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TVP1022 and Propargylamine Protect Neonatal Rat Ventricular Myocytes Against Doxorubicin-Induced and Serum Starvation-Induced Cardiotoxicity

Kleiner, Yana MSc*†; Bar-Am, Orit MSc; Amit, Tamar PhD; Berdichevski, Alexandra BSc*†; Liani, Esti PhD*†; Maor, Gila PhD; Reiter, Irina MSc*†; Youdim, Moussa B H PhD; Binah, Ofer PhD*†

Journal of Cardiovascular Pharmacology: September 2008 - Volume 52 - Issue 3 - p 268-277
doi: 10.1097/FJC.0b013e3181862441
Original Article
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We recently reported that propargylamine derivatives such as rasagiline (Azilect) and its S-isomer TVP1022 are neuroprotective. The aim of this study was to test the hypothesis that the neuroprotective agents TVP1022 and propargylamine (the active moiety of propargylamine derivatives) are also cardioprotective. We specifically investigated the protective efficacy of TVP1022 and propargylamine in neonatal rat ventricular myocytes (NRVM) against apoptosis induced by the anthracycline chemotherapeutic agent doxorubicin and by serum starvation. We demonstrated that pretreatment of NRVM cultures with TVP1022 or propargylamine attenuated doxorubicin-induced and serum starvation-induced apoptosis, inhibited the increase in cleaved caspase 3 levels, and reversed the decline in Bcl-2/Bax ratio. These cytoprotective effects were shown to reside in the propargylamine moiety. Finally, we showed that TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor interfered with the anti-cancer efficacy of doxorubicin. These results suggest that TVP1022 should be considered as a novel cardioprotective agent against ischemic insults and against anthracycline cardiotoxicity and can be coadministered with doxorubicin in the treatment of human malignancies.

From the *Department of Physiology, Rappaport Family Institute for Research in the Medical Sciences, and ‡Department of Anatomy and Cell Biology, the Ruth & Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

Received for publication December 13, 2007; June 12, 2008.

This work was supported by the Rappaport Family Institute for Research in the Medical Sciences, the Horowitz Foundation-Technion, and by the Alfred Mann Institute at the Technion (AMIT). TVP1022 and propargylamine were kindly donated by Teva Pharmaceutical, Israel Ltd.

The authors state that they have no financial interest in the products mentioned within this article.

Reprints: Ofer Binah, PhD, Rappaport Institute, P.O.Box 9697, Haifa 31096, Israel (e-mail: binah@tx.technion.ac.il).

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INTRODUCTION

Cardiac death is the leading cause of morbidity and mortality worldwide. For example, some 6 million people suffer from cardiac diseases in the United States alone, and there are a half a million deaths annually. Apoptosis has been implicated in a wide variety of physiological and pathological processes. Although myocardial infarction (MI) was long considered to be characterized by non-apoptotic (necrotic) cell death due to the breakdown of cellular energy metabolism, there is growing evidence that myocytes loss during the acute stage of MI involves both apoptotic and non-apoptotic cell death. Different studies have shown a reduction in the area of damage and improved clinical outcome in animals treated with caspase inhibitors, compared to untreated animals, during and after MI.1,2 Others have shown a correlation between the amount of apoptosis and the clinical severity of heart failure caused by ischemic attacks and the amount of apoptosis found adjacent to the scars.3 Altogether, these results support the major role of apoptosis in the pathogenesis of MI and thus suggest that inhibition of apoptosis can be a potential therapeutic tool for various forms of cardiovascular diseases.

