Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia
2016; Elsevier BV; Volume: 57; Issue: 7 Linguagem: Inglês
10.1194/jlr.m067389
ISSN1539-7262
AutoresSamy A.F. Morad, Terence E. Ryan, P. Darrell Neufer, Tonya N. Zeczycki, Traci S. Davis, Matthew R. MacDougall, Todd E. Fox, Su‐Fern Tan, David J. Feith, Thomas P. Loughran, Mark Kester, David F. Claxton, Brian M. Barth, Tye Deering, Myles C. Cabot,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers. The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers. The bioactive, tumor suppressor sphingolipid ceramide (1Zheng W. Kollmeyer J. Symolon H. Momin A. Munter E. Wang E. Kelly S. Allegood J.C. Liu Y. Peng Q. et al.Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy.Biochim. Biophys. Acta. 2006; 1758: 1864-1884Crossref PubMed Scopus (443) Google Scholar, 2Gangoiti P. Camacho L. Arana L. Ouro A. Granado M.H. Brizuela L. Casas J. Fabrias G. Abad J.L. Delgado A. et al.Control of metabolism and signaling of simple bioactive sphingolipids: implications in disease.Prog. 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In addition to glycosylation, ceramide metabolism via hydrolysis gives rise to sphingosine 1-phosphate (S1-P), a mitogenic sphingolipid that competes with ceramide's proapoptotic effects (11Payne S.G. Milstien S. Spiegel S. Sphingosine-1-phosphate: dual messenger functions.FEBS Lett. 2002; 531: 54-57Crossref PubMed Scopus (183) Google Scholar, 12Pyne N.J. McNaughton M. Boomkamp S. MacRitchie N. Evangelisti C. Martelli A.M. Jiang H.R. Ubhi S. Pyne S. Role of sphingosine 1-phosphate receptors, sphingosine kinases and sphingosine in cancer and inflammation.Adv. Biol. Regul. 2016; 60: 151-159Crossref PubMed Scopus (107) Google Scholar). Acute myelogenous leukemia (AML) is the most common type of leukemia in adults. Only ∼25% of patients who experience remission with cytotoxic chemotherapy remain disease free. Therapy for these patients generally consists of cytosine arabinoside plus anthracycline, a regimen that has been in use for more than three decades; thus, there exists a critical need to develop more effective therapies in AML. One novel approach involves the use of C6-ceramide, a ceramide mimic, which like its long-chain, natural counterpart has tumor-suppressor properties. An innovative feature presented herein, however, is the inclusion of the breast cancer drug tamoxifen, which we show magnifies the C6-ceramide effect in AML, and the use of C6-ceramide-tamoxifen nanoliposomes, composites that deliver both agents within the same particle. Although tamoxifen has long been utilized as a modulator of multidrug resistance, similar to verapamil and cyclosporin A, the impact of tamoxifen on sphingolipid metabolism has only more recently been demonstrated. Apropos in this context, tamoxifen can suppress ceramide metabolism via glycosylation (13Cabot M.C. Giuliano A.E. Volner A. Han T.Y. Tamoxifen retards glycosphingolipid metabolism in human cancer cells.FEBS Lett. 1996; 394: 129-131Crossref PubMed Scopus (75) Google Scholar) and inhibit hydrolysis (14Morad S.A. Levin J.C. Tan S.F. Fox T.E. Feith D.J. Cabot M.C. Novel off-target effect of tamoxifen—inhibition of acid ceramidase activity in cancer cells.Biochim. Biophys. Acta. 2013; 1831: 1657-1664Crossref PubMed Scopus (51) Google Scholar), thus reducing S1-P formation (15Morad S.A. Tan S.F. Feith D.J. Kester M. Claxton D.F. Loughran Jr., T.P. Barth B.M. Fox T.E. Cabot M.C. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia—impact on enzyme activity and response to cytotoxics.Biochim. Biophys. Acta. 2015; 1851: 919-928Crossref PubMed Scopus (22) Google Scholar). Therefore, when used in combination with a cell-deliverable ceramide, such as C6-ceramide, or ceramide-generating drugs, beneficial actions from a therapeutic standpoint can be realized. The development of ceramide-based therapy in cancer treatment is promising (6Barth B.M. Cabot M.C. Kester M. Ceramide-based therapeutics for the treatment of cancer.Anticancer. Agents Med. Chem. 2011; 11: 911-919Crossref PubMed Scopus (68) Google Scholar). In earlier work in HL-60/VCR cells, we demonstrated that tamoxifen enhanced the cytotoxicity of fenretinide, a ceramide-generating retinoid (16Maurer B.J. Metelitsa L.S. Seeger R.C. Cabot M.C. Reynolds C.P. