Cardiotoxicity of the Anticancer Therapeutic Agent Bortezomib
2010; Elsevier BV; Volume: 176; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.090690
ISSN1525-2191
AutoresDominika Nowis, Michał Mączewski, Urszula Mackiewicz, Marek Kujawa, Anna Ratajska, Mariusz R. Wiȩckowski, Grzegorz M. Wilczyński, Monika Malinowska, Jacek Bil, Paweł Salwa, Marek Bugajski, Cezary Wójcik, Maciej Siński, Piotr Abramczyk, Magdalena Winiarska, Anna Dąbrowska‐Iwanicka, Jerzy Duszyński, Marek Jakóbisiak, Jakub Gołąb,
Tópico(s)Pharmacological Receptor Mechanisms and Effects
ResumoRecent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca2+ fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs. Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca2+ fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs. The use of novel drugs targeting vital cellular pathways in tumor cells has markedly improved the treatment outcomes for neoplastic diseases. Particularly good results have been achieved in the management of hematological malignancies with drugs such as Bcr-Abl antagonists, monoclonal antibodies, and proteasome inhibitors. These successes translate into rising numbers of long-term surviving patients, but paradoxically they also increase the significance of accompanying diseases, including complications of cancer treatments. Clinical reports have documented unexpected cardiotoxicity associated with the use of novel drugs such as herceptin, imatinib, sunitinib, or sorafenib.1Slamon DJ Leyland-Jones B Shak S Fuchs H Paton V Bajamonde A Fleming T Eiermann W Wolter J Pegram M Baselga J Norton L Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2.N Engl J Med. 2001; 344: 783-792Crossref PubMed Scopus (9385) Google Scholar, 2Kerkela R Grazette L Yacobi R Iliescu C Patten R Beahm C Walters B Shevtsov S Pesant S Clubb FJ Rosenzweig A Salomon RN Van Etten RA Alroy J Durand JB Force T Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.Nat Med. 2006; 12: 908-916Crossref PubMed Scopus (969) Google Scholar, 3Mego M Reckova M Obertova J Sycova-Mila Z Brozmanova K Mardiak J Increased cardiotoxicity of sorafenib in sunitinib-pretreated patients with metastatic renal cell carcinoma.Ann Oncol. 2007; 18: 1906-1907Crossref PubMed Scopus (48) Google Scholar, 4Schmidinger M Zielinski CC Vogl UM Bojic A Bojic M Schukro C Ruhsam M Hejna M Schmidinger H Cardiac toxicity of sunitinib and sorafenib in patients with metastatic renal cell carcinoma.J Clin Oncol. 2008; 26: 5204-5212Crossref PubMed Scopus (535) Google Scholar Side effects of treatment with a reversible proteasome inhibitor, bortezomib, are mainly associated with neurological complications (peripheral neuropathy).5Aghajanian C Soignet S Dizon DS Pien CS Adams J Elliott PJ Sabbatini P Miller V Hensley ML Pezzulli S Canales C Daud A Spriggs DR A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies.Clin Cancer Res. 2002; 8: 2505-2511PubMed Google Scholar, 6Argyriou AA Iconomou G Kalofonos HP Bortezomib-induced peripheral neuropathy in multiple myeloma: a comprehensive review of the literature.Blood. 2008; 112: 1593-1599Crossref PubMed Scopus (320) Google Scholar Recent case reports provide alarming signals that treatment with bortezomib might be associated with cardiac events.7Voortman J Giaccone G Severe reversible cardiac failure after bortezomib treatment combined with chemotherapy in a non-small cell lung cancer patient: a case report.BMC Cancer. 2006; 6: 129Crossref PubMed Scopus (96) Google Scholar, 8Orciuolo E Buda G Cecconi N Galimberti S Versari D Cervetti G Salvetti A Petrini M Unexpected cardiotoxicity in haematological bortezomib-treated patients.Br J Haematol. 2007; 138: 396-397Crossref PubMed Scopus (146) Google Scholar, 9Hacihanefioglu A Tarkun P Gonullu E Acute severe cardiac failure in a myeloma patient due to proteasome inhibitor bortezomib.Int J Hematol. 