Rasagiline (N-propargyl-1R-aminoindan) is a novel, selective, and highly potent irreversible monoamine oxidase (MAO) B inhibitor that has been approved for treating Parkinsonian patients, whereas its S-isomer, TVP1022, is more than a thousand times less potent as a MAO-B inhibitor. Both drugs have effective neuroprotective and anti-apoptotic efficacies in neuronal cell cultures in response to various neurotoxins as well as i268-277n vivo (e.g. global ischemia, neurotrauma, head injury, and anoxia). In this regard, numerous studies have shown that cardiomyocytes and neurons are similar in that they share comparable mitochondrial-dependent and -independent mechanisms for death and survival.4-11 Indeed, the mechanisms underlying cell death (in neurons and cardiomyocytes) in a number of diseases, including Parkinson, Alzheimer, and cardiovascular diseases such as ischemia and infarction, are similar. Therefore, due to the similarity between neurons and cardiomyocytes respecting the mechanism underlying cell damage and death, the present study tested the hypothesis that the neuroprotective/cytoprotective agents TVP1022 and propargylamine (the active moiety of propargylamine derivatives) are also cardioprotective. To test the abovementioned hypothesis, the protective efficacies of TVP1022 and propargylamine were investigated in 2 common in vitro models of myocardial damage in neonatal rat ventricular myocytes (NRVM): doxorubicin and serum starvation-induced apoptosis. In brief, doxorubicin or adriamycin is a quinine-containing anthracycline and is among the most widely prescribed and effective chemotherapeutic agents used in oncology.12 However, the utility of doxorubicin is limited by cumulative, dose-related, potentially fatal, progressive and often irreversible cardiac toxicity that may lead to congestive heart failure.13 As for serum starvation, this experimental protocol is used as an in vitro model to simulate nutrient deprivation - a key component of ischemia.14-16 Moreover, since TVP1022 is being developed as a potential cardioprotective drug for anthracycline-treated cancer patients, we determined whether TVP1022 causes proliferation of human cancer cells and/or interferes with the anticancer efficacy of doxorubicin. In support of the hypothesis, this study demonstrates that both TVP1022 and propargylamine inhibit NRVM apoptasis induced by doxorubicin and serum starvation.

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MATERIALS AND METHODS

Preparation of Cell Cultures

Neonatal Rat Ventricular Myocytes

NRVM cultures were prepared from ventricles of 1- to 2-day-old Sprague-Dawley rats.17 Briefly, the ventricles of the excised hearts were dissociated with 0.1% RDB (Israel Institute for Biological Research, Ness-Ziona, Israel). The dispersed cells were resuspended in F-10 culture medium containing 1 mM CaCl2, 100 U/mL penicillin-streptomycin, 5% fetal calf serum (FCS), 5% donor horse serum, and 25 mg of BrdU. The cells were preplated for 1 hour to reduce fibroblasts content, and the cell suspension was diluted to a final desired concentration. Cells were seeded in 2-well Permanox Slide (12.5 × 104 cells/cm2) or in 6-well plates (16 × 104 cells/cm2) precoated with collagen type I from calf skin (C-8919; Sigma Chemical, St. Louis, MO), diluted 1:10 in 0.1 M acetic acid. Thereafter, the cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.

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Human Cancer Cell Lines

The present study used the human cancer cell lines cervical carcinoma HeLa T47D and breast carcinoma MDA-231. The cell lines were cultured in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% FCS, 100 U/mL penicillin-streptomycin, and 1% L-Glutamine and were incubated at 37°C in a humidified atmosphere containing 5% CO2.

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Experimental Protocols

Doxorubicin-induced Cardiotoxicity

At day 4 to 6 after plating NRVM, the regular culture medium was replaced with a culture medium containing 0.5% serum (0.25% FCS, 0.25% donor horse serum) with or without drugs for 24 hours. Thereafter, doxorubicin was added to a final concentration of 0.5 μM for 24 hours.

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Serum Starvation-induced Cardiotoxicity

To induce apoptosis, NRVM were incubated in serum-free medium (0% serum) for 24 hours. Drugs (TVP1022 or propargylamine, 1 μM) were added to the culture medium 24 hours before inducing serum starvation and were present throughout the apoptosis-inducing protocol.

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DAPI (4′,6-Diamidino-2-phenylindole) Staining

To measure the percent of apoptotic myocytes, NRVM cultures were counterstained with DAPI to visualize the nuclear morphology. It is noteworthy that this assay has certain limitations because it cannot distinguish apoptosis that could occur during the processing of the slides. Briefly, cultures were fixed for 20 minutes with 4% paraformaldehyde solution, permeabilized by 5 minutes incubation with Triton X-100 (0.1% in 0.1% sodium citrate), and washed 3 times with phosphate-buffered saline (PBS, pH 7.4). Thereafter, a drop of the mounting solution containing DAPI was added to each slide. The slides were visualized using an upright fluorescence microscope. In every DAPI-stained slide, 200-300 cells were examined in 4 to 6 fields counted. Altogether, 4 slides were viewed in at least 3 experiments from each group. Myocytes were scored as apoptotic only if they exhibited unequivocal nuclear chromatin condensation and fragmentation. The apoptotic rate was expressed as percentage of total counted nuclei.

