ToxSci Advance Access originally published online on July 29, 2008
Toxicological Sciences 2008 106(1):153-161; doi:10.1093/toxsci/kfn157
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Effect of the Multitargeted Tyrosine Kinase Inhibitors Imatinib, Dasatinib, Sunitinib, and Sorafenib on Mitochondrial Function in Isolated Rat Heart Mitochondria and H9c2 Cells
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* Exploratory Safety Differentiation, Pfizer, Inc., Groton, Connecticut 06340
Drug Safety Research and Development, Pfizer, Inc., San Diego, California 92121
MitoSciences, Inc., Eugene, Oregon 97403
Luxcel Biosciences, Ltd., Lee Maltings, Cork, Ireland
¶ Clinical Development Pfizer, Inc., San Diego, California 92121
1 To whom correspondence should be addressed at Drug Safety Research and Development, Pfizer, Inc., 10646 Science Center Drive, San Diego, CA 92121. Fax: (858) 678-8290. E-mail: Bart.Jessen{at}pfizer.com.
Received June 3, 2008; accepted July 21, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotoxicity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phosphorylation or inhibition of electron transport chain components, as well as identifying targets within the mitochondrial electron transport chain, can be accomplished in vitro. Here, we examined the effects of the tyrosine kinase inhibitor drugs imatinib, dasatinib, sunitinib, and sorafenib on ATP content in H9c2 cells grown under conditions where cells are either glycolytically or aerobically poised. Furthermore, we measured respiratory capacity of isolated rat heart mitochondria in the presence of the four kinase inhibitors and examined their effect on each of the oxidative phosphorylation complexes. Of the four kinase inhibitors examined, only sorafenib directly impaired mitochondrial function at clinically relevant concentrations, potentially contributing to the cytotoxic effect of the drug. For the other three kinase inhibitors lacking direct mitochondrial effects, altered kinase and other signaling pathways, are a more reasonable explanation for potential toxicity.
Key Words: mitochondria; kinase; cytotoxicity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tyrosine kinases (TKs) are involved in regulating multiple cellular processes, including cellular proliferation. Over expression of TKs or TK receptors ligands, or genetic mutations or rearrangements that lead to constitutive activation of TKs is often associated with neoplastic progression. Specific TKs have been linked to certain cancers making TK inhibitors (TKis) attractive targeted therapies for the treatment of cancer (Krause and Van Etten, 2005
). Although these targeted approaches may lack the desired broad-spectrum antitumor activity, their potential for remarkable efficacy, particularly in tumors that display absolute dependence on single TKs, has led to the increasing approval and application of TKi drugs in recent years. In addition, targeted approaches offer the potential for reduced toxicity toward nontumor tissue relative to traditional chemotherapies that offer no selectivity between normal proliferating tissues and tumor cells.
Despite the potential for improved safety profile with TKis, recent concern over cardiac toxicity has arisen. Besides the vascular effects observed with inhibition of the vascular endothelial growth factor (VEGF) pathway (i.e., hypertension and hemorrhage), direct functional effects on the heart have been observed for several small molecule TKis. The labeling information for imatinib (a multitargeted TKi inhibiting abelson proto-oncogene (ABL), PDGFR-
[platelet-derived growth factor receptor], and stem cell growth factor receptor/CD117 (KIT) mentions the occurrence of severe congestive heart failure and left ventricular dysfunction in less than 1% of patients (Gleevec USPI, 2006 revision), whereas sorafenib (a multitargeted TKi inhibiting KIT, VEGF, PDGFR, and RAF [serine/threonine-specific kinase] kinase families) has been associated with cardiac ischemic/infarct events in as many as 2.9% of patients (Nexavar USPI, 2007, November). Effects on cardiac function have also been associated with sunitinib (a multitargeted TKi inhibiting KIT, fms-like tyrosine kinase, VEGFR, PDGFR, CSF, and glial cell-derived neurotrophic factor receptor kinase families) which has been associated with increases in patients with left ventricular ejection fractions below the lower limit of normal leading to congestive heart failure in some cases (Sutent USPI, 2007, February revision).
