eur. J. Biochem. 1999; 264: 687-701Crossref PubMed Scopus (659) Google Scholar). The experiments of Fig. 4 document that the addition of 10 nm BH3I-2′ (A), 1 μm Chelerythrine (B), or 5 μm HA14-1 (C) caused a fluorescence decrease (A–C, closed symbols) that is consistent with in situ mitochondrial depolarization, as demonstrated by the similar effect elicited by the protonophore FCCP (all panels). As expected of a PTP-dependent event, the TMRM fluorescence decrease elicited by the BCL-2 ligands was effectively prevented by CsA, which desensitizes the PTP (A–C, open symbols). As the concentration was increased to 30 nm BH3I-2′ (A′), 10 μm Chelerythrine (B′), and 30 μm HA14-1 (C′) mitochondrial depolarization was faster (A′–C′, closed symbols) and could no longer be inhibited by CsA (A′–C′, open symbols). 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-2′ (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 (21Figueroa-Villar J.D. Cruz E.R. Tetrahedron. 1993; 49: 2855-2862Crossref Scopus (28) Google Scholar) 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 μm (Fig. 7A), but it did cause sensitization of th
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