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines.J. Natl. Cancer Inst. 1999; 91: 1138-1146Crossref PubMed Scopus (255) Google Scholar), and C6-ceramide; however, mechanisms supporting this enhancement had not been elucidated (15Morad S.A. Tan S.F. Feith D.J. Kester M. Claxton D.F. Loughran Jr., T.P. Barth B.M. Fox T.E. Cabot M.C. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia—impact on enzyme activity and response to cytotoxics.Biochim. Biophys. Acta. 2015; 1851: 919-928Crossref PubMed Scopus (22) Google Scholar). Herein, we demonstrate that the mechanisms promoting C6-ceramide-tamoxifen cytotoxicity in AML cells encompass a reduction in mitochondrial membrane potential, inhibition of complex I respiration, generation of reactive oxygen species (ROS), reduced glycolytic capacity, and decreases in ATP. Significant players in AML cell survival were also targeted, XIAP and survivin, members of the inhibitors of apoptosis protein (IAP) family. As glycolysis is an important cell-sustaining maneuver in cancer, this unique C6-ceramide-tamoxifen combination, which imparts substantial bioenergetic clout, could constitute an effective therapy in AML. Of note, N-desmethyltamoxifen (DMT), the primary tamoxifen metabolite in humans with little antiestrogenic activity, was as effective as tamoxifen, demonstrating that we are not working through traditional antiestrogen pathways. Human AML cell lines KG-1 and HL-60 were obtained from the American Type Culture Collection (Manassas, VA). HL-60/VCR cells were provided by A. R. Safa (Indiana University School of Medicine, Indianapolis, IN) and were grown in medium containing 1.0 μg/ml vincristine. Cells were maintained in RPMI-1640 medium (Life Technologies, Carlsbad, CA) with 10% FBS (Atlanta Biologicals, Atlanta, GA), 100 U/ml penicillin, and 100 μg/ml streptomycin. For experiments with HL-60/VCR cells, vincristine was removed from the medium. Cells were cultured in a humidified atmosphere, 95% air, 5% CO2, at 37°C. AML patient samples with 20% or greater blast count were collected with signed informed consent according to a protocol approved by the Institutional Review Board of the Milton S. Hershey Medical Center. Human granulocyte-colony stimulating factor mobilized peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from the Blood Bank (Milton S. Hershey Medical Center). PBMCs were enriched using the Ficoll-Paque gradient separation method (Pharmacia Biotech, Piscataway, NJ). C6-ceramide was from Avanti Polar Lipids (Alabaster, AL). Propidium iodide (PI), JC-10 mitochondria kit, α-tocopherol (vitamin E), tamoxifen, and DMT were from Sigma (St. Louis, MO). C6-ceramide, tamoxifen, and DMT were dissolved in DMSO (10 mM stock solutions) and stored at −20°C. MitoSOXTM Red, Pierce™ Protease Inhibitor cocktail (EDTA free), and HBSS were from Life Technologies. Cells were seeded into 6-well plates (0.5 × 106 cells/well, 5% FBS medium, 2.0 ml final volume), treated with indicated agents for times designated, collected by centrifugation, washed with PBS, and suspended in 0.1 ml PI buffer [PBS, pH 7.4, 50 μg/ml PI, 0.2% BSA (BSA)] for 10 min. PI-positive cells were determined by flow cytometry (Becton Dickinson FACS Calibur); data were assessed using FCS Express 4, De Novo Software (Glendale, CA). Viability was also determined using the CellTiter 96 Assay Kit (MTS) (Promega, Milwaukee, WI), according to instructions. Viability was calculated as the mean (n = 3 or n = 6) absorbance (minus vehicle control) at 490 nm, using a Bio-Tek Synergy H1 microplate reader. Pegylated nanoliposomes were prepared from specific lipids at particular molar ratios as previously described (17Tran M.A. Smith C.D. Kester M. Robertson G.P. Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development.Clin. Cancer Res. 2008; 14: 3571-3581Crossref PubMed Scopus (112) Google Scholar, 18Morad S.A. Levin J.C. Shanmugavelandy S.S. Kester M. Fabrias G. Bedia C. Cabot M.C. Ceramide–antiestrogen nanoliposomal combinations—novel impact of hormonal therapy in hormone-insensitive breast cancer.Mol. Cancer Ther. 2012; 11: 2352-2361Crossref PubMed Scopus (39) Google Scholar). Briefly, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and C6-ceramide (or tamoxifen) were dissolved in chloroform, dried to a film under a stream of nitrogen, and then hydrated by addition of 0.9% NaCl. Solutions were sealed, heated at 55°C for 60 min, vortex mixed, and sonicated for 5 min until light no longer diffracted through the suspension. The solution was quickly extruded at 55°C by passage through 100 nm polycarbonate filters in an Avanti Mini-Extruder (Avanti Polar Lipids). Ghost nanoliposomes were prepared in the same manner excluding C6-ceramide. Composite nanoliposomes were formulated as above, using C6-ceramide and tamoxifen. Apoptosis was assessed using the ApoDETECT Annexin V-FITC Kit (Life Technologies) as previously described, to detect annexin V binding by flow cytometry (15Morad S.A. Tan S.F. Feith D.J. Kester M. Claxton D.F. Loughran Jr., T.P. Barth B.M. Fox T.E. Cabot M.C. Modification of sphingolipid metabolism by tamoxifen and N-desmethyltamoxifen in acute myelogenous leukemia—impact on enzyme activity and response to cytotoxics.Biochim. Biophys. Acta. 2015; 1851: 919-928Crossref PubMed Scopus (22) Google Scholar). Caspase-3 proteolytic activity was measured using the Caspase-3 DEVD-R110 Fluorometric HTS Assay Kit (Biotium Inc., Hayward, CA), per manufacturer's instructions. High-resolution O2 consumption measurements were conducted at 30°C in Mir05 (110 mM sucrose, 0.5 mM EGTA, 60 mM K-lactobionate, 3 mM MgCl2-6H2O, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, pH 7.1, 1 mg/ml BSA) using the OROBOROS O2K Oxygraph (Innsbruck, Austria). HL60/VCR (2 × 106 cells/chamber) and KG-1 (4 × 106 cells/chamber) were added to the respirometer and permeabilized with digitonin (4 μg and 3 μg/106 cells for HL60/VCR and KG-1 cell lines, respectively). Optimal permeabilization was determined by titration of digitonin in the presence of maximal substrate (glutamate, malate, ADP). Substrate inhibitor titration protocol was conducted as follows: 2 mM malate + 10 mM glutamate (complex I supported state 2 respiration), followed by the addition of 4 mM ADP to initiate state 3 respiration supported by complex I substrates. Convergent electron flow was initiated by addition of 10 mM succinate, and 10 μM rotenone was subsequently added to inhibit complex I, followed by 10 μM cytochrome c to test the integrity of the outer mitochondrial membrane, and finally, uncoupled respiration was assessed with the addition of 0.5 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone. The rate of respiration, oxygen consumption, was calculated as pmol/s/106 cells and expressed as a percentage of control (DMSO treated) cells. To detect changes in mitochondrial ΔΨm after long-term treatment, cells were stained with JC-10 dye. Assays were conducted according the manufacturer's instructions. To assess alterations in mitochondrial ΔΨm after short-term exposure to drugs, KG-1 cells (2 × 106) were preincubated in 2 ml of a JC-10 dye loading solution (HBSS containing 20 µl JC-10) for 15 min. Cells were then washed in HBSS containing 0.1% BSA, suspended in FBS-free RPMI-1640 medium containing 0.1% BSA, and a 0.1 ml aliquot was added to wells of a 96-well plate. Cells were treated by adding 0.1 ml of a 2× drug solution (C6-ceramide-tamoxifen, in RPMI-1640 medium, 0.1% BSA) to achieve the desired drug concentration. Plates were read at excitation/emission wavelengths 490/525 and 540/590 nm using a microplate reader. Mitochondrial superoxide was assayed using MitoSOXTM Red. Cells (1 × 106/2 ml 5% FBS RPMI-1640 medium) were seeded in 6-well plates, treated with indicated agents for 24 h, then washed in HBSS and incubated in 0.25 ml staining buffer (HBSS