The proposed mechanisms of action for TKi-induced cardiac effects are varied. In the case of imatinib, evidence has been reported linking inhibition of one of the intended targets (ABL) to cardiac toxicity (Kerkelä et al., 2006). This mechanism would also extend to other Abl inhibitors such as dasantinib and nilotinib. The proposed molecular mechanisms of other TKis have been speculative and based predominantly on the potential pathways that may form a link between the published kinase affinity profiles for the compounds, both on and off-target, and cytotoxicity. One argument put forth is that cardiac tissue requires high-energy utilization and therefore is exquisitely sensitive to mitochondrial damage (Force et al., 2007). In a 2007 review by Force et al., hypotheses for drug-induced cardiomyocyte pathology are proposed for sunitinib, imatinib, and sorafenib, each of which involves either direct or indirect mitochondrial effects.
Although the potential mechanisms of TKi-related cardiac toxicity remain unclear, the hypothesis of direct mitochondrial toxicity warrants investigation. Other drugs, for example anthracyclines such as doxorubicin, have been shown to cause mitochondrial dysfunction and cardiotoxicity by selectively abstracting electrons from complex I of the electron transport chain (Wallace, 2007). In order address the potential for direct TKi-induced mitochondrial toxicity, we examined the effect of imatinib, dasatinib, sorafenib, and sunitinib on viability of H9c2 cells grown either in glucose or in galactose. Cells in glucose rely heavily on glycolysis for ATP generation, whereas cells grown in galactose generate ATP solely through utilization of the mitochondrial electron transport chain. In addition, we measured effects on respiration in isolated heart mitochondria, and identified possible mitochondrial targets.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Materials
All chemicals were purchased from Sigma-Aldrich (St Louis, MO) and Toronto Research Chemicals (Toronto, Canada) and were of the highest purity available. Phosphorescent oxygen-sensitive probe, type A65N-1, was from Luxcel Biosciences (Cork, Ireland). The BCA kit for protein determination was from Pierce (Rockford, IL). Black body clear bottom 96-well plates (Costar 3631) were purchased through VWR (Westchester, PA). Nunc Maxisorp clear bottom 96-well plates were purchased from Fisher (Waltham, MA). All monoclonal antibodies were from MitoSciences, Inc. (Eugene, OR). Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA), except for fetal bovine serum (FBS) which was purchased from Tissue Culture Biologics (Los Alamitos, CA). Cell culture flasks (Becton, Dickinson and Company 355001) and 96-well plates (Costar 3599) were purchased from VWR (Westchester, PA). CellTiter-Glo Luminescent Cell Viability Assay kits were purchased from Promega (Madison, WI).
Cell Culture Conditions for H9c2 Cells
High-glucose media.
High-glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen 11995-065) containing 25mM glucose and 1mM sodium pyruvate was supplemented with 5mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 10% FBS, and penicillin-streptomycin (100 U/ml final concentration).
Galactose media.
DMEM deprived of glucose (Invitrogen 11966-025) was supplemented with 10mM galactose, 2mM glutamine (plus 4mM prior to supplementation to yield a final concentration of 6mM), 5mM HEPES, 10% FBS, 1mM sodium pyruvate, and penicillin-streptomycin.
H9c2 cells (ATTC, Manassas, VA) were grown in either high-glucose or galactose-containing medium and kept in 5% CO2 at 37°C. Cells were maintained on 150-cm2 flasks and seeded onto 96-well plates for individual experiments.
Measurement of Cellular ATP Content in H9c2 Cells
Cells were plated at 220,000 cells/ml on clear 96-well plates. The final media volume was 100 µl. Cellular ATP concentrations were assessed by using the CellTiter-Glo Luminescent Cell Viability Assay as per manufacturer's instructions. For drug treatments, compound stock solutions were prepared in dimethyl sulfoxide (DMSO) and added to the wells to give the indicated final drug concentrations. Final DMSO concentration was 0.5%. Drugs were added 24 h before measurements. Data represent the mean ATP measurements of triplicate wells from two to three separate experiments for a total of six to nine wells.