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Western Blot Analysis

Lysates were prepared from NRVM cultures using RIPA (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% Sodium deoxycholate, 5 mM EDTA, 1% Phosphatase inhibitor) containing cocktail protease inhibitor (Roche Diagnostics, Mannheim, Germany), and the protein concentration was determined by the Bradford assay. A 20- to 25-μg sample of total cellular protein was loaded on 12% SDS-PAGE, followed by blotting into polyvinylidene difluoride membranes (Millipore, Watford, UK), which were stained by Ponceau concentrate to verify equal loading of protein. Membranes were blocked with 5% dry milk in DDW and 0.05% Tween 20 in TBS for 1 hour. Primary antibodies were diluted in TBS containing 0.05% Tween 20 and incubated with membranes for 24 hours at 4°C followed by incubation (1 hour at room temperature) in dilutions of horseradish peroxidase-conjugated secondary antibodies in the same buffer. After antibody incubations, membranes were washed in 0.05% Tween 20 in TBS. Detection was performed using the Western blotting detection reagent ECL (GE Healthcare, Little Chalfort Buckinghamshire, UK). Quantification of the results was accomplished by measuring the optical density of the labeled bands from the autoradiograms using the computerized imaging program Bio-1D (Vilber Lourmat Biotech. Bioprof, France). The values were normalized to β-actin intensity levels.

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Proliferation Assay of Human Cancer Cell Lines

HeLa and MDA-231 cells (150,000 cells/mL) were plated in 24-well plates and allowed to attach for 24 hours before treatment. [3H]Thymidine (1 μCi/mL; GE Healthcare, Little Chalfort Buckinghamshire, UK) was added for a 3-hour incubation period. Thereafter, the plates were placed on ice, quickly washed twice with ice-cold PBS, incubated twice for 5 minutes with methanol, incubated three times for 5 minutes with 10% trichloroacetic acid (TCA), and finally washed again in PBS. Precipitates were solubilized for 15 minutes in 200 μL of 0.3 M NaOH and neutralized. The radioactivity of the samples was measured in a Wallac 1409 liquid scintillation counter (Turku, Finland) and expressed as CPM/μg protein.

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Immunofluorescence Staining

Cultures were fixed for 10 minutes with 4% paraformaldehyde solution, permeabilized by 5 minutes incubation with Triton X-100 (0.1% in 0.1% sodium citrate), and washed 3 times with PBS. Subsequently the reaction was blocked in 10% Donkey serum in PBS for 1 hour at 37°C and incubated overnight at 4°C with either mouse-anti cytochrome C (1:200) or rabbit anti-cleaved caspase 3 (1:75), followed by incubation with cy-2 anti-mouse (1:150) or anti-rabbit (1:50) secondary antibody (donkey anti-mouse and donkey anti-rabbit IgG, respectively) for 1 hour at 37°C. Thereafter, a drop of the mounting solution containing DAPI was added to all slides, which were visualized using a Nikon fluorescence microscope TE 2000-S (Nikon, Tokyo, Japan).

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Drugs and Reagents

TVP1022 and propargylamine were kindly donated by Teva Pharmaceutical, Israel Ltd. Lab-Tek Chamber Slide system and culture plates were purchased from Nalge Nunc International (Rochester, NY), electrophoresis reagents from Invitrogen Corporation, (Carlsbad, CA), cell culture reagents from Biological Industries (Beth-Haemek, Israel), mounting medium for fluorescence with DAPI from Vector Laboratories Inc. (Burlingame, CA), antibodies against caspase 3 and Bax from Cell Signaling (Beverly, MA), Bcl-2 antibodies from BD, Biosciences Transduction Laboratories (Heidelberg, Germany), mouse anti-cytochrome C from Promega (Madison, WI), cy-2 anti-mouse and cy-2 anti-rabbit from Jackson Immunoresearch Laboratories (West Grove, PA), and β-actin antibodies and all other reagents were from Sigma (St. Louis, MO).