containing 5 μM MitoSOX) for 15 min at 37°C. Cells were washed in HBSS, resuspended at 1 × 106/ml HBSS, and a 0.1 ml aliquot was added to the wells of a 96-well plate. Fluorescence was measured at 510 nm excitation and 580 nm emission. Cells were collected, isolated by centrifugation, and deproteinated on ice by the addition of 0.5 N perchloric acid for 10 min. Extracts were neutralized on ice for 10 min with 1N KOH at a ratio of 2:1. HPLC was used to quantitate cellular ATP and ADP (19Brault J.J. Pizzimenti N.M. Dentel J.N. Wiseman R.W. Selective inhibition of ATPase activity during contraction alters the activation of p38 MAP kinase isoforms in skeletal muscle.J. Cell. Biochem. 2013; 114: 1445-1455Crossref PubMed Scopus (16) Google Scholar), using a Shimadzu Prominence HPLC system equipped with a Purospher STAR RP-18 end-capped column (4.6 × 150 mm 3 μm, EMD Millipore) at flow rate of 0.4 ml/min with a column temperature of 37°C. Cells were collected and lysed by sonication in 1 ml buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1.0% Triton X-100. Samples were heated to 95°C for 5 min and centrifuged at 500 g for 10 min at 4°C to remove debris. Concentrations of Mg-ATP in supernatants were determined using the hexokinase/glucose-6-phosphate coupled assay (20Zeczycki T.N. Menefee A.L. Jitrapakdee S. Wallace J.C. Attwood P.V. Maurice M.St. Cleland W.W. Activation and inhibition of pyruvate carboxylase from Rhizobium etli.Biochemistry. 2011; 50: 9694-9707Crossref PubMed Scopus (20) Google Scholar). The reduction of NADP+ to NADPH was monitored at 340 nm (ε340 = 6,200 cm−1 M−1). All reactions were determined at 25°C in 1 ml containing 50 mM Tris buffer (pH 7.5), 5 mM MgCl2, 50 mM glucose, 1 mM NADP+, 0.5 units hexokinase, and 5 units glucose-6-phosphate dehydrogenase and initiated by addition of 50 µl of cell lysate. Cells were seeded into an XF assay microplate (Seahorse Bioscience, North Billerica, MA). The assay cartridge was hydrated overnight, at 37°C, in a CO2-free incubator. On the day of assay, the treatment medium was discarded, and assay medium (Dulbecco's modified Eagle's medium, Life Technologies) containing 1.85 g/l NaCl, 3 mg/l phenol red, and 2 mM l-glutamine was added. Alterations in the extracellular acidification rate (ECAR) were measured with the XF24 Seahorse extracellular flux analyzer. Results were normalized based on the number of live cells at assay time. Cytochrome c release from mitochondria was assessed as described (21Christensen M.E. Jansen E.S. Sanchez W. Waterhouse N.J. Flow cytometry based assays for the measurement of apoptosis-associated mitochondrial membrane depolarisation and cytochrome c release.Methods. 2013; 61: 138-145Crossref PubMed Scopus (56) Google Scholar). Cells (2 × 106/2 ml 2.5% FBS RPMI-1640 medium) were seeded in 6-well plates and treated with agents for 18 h, after which 1 × 106 cells were removed and placed on ice with 0.1 ml of digitonin (100 μg/ml in PBS, 100 mM KCl) for 3–5 min until 95% of the cells were permeabilized (stained positive with 0.2% trypan blue). Cells were then fixed at room temperature in 4% paraformaldehyde in PBS for 20 min and resuspended in blocking buffer (PBS, 3% BSA, 0.05% saponin). Fixed cells were incubated for 30 min at 4°C in a 1:100 dilution of FITC conjugate (6H2) anti-cytochrome c antibody (Life Technologies) in blocking buffer and washed, and levels of cytochrome c were determined. Cell acquisition was performed on a Becton Dickinson FACSCalibur. Analysis was performed using FCS Express 4 from De Novo Software. RIPA buffer and antibodies were from Cell Signaling Technology (Danvers, MA). Second mitochondria-derived activator of caspase (SMAC) release was performed as described (22Ng H. Smith D.J. Nagley P. Application of flow cytometry to determine differential redistribution of cytochrome c and Smac/DIABLO from mitochondria during cell death signaling.PLoS One. 2012; 7: e42298Crossref PubMed Scopus (16) Google Scholar) with modifications. After treatment, KG-1 cells were harvested in RIPA