Animals
Care and Maintenance were in accordance with the principles described in the Guide for Care and Use of laboratory Animals (NIH Publication 85-23, 1985). Male Sprague-Dawley Rats (150–180 g) were purchased from Charles River (Wilmington, MA). Animals were housed in pairs in a controlled environment with constant temperature (21 ± 2°C) and a 12-h light/dark cycle. Food and water were available ad libitum. Animals were euthanized with an overdose of carbon dioxide. Organs were rapidly excised and placed into ice-cold mitochondrial isolation buffers (see below).
Isolation of Heart Mitochondria
Heart mitochondria were isolated as previously described (Messer et al., 2004). Two rat hearts were freed of blood and connective tissue and placed in nine volumes of solution I (100mM KCl, 40mM Tris-HCl, 10mM Tris-base, 5mM MgCl2, 1mM EDTA [ethylenediaminetetraacetic acid], and 1mM ATP, pH 7.4). Tissue was finely minced using scissors with Type XXIV protease (Sigma-P8038) added at 5 mg/g wet tissue, and then incubated for 7 min with mixing and additional mincing. Adding an equal volume of solution I terminated the protease digestion. The mixture was then homogenized for 30 s with an Ultra-Turrax tissue homogenizer (IKA, T25) at 11,000 rpm (setting I). The homogenate was centrifuged at 4°C for 10 min at 700 x g; the supernatant was rapidly filtered through two layers of cheesecloth and recentrifuged at 14,000 x g for 10 min at 4°C. The resulting supernatant was discarded, and the mitochondrial pellet was resuspended in solution II (100mM KCl, 40mM Tris-HCl, 10mM Tris-base, 1mM MgSO4, 0.1mM EDTA, 0.2mM ATP, and 2% bovine serum albumin, pH 7.4) and centrifuged at 7000 x g for 10 min at 4°C. The supernatant was discarded and the mitochondria were subjected to washing with 20 ml of solution III (100mM KCl, 40mM Tris-HCl, 10mM Tris-base, 1mM MgSO4, 0.1mM EDTA, and 0.2mM ATP, pH 7.4) and centrifugation at 3500 x g. Finally, the mitochondrial pellet was resuspended in a minimal volume of solution IV (220mM mannitol, 70mM sucrose, 10mM Tris-HCl, and 1mM ethylene glycol tetra-acetic acid (EGTA), pH 7.4) for further use. Protein concentration was determined using the BCA kit according to the manufacturer's protocol. Respiration buffer consisted of 100mM KCl, 50mM MOPS, 10mM K2HPO4, 10mM MgCl2, and 1mM EGTA, pH 7.4. Mitochondrial quality was confirmed by measuring respiratory control ratios (RCR) (Hynes et al., 2006). The mean RCR was 6.56 ± 0.24 for three independent mitochondrial preparations.