2008; 88: 219-222Crossref PubMed Scopus (84) Google Scholar In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Our findings indicate that bortezomib treatment leads to impaired cardiac function associated with dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria. Rat cardiomyoblastic (H9c2), human colorectal carcinoma (SW480), human breast cancer (MDA-MB 231), and human ovarian carcinoma (MDAH2774) cell lines were purchased from ATCC (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum, antibiotics, 2-mercaptoethanol (50 mmol/L), and l-glutamine (2 mmol/L) (all from Invitrogen, Carlsbad, CA). MG132, epoxomycin, carbobenzoxyl-lle-Glu-(O-t-butyl)-Ala-Leucinal (PSI), and tunicamycin were purchased from Calbiochem/EMD (San Diego, CA), and were dissolved in dimethyl sulfoxide. Imatinib mesylate was from Novartis Pharma AG (Basel, Switzerland) and was dissolved in dimethyl sulfoxide. Bortezomib (Millenium Pharmaceuticals, Cambrigde, MA) was dissolved in 0.9% NaCl. Male Wistar rats (250 to 350 g) were used in the experiments. Breeding pairs were obtained from the Animal House of the Polish Academy of Sciences Medical Research Center (Warsaw, Poland). All in vivo experiments were performed in accordance with the guidelines approved by the Ethical Committee of the Medical University of Warsaw, based on national laws that are in full agreement with the European Union directive and US National Institutes of Health guidelines on animal experimentation. Rats were injected i.p. with 0.2 mg/kg of bortezomib three times a week for 1 to 3 weeks. Wash-out indicates time after treatment termination. Echocardiography was performed using MyLab25 (Esaote, Italy) with a 13 MHz linear array transducer. Each rat was examined at baseline, 24 hours, 3 days, 7 days, 14 days, and 21 days after treatment initialization, and 1 week and 3 weeks after termination of treatment (wash-out). Under light anesthesia (i.p. ketamine HCl and xylazine, 75 and 3.5 mg/kg body weight, respectively,) left ventricular (LV) end-diastolic and end-systolic diameters were determined from the short-axis view at the midpapillary level and fractional shortening was calculated as (LV diastolic − LV systolic diameter)/LV diastolic diameter. LV end-diastolic and end-systolic areas were planimetered from the parasternal long-axis view. LV ejection fraction was calculated as (LV diastolic area − LV systolic area)/LV diastolic area. For immunofluorescence microscopy H9c2 cells were dispensed in 8-well chamber slides (Nunc, Roskilde, Denmark) and cultured with 10 nmol/L bortezomib for 24 hours. Then, the cells were washed with PBS, slides were methanol-fixed for 30 minutes at −20°C, blocked with 5% normal donkey serum and incubated overnight at 4°C with primary anti-ubiquitin FK2 antibody (Assay Designs, Ann Arbor, MI) in 5% normal donkey serum in PBS. Slides were washed thrice in PBS and incubated with donkey anti-mouse Alexa555-conjugated antibody (Invitrogen; 1:200 for 2 hours at room temperature). The slides were washed, mounted in 4,6-diamidino-2-phenylindole-enriched Vectashield medium (Vector Laboratories, Burlingame, CA), and observed under fluorescence confocal microscope (Leica TCS SP2). pEGFP-LC3m plasmid encoding rat LC3 gene cloned into a green fluorescent protein (GFP) fusion protein expression vector was a generous gift from Prof. Noboru Mizushima and Dr. Tamotsu Yoshimori and has been described.10Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5457) Google Scholar H9c2 cells were seeded into 2-well chamber slides (Nunc) at 1 × 105 cells per well. After 24 hours, the cells were transfected with pEGFP-LC3m plasmid using a standard LipofectAMINE2000 protocol (Invitrogen). Six hours post-transfection, growth medium supplemented with 