buffer supplemented with 1 mM PMSF, centrifuged, washed in PBS, and suspended in 0.2 ml of ice-cold buffer A [250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 20 mM HEPES (pH 7.5)] containing protease inhibitor cocktail, for 15 min followed by addition of digitonin to a final concentration of 200 µg/ml. Samples were mixed for 30 s, and permeabilization of cells was confirmed by staining with 0.2% trypan blue. Cells were centrifuged at 16,000 g for 5 min at 4°C. The supernatant (cytosolic fraction) was retained, and the pellets were washed with buffer A, without digitonin. After centrifugation (16,000 g, 5 min), pellets containing mitochondria were solubilized in mitochondrial lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40) containing protease inhibitor cocktail, followed by centrifugation (16,000 g, 5 min, 4°C). The supernatants (solubilized mitochondria) were retained for analysis. Protein in cellular lysates, cytosolic fractions, and solubilized mitochondria was measured using a BCA Protein Assay Kit. Equal amounts of protein were loaded onto 4%–12% NuPAGE gels (Invitrogen) for electrophoresis. Protein was transferred to polyvinylidene difluoride membranes and probed with antibodies against SMAC, survivin, XIAP, voltage-dependent anion channel, and β-actin. Blots were imaged using a LI-COR Biosciences Odyssey Infrared Fluorescent scanner (Lincoln, NE). Total lipids were extracted from cells using ethyl acetate-isopropanol-water (60:30:10, v/v) without phase partitioning, and the solvents were evaporated (azeotroph) under a stream of nitrogen. Internal standards were added, and separations were conducted using electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS, using a Waters I-class Acquity LC and the Waters Xevo TQ-S mass spectrometer, as previously described (23Fox T.E. Bewley M.C. Unrath K.A. Pedersen M.M. Anderson R.E. Jung D.Y. Jefferson L.S. Kim J.K. Bronson S.K. Flanagan J.M. et al.Circulating sphingolipid biomarkers in models of type 1 diabetes.J. Lipid Res. 2011; 52: 509-517Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The results are expressed as the mean ± SE and were analyzed by ANOVA. Differences among treatment groups were assessed by Tukey's test. Differences were considered significant at P ≤ 0.05. An asterisk (*) used in specific figures denotes significance. Replicates ranged from n = 3 to n = 6, and repeated experiments yielded similar results. The C6-ceramide-tamoxifen regimen was active in three cell lines, KG-1, HL-60, and HL-60/VCR, and in patient-derived AML cells. The interdependent activity of this regimen is illustrated in Fig. 1A–C, which shows that exposure to C6-ceramide yielded only slight but significant responses in HL-60 and in HL-60/VCR cells and produced no response in KG-1 cells. However, whereas tamoxifen was without significant impact in all cell lines, the combination resulted in 70% or greater cell death. Moreover, the regimen was effective in multidrug-resistant HL-60/VCR cells (Fig. 1C), and DMT, the most prominent tamoxifen metabolite in humans having <1% of the antiestrogenic activity of tamoxifen (24Fabian C. Tilzer L. Sternson L. Comparative binding affinities of tamoxifen, 4-hydroxytamoxifen, and desmethyltamoxifen for estrogen receptors isolated from human breast carcinoma: correlation with blood levels in patients with metastatic breast cancer.Biopharm. Drug Dispos. 1981; 2: 381-390Crossref PubMed Scopus (89) Google Scholar), was as effective as tamoxifen when combined with C6-ceramide. Noteworthy, the addition of antioxidant vitamin E totally mitigated cytotoxicity. The regimen was not cytotoxic in PBMCs (Fig. 1D). Treatment of KG-1 cells with the combination promoted significant increases over single agents in annexin V binding (Fig. 1E) and elicited dose-dependent caspase activation (Fig. 1F). Similar responses were observed in HL-60 and HL-60/VCR cells (data not shown). The regimen also induced apoptosis in AML cells derived from patients (Fig. 1G, left). In this experiment, nanoliposomal formulations of C6-ceramide and tamoxifen were used, as this would be the preferred mode of in vivo administration. Approximately 50% of patients demonstrated strong apoptotic response to the C6-ceramide-tamoxifen regimen. The disparate distribution in responses is perhaps reflective of the heterogeneity in AML and the difficulties inherent in treating this disease. Of the emerging areas in cancer therapy, mitochondria have been the object of much attention (25Wenner C.E. Targeting mitochondria as a therapeutic target in cancer.J. Cell. Physiol. 