Measurement of Mitochondrial Respiration Using an Oxygen-Sensitive Probe
Oxygen consumption in the isolated heart mitochondria was monitored in 96-well plate format using a phosphorescent oxygen-sensitive probe as previously described (Hynes et al., 2006; Nadanaciva et al., 2007b; Will et al., 2006) with minor modifications. Briefly, A65N-1 oxygen probe was reconstituted in 10.5 ml of mitochondrial incubation buffer to a concentration of approximately 100nM. One hundred microliters of this solution were pipetted into each well of a 96-well plate (10 pmol of probe per well). For drug treatments, compound stock solutions were prepared in DMSO and added to the wells to give the indicated final concentrations (the final DMSO content was not more than 0.5%). All drug concentrations are presented as nmol/mg of mitochondrial protein. After drug or vehicle addition, 50 µl of mitochondria stock solutions were added to each well giving the desired final concentration of mitochondria, followed by 50 µl of substrate (12.5/12.5mM glutamate/malate final concentration) without or with ADP (1.65mM final concentration) in incubation buffer. Finally, 100 µl of heavy mineral oil was added to each well to seal the samples from ambient oxygen, and the plate was placed in a Safire2 fluorescence plate reader (Tecan, Austria) equilibrated at 30°C and monitored over a period of 20 min measuring probe fluorescence signal in each well every 1.5 min in kinetic mode. Instrument settings were: 380/650-nm excitation/emission filters, a delay time of 30 µs, a measurement window of 100 µs, and active temperature control of the microplate compartment at 30°C. To ensure gas and temperature equilibration of samples at the start of the assay, all the dispensing steps were carried out at 30°C using prewarmed solutions and a Multi-Blok heater (Barnstead/LabLine, Melrose Park, IL) holding the microplate. Intensity measurements were linearized (Ogurtsov et al., 2008) and data were presented as percent of DMSO-treated mitochondria.
Measurement of Activities of Individual OXPHOS Complexes
Bovine heart mitochondria were isolated according to Smith (1967). Activities of Complex I (NADH-Ubiquinone oxidoreductase), Complex II + III (Succinate-cytochrome c oxidoreductase), Complex IV (Cytochrome c oxidase), and Complex V (F1Fo-ATPase) were all performed as previously described (Nadanaciva et al., 2007a, b).
For drug treatments, compound stock solutions were prepared in DMSO, added to multichannel Dilux Dilution Reservoirs (ISC BioExpress, Kaysville, UT) containing the appropriate assay solution and then dispensed into each 96 well plate in quadruplicate wells: in the Complex I, IV, and V activity assays, measurements for a compound at a given concentration were done in triplicate wells coated with the appropriate immunocapture monoclonal antibody and a single well containing a null capture antibody (negative control); in the Complex II + III activity assay, measurements for a compound at a given concentration were done in triplicate wells with bovine heart mitochondria and a single well with no mitochondria (negative control). The final DMSO concentration in all these activity assays was 1.5% (vol/vol). This concentration of DMSO did not have an inhibitory effect in any of the assays. Each assay was read in a SpectraMax Plus384 plate reader (MDS Sciex; Concord, Ontario, Canada) immediately after addition of the assay solution (containing the drugs) to the 96-well plates. Absorbance values obtained during all activity assays on the SpectraMax Plus384 plate reader were exported from SoftmaxPro to SigmaPlot (Systat Software, Inc., San Jose, CA). One hundred percent activity (i.e., no inhibition) for each complex was determined as the mean of the triplicate measurement in absence of compound—negative control value in absence of compound. IC50 values were generated using a four-parameter logistic equation.
Induction of Mitochondrial Permeability Transition
Mitochondrial swelling was measured spectrophotometrically using a plate reader Safire2 (Tecan, Austria) by monitoring the decrease in absorbance at 560 nm over 12 minutes in a manner similar to previously described methods (Berman et al., 2000, Zhou et al., 2001). Liver and heart mitochondria (500 µg/ml) were incubated in media containing 213mM mannitol, 70mM sucrose, 3mM Hepes (pH 7.4), 10mM succinate, 1µM rotenone, and 1 µg/ml oligomycin. CaCl2 (50 or 260µM for liver and heart, respectively) was added 30 s after addition of mitochondria and the indicated compounds were added after 2 min. When Cyclosporin A (1µM) was used, it was added prior to the addition of mitochondria.