10 nmol/L bortezomib was added after removal of transfection mixture. Cells were washed with PBS 48 hours later, and slides were methanol-fixed for 30 minutes at −20°C, blocked with 5% normal donkey serum, and incubated for 2 hours at room temperature with primary monoclonal anti-GFP antibody (Convance, Emeryville, CA) in 5% normal donkey serum in PBS. Slides were washed thrice in PBS and incubated with donkey anti-mouse Alexa488-conjugated antibody (Invitrogen, 1:200 for 1 hour at room temperature). The slides were washed, stained with TO-PRO (Invitrogen, 1:3000, 15 minutes at room temperature), mounted in Vectashield (Vector Laboratories), and observed under a fluorescence confocal microscope (Leica TCS SP2). The morphological studies were performed in Wistar rats (250 to 350 g). Rats received i.p. injections of bortezomib at the dose of 0.2 mg/kg for 7 days (total three injections) or for 3 weeks (total nine injections). Control rats were injected with the diluent (0.9% saline). Rats were anesthetized with i.p. injection of a lethal dose of pentobarbital/pentobarbital nitrite solution (7 mg and 35 mg per rat, respectively), the aorta was cannulated, and pre-warmed PBS containing 12 IU/ml heparin was injected through the cannula followed by injection of 2% paraformaldehyde in PBS. The hearts were excised, cut transversally into four rings, and immersed in buffered 4% paraformaldehyde for subsequent 12 hours. After washing in distilled water, the hearts were dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in paraffin. Paraffin sections were deparaffinized on a hot plate, followed by three changes of xylene and three changes of ethanol and stained with H&E or Picrosirius red. Sections were analyzed on a Nikon Labophot light microscope equipped with a digital camera. For transmission electron microscopy (TEM), Wistar rats (treated with bortezomib for 1 or 3 weeks) or C57BL/6 mice (treated with bortezomib for 3 weeks) and control animals were perfused transcardially with 3% glutaraldehyde in 0.1 mol/L cacodylate buffer. For TEM studies in H9c2 cells, 1 × 106 of control cells or cells incubated for 48 hours with 10 nmol/L bortezomib were trypsinized, washed with ice-cold PBS, and suspended in 3% glutaraldehyde in 0.1 mol/L cacodylate buffer. Fragments of heart left ventricle or cell pellets were postfixed with 1% OsO4 in the cacodylate buffer, and then dehydrated in increasing concentrations of ethanol and propylene oxide and embedded in Poly/Bed 812 (Polysciences, Inc., Warrington, PA). Resin blocks were cut with a diamond knife on an RMC type MTXL ultramicrotome. Ultrathin sections were mounted on Formvar carbon-coated grids, stained with lead citrate and uranyl acetate, and observed in a Jeol JEM-100S transmission electron microscope (Jeol, Tokyo, Japan). Heart mitochondria were prepared from 250 to 350 g male Wistar rats, as described by Schaller et al11Schaller H Letko G Kunz W Influence of Mg2+-ions on the properties of rat heart mitochondria in dependence on the preparation.Acta Biol Med Ger. 1978; 37: 31-38PubMed Google Scholar using the trypsin-digestion procedure. In brief, pieces of minced hearts were trypsinized (1 mg trypsin/heart) for 20 minutes in 180 mmol/L KCl, 25 mmol/L Tris/HCl, 10 mmol/L EDTA (pH 7.4) under gentle agitation in an ice bath. Next, the material was mixed with the same buffer containing albumin and Complete protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany). Trypsinized hearts were then gently homogenized at 4°C, debris, undisturbed cells and nuclear fraction were removed by centrifugation at 350 × g for 3 minutes, and mitochondria were sedimented at 2500 × g for 10 minutes and washed in 180 mmol/L KCl without EDTA and albumin. Only mitochondrial preparations with mostly intact outer membranes were