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Statistical Analyses

All data were from 3 separate experiments and were expressed as mean ± SEM. Comparisons among groups were performed using 1-way ANOVA followed by Tukey post hoc test, using Prism v.5.00 for Windows, GraphPad Software Inc. A level of P < 0.05 was accepted as statistically significant.

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RESULTS

Doxorubicin Induces Apoptosis in NRVM

Two experimental considerations are relevant to the doxorubicin study, the first being the serum content of the culture medium. Although NRVM cultures were regularly grown at 10% serum, the doxorubicin experiments were performed in a culture medium containing 0.5% serum. Our previous experience and preliminary studies have shown that full serum (10%) confers protection to cardiomyocytes against doxorubicin-induced cardiotoxicity. In addition, the protocol we used is in agreement with previous reports in which doxorubicin cardiotoxicity was investigated under serum starvation conditions (in the absence of FCS)18-19 or in a medium containing 0.5% or 1% serum.18,21 However, the complete absence of serum induces apoptosis as was demonstrated in the present work; therefore, the doxorubicin experiments were performed in the presence of 0.5% serum, which does not confer protection against doxorubicin, but it is still a sufficient amount of serum not to induce apoptosis. As seen in Figure 1A, apoptosis (expressed as the ratio between the numbers of apoptotic nuclei to total counted nuclei) in the presence of medium containing 10% serum (FS) and 0.5% serum (Control group for the doxorubicin experiments) were not significantly different.

FIGURE 1

FIGURE 1

The second consideration is the doxorubicin concentration. In the present work, doxorubicin at 0.5 μM was chosen on the basis of our preliminary experiments (data not shown) and on previous studies using a concentration range of 0.1 to 1 μM.18-25 The concentration used here (0.5 μM) is above the threshold for doxorubicin-induced apoptosis (0.01 to 0.1 μM25), and is sufficient to cause measurable apoptosis (see below), which enabled us to study the protective efficacies of the tested drugs.

After establishing the appropriate experimental conditions, we characterized the apoptotic effects of doxorubicin in NRVM by (1) inspecting nuclear morphology for measuring the percent of apoptotic myocytes, (2) immunostaining for cleaved caspase 3 and cytochrome C, and (3) determining the expression levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic proteins cleaved caspase 3 and Bax.6 In agreement with previous reports,18-19,25,27-28 incubation of NRVM with doxorubicin (0.5 μM) for 24 hours was associated with apoptosis, indicated by massive nuclear fragmentation (Figure 1, B and C). In summary, doxorubicin caused prominent apoptosis of NRVM, expressed either as the apoptotic ratio (Figure 1A) or as fold of control (P < 0.001) (Figure 2A). To support the DAPI measurements, we used fluorescent antibodies for cleaved (activated) caspase 3 and cytochrome C, which are known to actively participate in the apoptotic cascade. Specifically, during the apoptotic process, cytochrome C and other apoptotic proteins such as Smac/DIABLO, Omi-HTRA, AIF, ARTS29-31 are released from mitochondria to the cytosol, where they activate executioner caspases, mainly caspase 3. Hence, as seen in Figures 1D and 1E, in addition to the presence of typical apoptotic myocytes characterized by fragmented and condensed nuclei (Figure 1C, stained in blue with DAPI), the apoptotic myocytes were stained positively (in green) for cleaved (activated) caspase 3 and cytochrome C. Altogether, similar observations were monitored in at least 3 NRVM cultures from different experiments. In agreement with the immunofluorescence data, doxorubicin markedly increased cleaved caspase 3 protein expression (Figure 2B). Further, doxorubicin diminished the ratio Bcl-2/Bax by decreasing Bcl-2 protein expression while not affecting Bax protein expression (Figure 3).