Statistics
IC50 values and one-way ANOVA followed by Tukey's post hoc test for the mitochondrial respiration, ATP measurements, and the mitochondrial swelling were analyzed via GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of TKis on ATP Content in H9c2 Cells
The myoblastic cell line H9c2 was used as it is a well established in vitro model to examine cardiac related findings (L'Ecuyer et al., 2001
; Sardão et al., 2008). Cells were incubated for 24 h with the four TKis at concentrations up to 100µM in media containing either glucose (glycolytic conditions) or galactose (mitochondrial-dependent conditions). As previously reported, the system is capable of identifying drugs with primary mitochondrial liabilities (Marroquin et al., 2007). Briefly, cell lines grown in medium containing high concentrations of glucose generate ATP through glycolysis and are less reliant on oxidative phosphorylation, rendering them less sensitive to mitochondrial toxicants. In contrast, cells grown in galactose-containing medium generate ATP solely through oxidative phosphorylation as galactose yields no net ATP through the glycolytic pathway, and hence these cells are more sensitive to mitochondrial toxicants than glucose-grown cells (Marroquin et al., 2007). Under these conditions, sorafenib was significantly more toxic to galactose-grown cells than glucose-grown cells, demonstrating a greater than fourfold shift in the IC50 (10.1µM vs. 44.7µM respectively; Fig. 1D). In contrast, imatinib, dasatinib, and sunitinib were equally cytotoxic to both glucose and galactose-grown cells (Figs. 1A–C). The IC50 values for all four kinase inhibitors are reported in Table 1.
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The cell data suggested direct mitochondrial impairment to be a mechanism for the cytotoxicity observed with sorafenib. To investigate this hypothesis further, we examined the effect of all four TKis on respiration (oxygen consumption) in mitochondria isolated from rat hearts.
Effect of TKis on Respiration Measured in Isolated Rat Heart Mitochondria
Mitochondrial oxygen consumption measurements were performed in two different states: State 2, in which oxidizable substrate (glutamate/malate) was provided in the absence of exogenous ADP, and State 3, where both substrates and ADP were provided. Effects on mitochondrial function by the four kinase inhibitors were evaluated at 250 nmol/mg mitochondrial protein for imatinib, dasatinib, and sunitinib and 100 nmol/mg mitochondrial protein for sorafenib, in accord with sorafenib's higher potency in the cell-based ATP depletion assay. None of the four drugs had a statistically significant effect on basal respiration at the highest dose tested (Fig. 2A). In contrast, sorafenib inhibited ADP-stimulated respiration > 80% at 100 nmol/mg protein (Fig. 2B). None of the other three Tki's showed any effect at the highest concentration tested on ADP-stimulated respiration.
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Sorafenib was further evaluated over an 8-point dose range using twofold dilutions.
Figure 3 shows the dose-response relationship for sorafenib expressed in percent of DMSO control-treated heart mitochondria. Uncoupling was observed at concentrations above 1.56 nmol/mg heart mitochondria protein, reaching a maximum uncoupling (fourfold over controls) at 12.5 nmol/mg protein. At concentrations > 12.5 nmol/mg protein, maximum uncoupling decreased due to concurrent inhibition (Fig. 3A). Such inhibition was also revealed in ADP-stimulated state 3 respiration (Fig. 3B) in which only modest inhibition was observed at the lower 4 concentrations. Beyond the inflection point at 6.25 nmol/mg protein, inhibition was much more profound, resulting in an IC50 value of 
30 nmol/mg protein.
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Target Identification
The four TKis were tested for their inhibition of Complex I, II + III, IV, and V of the electron transport chain. Imatinib (Fig. 4) caused inhibition of Complex V with an IC50 value of 190µM and negligible inhibition of the other complexes, the IC50 values being greater than 300µM in each case. Dasatinib (Fig. 5) inhibited Complex IV and Complex V with IC50 values of 124 and 96µM, respectively and caused negligible inhibition of Complex I and Complex II+III (IC50 values > 300µM). Sunitinib (Fig. 6) inhibited Complex V with an IC50 of 124µM and caused slight inhibition of the other complexes, the IC50 values exceeding 200µM in each case. Sorafenib (Fig. 7) was the most potent of the four TKis, inhibiting Complex V with an IC50 of 5.1µM. It also potently inhibited Complex II+III (IC50, 3µM) and Complex I and Complex IV to a lesser degree (IC50s of 31 and > 75µM, respectively).