used. Protein concentration in the mitochondrial fraction was determined according to Bradford’s method using Bio-Rad Protein Assay (BioRad). Respiration of the rat heart mitochondria was measured polarographically using a Clark oxygen electrode (Yellow Springs Instruments, Yellow Springs, OH) in a thermostatic water-jacketed vessel at 25°C. The respiratory buffer contained 180 mmol/L KCl, 25 mmol/L Tris/HCl, and 0.5 mmol/L EGTA (pH 7.4). Total volume was 1.0 ml and the amount of mitochondria was 1 mg. Respiratory substrates were used at the following concentrations: 5 mmol/L glutamate, 5 mmol/L malate, 5 mmol/L succinate, 2 mmol/L ascorbate, 100 μmol/L tetramethylphenylenediamine (TMPD). The concentration of oligomycin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP), when added, was 1 μg/ml and 1 μmol/L, respectively. To determine respiration under state III (the capability of ATP production by mitochondria), 2 mmol/L solution of ADP was added to the suspension of mitochondria and oxygen consumption was measured polarographically as described above. The cytostatic/cytotoxic effects in H9c2 and cancer cell lines were measured using crystal violet staining as described.12Nowis D Legat M Grzela T Niderla J Wilczek E Wilczynski GM Glodkowska E Mrowka P Issat T Dulak J Jozkowicz A Was H Adamek M Wrzosek A Nazarewski S Makowski M Stoklosa T Jakobisiak M Golab J Heme oxygenase-1 protects tumor cells against photodynamic therapy-mediated cytotoxicity.Oncogene. 2006; 25: 3365-3374Crossref PubMed Scopus (161) Google Scholar Briefly, tumor cells were dispensed into 96-well plates (Sarstedt, Numbrecht, Germany) at 3 × 103 cells per well and allowed to attach overnight. The following day the investigated agents were added at indicated concentrations. After incubation the cells were rinsed with PBS and stained with 0.5% crystal violet in 2% ethanol for 10 minutes at room temperature. Plates were washed four times with tap water and cells were lysed with 1% SDS solution. Absorbance was measured at 595 nm using an enzyme-linked immunosorbent assay reader (Bio-Rad, Hercules, CA). Cryopreserved ready-to-use contractile neonatal ventricular rat cardiomyocytes (P1–2) were purchased from Lonza (Walkersville, MD) and seeded into 96-well plate in Clonetics Rat Cardiac Myocyte Basal Medium supplemented with Clonetics SingleQuots, 10% horse serum, 10% fetal bovine serum, and antibiotics/antimycotics mixture (all from Lonza), according to the manufacturer’s protocol. The wells were previously coated with nitrocellulose (BioRad) dissolved in methanol and left for 10 minutes to evaporate alcohol. The medium was replaced with the fresh one containing tested drugs 4 hours post-seeding and supplemented with 200 μmol/L 5-bromo-2′-deoxyuridine (Sigma) to prevent proliferation of non-cardiomyocyte cells. After 24 hours of incubation with drugs 50 μg of 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) and 0.38 μg of phenazine methosulfate (all from Sigma) per well were added for a 24-hour co-incubation. Next, the absorbance was measured at the wavelength of 450 nm. Cytotoxicity was expressed as relative viability of cells (% of control cultures incubated with medium only) and was calculated as follows: relative viability = (Ae − Ab) × 100/(Ac − Ab), where Ab is the background absorbance, Ae is experimental absorbance, and Ac is the absorbance of untreated controls. Series (at least three) of independent experiments were performed and the results presented are representative. Female C57BL/6 mice 8 to12 weeks old and were used in the experiments. Breeding pairs were obtained from the Animal House of the Polish Academy of Sciences Medical Research Center (Warsaw, Poland). All in vivo experiments were performed in accordance with the guidelines approved by the Ethical Committee of the Medical University of Warsaw, based on national laws that are in full agreement with the European Union directive on animal experimentation. Mice were treated with i.p. injections of bortezomib at a dose of 1 mg/kg three times a week for 3 weeks. Cells were isolated as described previously, with some modifications.13Mackiewicz U Emanuel K Lewartowski B Voltage dependent activation of tonic contraction in cardiac myocytes.J Physiol Pharmacol. 