FIGURE 2

FIGURE 2

FIGURE 3

FIGURE 3

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TVP1022 and Propargylamine Protect NRVM Against Doxorubicin-induced Apoptosis

To determine the ability of TVP1022 to attenuate doxorubicin-induced apoptosis, NRVM were treated with the neuroprotective concentration of the drug (1 μM)32 for 24 hours before adding doxorubicin. Since TVP1022 is being developed as a cardioprotective drug to be administered to cancer patients before doxorubicin treatment; NRVM cultures were pretreated with TVP1022 before the exposure to doxorubicin. As depicted in Figure 2A, TVP1022 attenuated (P < 0.05) doxorubicin-induced apoptosis. Accordingly, TVP1022 inhibited doxorubicin-induced increase in cleaved caspase 3 (P < 0.05) (Figure 2B) and prevented the reduction in doxorubicin-induced Bcl-2/Bax ratio (P < 0.05) (Figure 3C), which resulted from preventing the decrease in Bcl-2 levels (P < 0.05) (Figure 3A). Bax expression was not changed by doxorubicin alone or by the combination of TVP1022 + doxorubicin or TVP1022 + propargylamine (Figure 3B).

To determine the importance of the propargyl moiety in the cardioprotective efficacy of TVP1022, we investigated the ability of propargylamine to attenuate doxorubicin-induced apoptosis. As depicted in Figure 2A, propargylamine reduced doxorubicin-induced apoptosis (P < 0.05) and attenuated doxorubicin-induced increase in cleaved caspase 3 level (P < 0.05) (Figure 2B). Accordingly, similar to TVP1022, propargylamine prevented doxorubicin-induced decrease in Bcl-2/Bax ratio (P < 0.05) (Figure 3C), caused by increasing Bcl-2 expression (P < 0.05) (Figure 3A). Like TVP1022, propargylamine did not affect Bax expression. Finally, neither TVP1022 nor propargylamine caused apoptosis on their own (Figure 2A).

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TVP1022 and Propargylamine Protect NRVM Against Serum Starvation-induced Apoptosis

To determine whether TVP1022 can attenuate apoptosis induced by an ischemic insult, NRVM were grown in serum-free medium in the absence or presence of TVP1022. As control, NRVM were grown in full serum. The pro-apoptotic effect of serum starvation is reflected by the increased apoptosis level determined by DAPI; ~ 7 folds (P < 0.001) in serum-free compared to full serum (Figure 4A). The apoptotic ratio of 8.5 ± 0.6% (Figure 1A) caused by serum starvation in NRVM, is in agreement with the 11.6% (range, 1.6 to 26%) apoptosis observed in postmortem human tissues collected within 10 days after acute MI.19 Additionally, Abbate et al showed that the apoptotic ratio in postinfarcted left ventricular tissue ranged from 0.05% to 8.9%,20 which is well in the range of reported amount of apoptosis detected in ischemic heart tissue. In accordance with the DAPI measurements, in NRVM grown in serum-free medium for 24 hours, cleaved caspase 3 levels were markedly increased compared to FS (Figure 4B). Additionally, in serum free cultures, the level of Bcl-2 was also reduced (Figure 5A), whereas the level of the pro-apoptotic protein Bax was unchanged (Figure 5B), collectively resulting in a reduction of the Bcl-2/Bax ratio (Figure 5C). In agreement with the protective efficacy of TVP1022 against doxorubicin-induced apoptosis, TVP1022 also attenuated the apoptosis caused by serum starvation (Figure 4A). Accordingly, TVP1022 diminished the serum starvation-induced increase in cleaved caspase 3 (Figure 4B), it attenuated the decrease in Bcl-2 (Figure 5A) and decreased Bax expression below the control level (Figure 5B), collectively resulting in preventing the decrease in Bcl-2/Bax ratio (Figure 5C). Finally, in accordance with its protective efficacy against doxorubicin-induced apoptosis, propargylamine also attenuated (although to a lesser extent than TVP1022) apoptosis induced by serum starvation (Figure 4A). This anti-apoptotic effect was associated with attenuation of the increase in cleaved caspase 3, while serum starvation-induced reduction in the Bcl-2/Bax ratio was not significantly affected (Figures 4 and 5).