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Effect of TKis on the Mitochondrial Permeability Transition Pore
The four TKis were tested for their potential to enhance calcium-induced opening of the mitochondrial permeability transition pore (MPTP). This was achieved by monitoring swelling of mitochondria in the presence of calcium and the TKis. Cyclosporin A was used as a classical inhibitor that blocks opening of the MPTP. In the presence of 50µM calcium, both phosphate as well as troglitazone statistically enhanced opening of the MPTP in liver mitochondria, and this opening could be inhibited by cyclosporin A (Figs. 8A and 8B). None of the four TKis statistically enhanced calcium-induced opening of the MPTP in liver mitochondria (Figs. 8A and 8B). The same experiment was also performed with isolated heart mitochondria (Fig. 8C), where both phosphate as well as troglitazone enhanced calcium-induced MPTP opening whereas none of the TKis were able to do so. Figure 8C represents the same data as Figure 8D with an enlarged scale for better demonstration purposes. Statistical analysis was not performed on the isolated heart mitochondria due to the small magnitude of changes in absorbance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent reports indicate possible mechanistic or pharmacological evidence for cardiac effects observed with the multitargeted TKis, imatinib, and sunitinib. In the imatinib example, evidence of mitochondrial abnormalities and sarcoplasmic reticular membrane swirls were observed in human biopsies and in treated mice (Kerkelä et al., 2006
). In vitro experiments with imatinib demonstrated activation of ER stress, loss of mitochondrial membrane potential, cytochrome c release, ATP depletion and cell death. Furthermore, these effects were linked to inhibition of Abl kinase in vitro. Association of Abl with cardiac toxicity would implicate all Abl inhibitors (dasatinib and nilotinib, in addition to imatinib) as raising the risk for cardiac dysfunction. In the sunitinib case, mitochondrial damage was observed in human biopsies and in mice treated with sunitinib, and cardiomyocyte apoptosis was demonstrated in vitro (Chu et al., 2007).
Based on the findings of these two examples and the available kinase profiling of several drugs (Fabian et al., 2005), some of the coauthors from the sunitinib and imatinib reports proposed potential molecular mechanisms for the cardiac toxicity of several TKi drugs, including sunitinib, imatinib, and sorafenib (Force, 2007). In the case of sorafenib, it has been suggested that inhibition of the intended target RAF1 could interfere with cardiac survival and apoptotic pathways through interaction with MEK and possible interactions with ASK1 and MST2, leading to BAX-mediated mitochondrial cytochrome c release and cell death. For the sunitinib example, off-target inhibition of ribosomal S6 kinase (RSK) and AMP-activated protein kinase (AMPK) (Fabian et al., 2005) forms the basis for the hypothesis of sunitinib cardiac toxicity. Inhibition of AMPK is thought to lead to ATP depletion, whereas RSK inhibition could lead to activation of bcl-2-associated death promoter-induced mitochondrial cytochrome c release. In addition, the authors suggested a possible direct interaction of sunitinib with the mitochondria that may lead to cell death. Finally, as mentioned above, it is the inhibition of Abl by imatinib that is suggested to initiate ER stress leading pathway, leading to BAX or PKC
-mediated mitochondrial cytochrome c release.