2003; 54: 409-421PubMed Google Scholar After perfusion of the heart with Tyrode solution containing collagenase (Boehringer, Ingelheim, Germany) and protease (Sigma), right ventricle was separated and discarded. For Ca2+ transient and cell shortening recordings, myocytes of the LV tissue were resuspended in Tyrode solution, (144 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 0.43 mmol/L NaH2PO4, 10 mmol/L N-2-hydroxyethylpiperazine-N′−2-ethanesufonic acid, 11 mmol/L glucose) placed in a superfusion chamber mounted on the stage of an inverted microscope (Nikon), and superfused with Tyrode solution containing 2 mmol/L Ca2+ at 37°C. For cell culture, myocytes of LV tissue were resuspended in the myocyte plating medium, containing modified Eagle’s medium with Hank’s balanced salt solution supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin-G (Sigma), 10 mmol/L butanedione monoxime (BDM) (Sigma), and 2 mmol/L l-glutamine (Sigma), and plated into 96-well plates freshly coated with 10 μg/ml laminin (BD Biosciences, Bedford, MA). One hour post-plating, the plating medium was replaced with culture medium composed of modified Eagle’s medium with Hanks balanced salt solution supplemented with 10% fetal bovine serum (Hyclone), 0.1% bovine serum albumin (Sigma), ITS medium supplement (insulin, transferrin, selenium; Sigma), 100 U/ml penicillin-G (Sigma), 10 mmol/L butanedione monoxime (BDM) (Sigma), 2 mmol/L l-glutamine (Sigma), and supplemented with bortezomib. After 24 or 48 hours of treatment, the cytostatic/cytotoxic effects were tested in a standard 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide assay. The LV myocytes were incubated for 15 minutes with 10 μmol/L Indo-1 acetoxymethyl ester as described.14Spurgeon HA Stern MD Baartz G Raffaeli S Hansford RG Talo A Lakatta EG Capogrossi MC Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes.Am J Physiol. 1990; 258: H574-H586PubMed Google Scholar The ratio of Indo-1 fluorescence at 405 and 495 nm for the diastolic and systolic Ca2+ concentration was measured on a Dual Channel Ratio Fluorometer (Biomedical Instrumentation Group, University of Pennsylvania, PA). The difference between the systolic and diastolic Indo-1 ratios was used as a measure of the amplitude of Ca2+ transients. The rate constant (r) of monoexponential curve (described by equation: y = Ae−xr) fitted to the decaying part of Ca2+ transient was taken as an index of the rate of Ca2+ transient decay. Cell shortening was recorded with a video edge detector (Cardiovascular Laboratories, School of Medicine, UCLA). Ca2+ transport by sarcoplasmic reticulum Ca2+-ATP-ase (SERCA2a) and Na+/Ca2+ exchanger NCX was estimated from the rate constants (r) of single exponential curves fitted to the decaying part of electrically evoked and caffeine-evoked Ca2+ transients according to Choi and Eisner.15Choi HS Eisner DA The role of sarcolemmal Ca2+-ATPase in the regulation of resting calcium concentration in rat ventricular myocytes.J Physiol. 