FIGURE 4

FIGURE 4

FIGURE 5

FIGURE 5

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TVP1022 Neither Causes Proliferation of Human Cancer Cells nor Interferes With the Anti-cancer Efficacy of Doxorubicin

Since TVP1022 is being developed as a cardioprotective drug for anthracycline-treated cancer patients, we investigated whether TVP1022 causes proliferation of the human cancer cell lines HeLa and MDA-231 or interferes with the anti-cancer activity of doxorubicin. In these experiments, the cancer cell lines were treated with doxorubicin and/or TVP1022 (as described below), and cell proliferation was determined by means of the [3H]Thymidine incorporation assay. As shown in Figure 6, TVP1022 at 0.01, 0.1, and 1 μM did not cause proliferation of HeLa and MDA-231. Further, as expected, the proliferation of both cancer cell lines was markedly (P < 0.01) reduced by doxorubicin, demonstrating its anti-cancer efficacy. Importantly, TVP1022 (at 0.01, 0.1, and 1.0 μM) did not interfere with the anti-cancer effect of doxorubicin (Figure 6), suggesting that TVP1022 may be safe for clinical use.

FIGURE 6

FIGURE 6

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DISCUSSION

In the present study we investigated in NRVM the cardioprotective efficacy of TVP1022 and the active moiety propargylamine against apoptosis induced by doxorubicin and serum starvation. The major findings are (1) TVP1022 and propargylamine inhibited apoptosis by attenuating the increase in cleaved caspase 3 and preventing the decrease in Bcl-2/Bax ratio (only TVP1022), and (2) TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor interfered with the anti-cancer effect of doxorubicin against these cancer cells.

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Doxorubicin-induced Cardiotoxicity

Doxorubicin is a quinine-containing anthracycline and is of the most widely prescribed and effective chemotherapeutic agent used in oncology. All anthracyclines contain a common quinone moiety, readily participating in oxidation-reduction reactions that ultimately generate highly reactive oxygen species thought to be responsible for anthracycline-induced cardiomyopathy.38 Doxorubicin is indicated in a wide range of human malignancies, including tumors of the bladder, stomach, ovary, lung, and thyroid.39 It is one of the most active agents available for the treatment of breast cancer and other indications, including acute lymphoblastic and myelogenous leukemias, Hodgkin's and non-Hodgkin's lymphomas, Ewing's and osteogenic bone tumors, soft tissue sarcomas, and pediatric cancers such as neuroblastoma and Wilms' tumors.12 However, the utility of doxorubicin is limited by cumulative, dose-related, potentially fatal, progressive and often irreversible cardiac toxicity that may lead to congestive heart failure.21 Anthracycline cardiotoxicity may be either acute or chronic. Acute effects include electrocardiographic changes, such as sinus tachycardia, ectopic contractions, T-wave changes, decreased QRS voltage, prolonged Q-T intervals, and heart block. These acute toxicities are generally reversible and clinically insignificant, and they do not predict future cumulative drug-related cardiac complications. In contrast, chronic anthracycline-induced cardiotoxicity is characterized by myocardial dysfunction and congestive heart failure, most often starting after 1 year of treatment. It is typically irreversible and associated with cumulative drug exposure. Nevertheless, despite these side effects, the benefits of anti-cancerous therapies, including anthracyclines such as doxorubicin, outweighs the risks, so that drugs aimed at minimizing cardiomyocytes damage are actively sought.

In agreement with previous studies,18-19,23,25,40-42 doxorubicin caused prominent apoptosis, indicated by nuclear fragmentation, increased cytochrome C release, increased cleaved caspase 3 (represented both by increased protein expression and immuofluorescent staining), and a reduction in the Bcl-2/Bax ratio resulting from a decrease in the Bcl-2 expression level. Recent studies implicated mitochondrial dysfunction as an early event in doxorubicin-induced cardiotoxicity and demonstrated that doxorubicin increases cytochrome C release.40 It is well established that cytochrome C binds to apoptotic protease-activating factor 1 (Apaf1) and to pro-caspase 9 once it is released to the cytosol, leading to generation of activated caspase 9. Active caspase 9 then activates executioner caspases, mainly caspase 3, which leads to apoptosis.30 In addition, in view of the importance of the Bcl-2 family proteins in regulating the mitochondrial apoptotic pathway,22,23 we demonstrated that doxorubicin markedly decreased Bcl-2 protein expression without changing Bax, thus decreasing the Bcl-2/Bax ratio, which predisposes the cell to apoptotic stimuli. These data confirm previous results showing a decrease in Bcl-2 protein expression after doxorubicin treatment.24,25 Indeed, apoptotic-like cell death is known to play a role in cardiomyopathy induced by doxorubicin,26-28 indicating that inhibitors of apoptosis may provide hope for the prevention/treatment of doxorubicin-induced cardiomyopathy.