Because mitochondrial dysfunction was implicated in the toxic mechanisms of several TKi drugs, and direct mitochondrial toxicity was suggested for sunitinib, we investigated the hypothesis that direct inhibitory effects on mitochondrial function are involved in the mechanism of cytotoxicity of the TKi drugs sunitinib, imatinib, sorafenib, and dasatinib. We initially tested the four drugs for their effect on ATP depletion in H9c2 cells. H9c2 is a myoblast cell line derived from embryonic rat heart myocardium and is a model for cardiac and skeletal muscle. It should be emphasized that this model is designed to test the contribution of direct mitochondrial functional impairment to cytotoxicity. As has been described previously (Marroquin et al., 2007), a difference in the toxicity between the glucose and galactose conditions indicates a direct effect on mitochondrial function, whereas the actual IC50 value in each condition is related to the individual compound's toxic mechanism and is not relevant to interpretation of the assay. We found that all four drugs depleted ATP, both in glucose-grown cells and galactose-grown cells, at concentrations above 10µM (Table 1) but only sorafenib rendered the galactose-grown cells to be more sensitive to ATP depletion than glucose-grown cells. This suggested that sorafenib impaired mitochondrial function in H9c2 cells. In contrast, imatinib, dasatinib and sunitinib, each depleted ATP to the same extent in both glucose- and galactose-grown cells, suggesting that direct mitochondrial toxicity was not the primary event in their cytotoxic pathway.
Oxygen consumption (respiration) measurements with rat heart mitochondria in the presence of the substrates, glutamate/malate, and ADP showed that sorafenib was the most potent inhibitor of the four drugs. Sorafenib acted as both an uncoupler and inhibitor in State 2 respiration. The uncoupling effects are most apparent at lower concentrations in State 2, whereas at higher concentrations the inhibitory effects become more apparent (Fig. 3). Such a pattern of uncoupling and inhibition at different concentrations is reminiscent of the classical uncoupler FCCP (Hynes et al., 2006). Sorafenib also inhibited State 3 respiration with an IC50 of 30 nmol/mg protein. Analysis of sorafenib's effects on the oxidative phosphorylation complexes showed that it inhibited Complex V and Complexes II + III with IC50 values less than 10µM (Fig. 7). The other TKis (imatinib, dasatinib, and sunitinib; Figs. 4–6
, respectively) inhibited the oxidative phosphorylation complexes only at concentrations well above clinical Cmax values (Table 2), correlating well with their lack of effects on respiration (Fig. 2). Our findings also suggest that sorafenib as well as the other TKis do not enhance calcium-induced opening of the MPTP in liver or heart mitochondria demonstrated by the lack of measurable mitochondrial swelling (Fig. 8).
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The findings here do not necessarily conflict with previous reports of imatinib and sunitinib cardiomyocyte toxicity or the proposed molecular mechanism involving Abl. Evidence that mitochondrial impairment is not the primary contributing factor in cytotoxicity is not inconsistent with the hypothesis that ER stress leads to downstream events that involve mitochondrial-induced cell death. Mitochondria are a sensitive index of a host of cytotoxic stressors and are typically disrupted during necrosis or apoptosis regardless of cause. Evidence presented here would tend to argue against the suggestion that a direct interaction of sunitinib with mitochondria plays a major role in its potential cardiac toxicity. Given this finding, the roles of RSK and AMPK inhibition, as well as other cellular mechanisms, are worth further investigation. Finally, although the suggested role of RAF as a contributing factor in sorafenib-induced cardiomyocte toxicity cannot be ruled out, the evidence presented here suggests the more likely mechanism involving direct effects on the mitochondria.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Berman SB, Watkins SC, Hastings TG. Quantitative biochemical and ultrastructural comparison of mitochondrial permeability transition in isolated brain and liver mitochondria: Evidence for reduced sensitivity of brain mitochondria. Exp. Neurol. (2000) 164:415–425.[CrossRef][Web of Science][Medline]
Chu TF, Rupnick MA, Kerkela R, Dallabrida SM, Zurakowski D, Nguyen L, Woulfe K, Pravda E, Cassiola F, Desai J, et al. Cardiotoxicity associated with tyrosine kinase inhibitor sunitinib. Lancet (2007) 370:2011–2019.[CrossRef][Web of Science][Medline]
Dasatinib (BMS-354825). ODAC briefing document, NDA 21-986 02-June-2006 Applicant. (2006) Bristol Myers Squibb Company, Wallingford, CT.