1999; 515: 109-118Crossref PubMed Scopus (86) Google Scholar The rate constant of decay of electrically evoked Ca2+ transients (r) reflects the rate of combined Ca2+ transport executed mainly by SERCA2a, which pumps Ca2+ back to sarcoplasmic reticulum, and by NCX, which extrudes Ca2+ out of the cell. Ca2+ transport by sarcolemmal Ca2+ ATP-ase and mitochondrial uptake are of minor importance. Caffeine releases Ca2+ from the sarcoplasmic reticulum and prevents its reaccumulation in sarcoplasmic reticulum (Ca2+ transport by SERCA2a under caffeine perfusion = 0). Thus, the rate constant of decline of caffeine-evoked Ca2+ transient (rcaff) approximately reflects the rate of Ca2+ transport by NCX (rcaff = rNCX). The rate constant of Ca2+ transport by SERCA2a was estimated by subtracting rcaff from r (rSERCA = r-rcaff), while rcaff was taken as an index of the rate of Ca2+ transport by NCX. For Western blotting, H9c2 cells were treated with 10 nmol/L bortezomib for time indicated. Rat heart mitochondria for this procedure were isolated as described above. The samples were washed with PBS and lysed with radioimmunoprecipitation assay buffer containing 50 mmol/L Tris base, 150 mmol/L NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mmol/L EDTA supplemented with Complete protease inhibitor cocktail tablets (Roche Diagnostics). Protein concentration was measured using Bio-Rad Protein Assay. Equal amounts of protein were separated on 12% SDS polyacrylamide gel, transferred onto Protran nitrocellulose membranes (Schleicher and Schuell BioScience Inc., Keene, NH), blocked with Tris-buffered saline (pH 7.4) and 0.05% Tween 20, supplemented with 5% nonfat milk and 5% fetal bovine serum. The following antibodies at 1:1000 dilution were used for 24-hour incubation at 4°C: mouse monoclonal anti-porin, mouse monoclonal anti-complex V α subunit, mouse monoclonal anti-complex V β subunit, mouse monoclonal anti-complex V IF1 subunit, mouse monoclonal anti-complex I NDUFA9 subunit, mouse monoclonal anti-PDH E1 α subunit, and MitoProfile Total OXOPHOS Antibody Cocktail (all from Mitosciences Inc., Eugene, OR); mouse monoclonal anti α-tubulin; rabbit anti-β-actin antiserum and rabbit polyclonal anti-LC3B (Sigma); mouse monoclonal anti-KDEL/BiP (Stressgen). After washing with Tris-buffered saline (pH 7.4) and 0.05% Tween 20, membranes were incubated for 45 minutes with corresponding alkaline phosphatase-coupled secondary antibodies (Pierce). The reaction was developed using 1-Step nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate Reagent (Pierce). After scanning, densitometric analysis of Western blots was performed using the Image Quant 5.2 software (Amersham Bioscience, Piscataway, NJ). Data were analyzed using Microsoft Excel 2007. Differences in cytostatic/cytotoxic effects, band densities, Ca2+ transient, and cell shortening, as well as in parameters measured in echocardiography were analyzed for significance by Student’s t-test. Significance was defined as two-sided P < 0.05. Bortezomib and three other proteasome inhibitors (MG132, carbobenzoxyl-lle-Glu-(O-t-butyl)-Ala-Leucinal (PSI) and epoxomycin) reduced survival of H9c2 rat cardiomyoblastic cells (Figure 1A). These effects were time- and dose-dependent and were observed at 5 to 10 nmol/L concentrations, which are within the range inducing cytostatic/cytotoxic effects in human tumor cells (see Supplemental Figure S1A at http://ajp.amjpathol.org) and are found in the plasma of bortezomib-treated patients.16Leveque D Carvalho MC Maloisel F Review. Clinical pharmacokinetics of bortezomib.In Vivo. 2007; 21: 273-278PubMed Google Scholar Although bortezomib did not affect survival of primary adult rat ventricular cardiomyocytes (Figure 1B), it was cytotoxic to contractile neonatal ventricular rat cardiomyocytes at 20 nmol/L concentration (Figure 1C). Moreover, bortezomib at 10 nmol/L concentration potentiated cytotoxic effects of melphalan and doxorubicin against neonatal rat cardiomyocytes (Figure 1C). Dasatinib, imatinib, sorafenib, and sunitinib, other approved anticancer agents that were recently shown to induce cardiotoxic effects also induced cytostatic/cytotoxic