2012; 227: 450-456Crossref PubMed Scopus (70) Google Scholar). As mitochondria play crucial roles in generation and maintenance of cellular energy and redox charge, we investigated the impact of C6-ceramide and tamoxifen on bioenergetics. Treatment of KG-1 and HL-60/VCR cells with the combination as opposed to the single agents decreased basal (nonphosphorylating, state 4) respiration by ∼50%, and maximal (phosphorylation under conditions with saturating [ADP], state 3) respiration supported by complex I (glutamate + malate) by ∼60%, but not complex II only (succinate in the presence of complex I inhibitor rotenone) respiration (Fig. 2A, B). Acute treatment was also evaluated in which cells were permeabilized and exposed to a higher dose regimen (20 µM C6-ceramide plus 10 µM tamoxifen) for only 20 min. Results revealed that both basal and complex I-supported respiration were decreased by 50% and 75%, respectively, in HL-60/VCR cells and by 25% and 75%, respectively, in KG-1 cells (data not shown). These data highlight a crippling reduction in respiration specifically supported by electron transfer from NADH through complex I, contributing to decreased capacity for oxidative metabolism. Inhibition of complex I has been shown to promote mitochondrial ROS production (26Zigdon H. Kogot-Levin A. Park J.W. Goldschmidt R. Kelly S. Merrill Jr., A.H. Scherz A. Pewzner-Jung Y. Saada A. Futerman A.H. Ablation of ceramide synthase 2 causes chronic oxidative stress due to disruption of the mitochondrial respiratory chain.J. Biol. Chem. 2013; 288: 4947-4956Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 27Radad K. Rausch W.D. Gille G. Rotenone induces cell death in primary dopaminergic culture by increasing ROS production and inhibiting mitochondrial respiration.Neurochem. Int. 2006; 49: 379-386Crossref PubMed Scopus (184) Google Scholar), and as vitamin E mitigated cytotoxicity, we evaluated the levels of ROS. Exposure to the regimen produced a 2-fold increase over control in mitochondrial ROS levels (Fig. 2C). Successful apoptosis is allied with dissipation of mitochondrial membrane potential (ΔΨm) that elicits transitions in mitochondrial outer membrane permeability, a process that can be accompanied by release of cytochrome c, key in downstream signaling events. Consistent with this scenario, the C6-ceramide-tamoxifen regimen induced dose- and time-dependent decreases in ΔΨm (Fig. 3A); decreases were evident as early as 10 min after exposure. Longer-term exposures showed that single agents were not as effective as the combination, which resulted in a 75% decrease in ΔΨm (Fig. 3B). Membrane permeability changes were accompanied by release of proapoptotic cytochrome c that was largely driven by the combination (Fig. 3C, left to right, histogram, density plot, bar graph). For example, whereas C6-ceramide and tamoxifen treatment resulted in cytochrome c release that was ∼9% and 6% over control, respectively, the combination produced a 40% (Fig. 3C), supporting the view that tamoxifen magnifies the effect of C6-ceramide on mitochondrial function. Exposure to the regimen also promoted release of SMAC and downregulated the expression of the IAPs survivin and XIAP (Fig. 3D), two important mediators of the intrinsic mitochondrial pathway of apoptosis. The data in Fig. 3D (top) show a dose-dependent decrease in mitochondrial SMAC accompanied by an increase in cytosolic SMAC, indicative of mitochondrial membrane
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