Fabian MA, Bigg WH 3rd, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M, et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. (2005) 23:329–336.[CrossRef][Web of Science][Medline]
Force T, Krause DS, Van Etten RA. Molecular mechanisms of cardiotoxicity of tyrosine kinase inhibition. Nat. Rev. Cancer. (2007) 7:332–344.[CrossRef][Web of Science][Medline]
Gleevec USPI. (2006) November revision. www.gleevec.com/hcp/index.jsp.
Hynes J, Marroquin LD, Ogurtsov VI, Christiansen KN, Stevens GJ, Papkovsky DB, Will Y. Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes. Toxicol. Sci. (2006) 92:186–200.
Kerkelä R, Grazette L, Yacobi R, Iliescu C, Patten R, Beahm C, Walters B, Shevtsov S, Pesant S, Clubb FJ, et al. Cardiotoxicity of the cancer therapeutic agent imatinib mesylate. Nat. Med. (2006) 12:908–916.[CrossRef][Web of Science][Medline]
Krause DS, Van Etten RA. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. (2005) 353:172–187.
L'Ecuyer T, Horenstein MS, Thomas R, Vander Heide R. Anthracycline-induced cardiac injury using a cardiac cell line: Potential for gene therapy studies. Mol. Genet. Metab. (2001) 74:370–379.[CrossRef][Web of Science][Medline]
Marroquin LD, Hynes J, Dykens JA, Jamieson JD, Will Y. Circumventing the Crabtree effect: Replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. Toxicol. Sci. (2007) 97:539–547.
Messer JI, Jackman MR, Willis WT. Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria. Am. J. Physiol. Cell Physiol. (2004) 286:C565–C572.
Nadanaciva S, Bernal A, Aggeler R, Capaldi R, Will Y. Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays. Toxicol. In Vitro (2007a) 21:902–911.[CrossRef][Web of Science][Medline]
Nadanaciva S, Dykens JA, Bernal A, Capaldi RA, Will Y. Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration. Toxicol. Appl. Pharmacol. (2007b) 223:277–287.[CrossRef][Web of Science][Medline]
Nexavar USPI. (2007) November revision. www.nexavar.com/hcp_REACH.
Nikolova Z, Peng B, Hubert M, Sieberling M, Keller U, Ho YY, Schran H, Capdeville R. Bioequivalence, safety, and tolerability of imatinib tablets compared with capsules. Cancer Chemother. Pharmacol. (2004) 53:433–438.[CrossRef][Web of Science][Medline]
Ogurtsov VI, Hynes J, Will Y, Papkovsky DB. Data analysis algorithm for high throughput enzymatic oxygen consumption assays based on quenched-fluorescence detection. Sensors Actuators B (2008) 129:581–590.[CrossRef]
Sardão VA, Oliveira PJ, Holy J, Oliveira CR, Wallace KB. Morphological alterations induced by doxorubicin on H9c2 myoblasts: Nuclear, mitochondrial, and cytoskeletal targets. Cell Biol. Toxicol. (2008).
Smith AL. Preparation, properties, and conditions for assay of mitochondria: Slaughterhouse material, small-scale. Methods Enzymol. (1967) 10:81–86.[CrossRef]
Sutent USPI. (2007) February. www.pfizer.com/files/products/uspi_sutent.pdf.
Strumberg D, Clark JW, Awada A, Moore MJ, Richly H, Hendlisz A, Hirte HW, Eder JP, Lenz HJ, Schwartz B. Safety, pharmacokinetics, and preliminary antitumor activity of sorafenib: A review of four phase I trials in patients with advanced refractory solid tumors. Oncologist (2007) 12:426–437.
Wallace KB. Adriamycin-induced interference with cardiac mitochondrial calcium homeostasis. Cardiovasc. Toxicol. (2007) 7:101–107.[CrossRef][Web of Science][Medline]
Will Y, Hynes J, Ogurtsov VI, Papkovsky DB. Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat. Protoc. (2006) 1:2563–2572.[CrossRef][Medline]
Zhou S, Starkov A, Froberg M, Leino R, Wallace K. Cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin. Cancer Res. (2001) 61:771–777.
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