effects in H9c2 cells, but at concentrations 2 to 3 orders of magnitude higher than bortezomib (see Supplemental Figure S1B at http://ajp.amjpathol.org). The imatinib-associated cardiotoxicity was proposed to result from induction of endoplasmic reticulum (ER) stress.2Kerkela R Grazette L Yacobi R Iliescu C Patten R Beahm C Walters B Shevtsov S Pesant S Clubb FJ Rosenzweig A Salomon RN Van Etten RA Alroy J Durand JB Force T Cardiotoxicity of the cancer therapeutic agent imatinib mesylate.Nat Med. 2006; 12: 908-916Crossref PubMed Scopus (969) Google Scholar Proteasome inhibition is also associated with induction of ER stress,17Fribley A Zeng Q Wang CY Proteasome inhibitor PS-341 induces apoptosis through induction of endoplasmic reticulum stress-reactive oxygen species in head and neck squamous cell carcinoma cells.Mol Cell Biol. 2004; 24: 9695-9704Crossref PubMed Scopus (368) Google Scholar which results from inhibition of protein retrotranslocation from the ER leading to accumulation of undegraded proteins. Bortezomib induced marked accumulation of polyubiquitinated proteins in H9c2 cells (Figure 2A), accompanied by induction of ER stress with induction of binding protein (BiP) expression (Figure 2B) and pronounced widening of ER lumen observed under TEM (Figure 2C). Impaired proteasomal removal of damaged or misfolded proteins was reported to induce formation of large protein aggregates and compensatory induction of autophagy in primary rat cardiomyocytes.18Tannous P Zhu H Nemchenko A Berry JM Johnstone JL Shelton JM Miller Jr, FJ Rothermel BA Hill JA Intracellular protein aggregation is a proximal trigger of cardiomyocyte autophagy.Circulation. 2008; 117: 3070-3078Crossref PubMed Scopus (196) Google Scholar Incubation of H9c2 cardiomyoblasts with bortezomib led to the formation of multilamellar and lysosomal/autophagosomal structures observed under TEM (Figure 2C). Aggregation of GFP-tagged LC3 (Figure 2D) and increased LC3 processing (Figure 2E) in H9c2 cells further confirmed that bortezomib induces autophagy in cardiomyoblasts.Figure 2Bortezomib induces ER stress and autophagy in H9c2 cells.A: H9c2 cells were incubated for 24 hours with 10 nmol/L bortezomib and subjected to indirect immunofluorescence microscopy using anti-FK2 (multiubiquitin) antibody (red) and 4,6-diamidino-2-phenylindole staining (blue). Scale bars = 75 μm. B: H9c2 cells were incubated with 10 nmol/L bortezomib for indicated periods. Total cell lysates were prepared and Western blot analysis was performed using anti-BiP and anti-α tubulin antibodies. Cells incubated for 16 hours with 10 μg/ml tunicamycin were used as a positive control. Data are mean ± SE *P < 0.05 versus controls (two-tailed Student’s t-test). C: H9c2 cells were incubated for 48 hours with 10 nmol/L bortezomib, collected, and fixed as described in Materials and Methods, and observed by TEM. Thin red arrows indicate multilamellar and/or lysosomal/autophagosomal structures; thick red arrows point to widened endoplasmic reticulum. Scale bars = 200 nm. D: H9c2 cells were transiently transfected with pEGFP-LC3m plasmid encoding EGFP-LC3 fusion protein, incubated for 48 hours with 10 nmol/L bortezomib, fixed, stained with anti-GFP antibody and TO-PRO nuclear stain and observed under fluorescence microscopy. Scale bars = 75 μm. E: H9c2 cells were incubated with 10 nmol/L bortezomib for the indicated periods. Then total cell lysates were prepared and Western blot analysis was performed using anti-LC3B antibody against LC3B-I and LC3B-II fragments (upper panel). Densitometric analysis of LC3B-II to LC3B-I ratio in H9c2 cells (lower panel). Data are mean ± SE *P < 0.05 versus controls (two-tailed Student’s t-test).View Large Image Figure ViewerDownload Hi-res
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