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Serum Starvation-induced Cardiotoxicity

Serum starvation is a common experimental procedure that simulates in vitro a key component of myocardial ischemia (growth factors deprivation) and induces apoptosis. In the present work we showed that in agreement with previous studies,29-32 serum starvation induced apoptosis in NRVM, increased cleaved caspase expression, and decreased the ratio of Bcl-2/Bax. In support of our findings, overexpression of Bcl-2 was shown to protect against apoptosis during oxidative stress,52 and a protective effect of ischemic preconditioning was suggested to involve upregulation of Bcl-2.53 Furthermore, several studies demonstrated that a decreased Bcl-2/Bax ratio might contribute to an increased rate of apoptosis in cardiac myocytes.33-37

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The Protective Efficacies of TVP1022 and Propargylamine Against Doxorubicin and Serum Starvation-induced Apoptosis

Previous studies showed that propargylamine derivatives such as rasagiline and TVP1022 exhibit broad cytoprotective efficacy against a variety of neurotoxins in neuronal cell cultures and in in vivo models. Moreover, propargylamine, the free propargyl moiety of rasagiline and TVP1022, exerts neuroprotective activity against N-methyl-R-salsolinol and serum deprivation-induced cell death, suggesting it essentiality for neuroprotection.38,39 On the basis of the notion that the mechanisms of apoptotic cell death of neurons and cardiomyocytes are similar,40,41 the present study tested the hypothesis that TVP1022 is anti-apoptotic in NRVM. In support of the hypothesis we show here for the first time that TVP1022 can attenuate the apoptotic process induced in NRVM by doxorubicin and serum starvation. The inhibition of apoptosis by TVP1022 was correlated with its inhibitory effects on doxorubicin and serum starvation-induced caspase 3 activation. In addition, TVP1022 almost completely prevented doxorubicin and serum starvation-induced reduction in the expression of anti-apoptotic Bcl-2 protein, thus increasing Bcl-2/Bax ratio and thereby protecting myocytes from mitochondria-mediated apoptosis. These observations are consistent with our recent studies demonstrating that activation/regulation of PKC in association with Bcl-2 protein family promotes neuronal survival by rasagiline and by its propargyl moiety.48 In this regard, rasagiline was shown to suppress cell death by preventing the activation of the mitochondrial apoptotic cascade in response to the neurotoxins SIN-1 and N-methyl-R-salsolinol.42,43 Importantly, previous studies provided clear evidence that the neuroprotective efficacy of rasagiline does not depend on inhibition of MAO-B; rather, it is associated with some intrinsic pharmacological action of the propargyl moiety acting on mitochondria cell survival proteins.60-62

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The Involvement of the Propargyl Moiety in the Cardioprotective Efficacy of TVP1022

To determine the role of the propargyl moiety in the cardioprotective efficacy of TVP1022, we tested the ability of propargylamine to prevent doxorubicin and serum starvation-induced apoptosis. In agreement with previous studies in neuronal models,38,44 we show here that the cytoprotective effect of TVP1022 in doxorubicin-treated and serum-starved NRVM resides in the propargyl moiety; both molecules (TVP1022 and propargylamine) diminished (to a different extent) the apoptotic process, decreased the elevation in cleaved caspase 3, and prevented the decrease in Bcl-2/Bax ratio (only TVP1022) by increasing Bcl-2 expression. In this regard, the neuroprotective efficacy of propargylamine was recently attributed to stabilization of the mitochondrial membrane potential (ΔΨm), prevention of permeability transition (PT) pore, induction of Bcl-2 that regulates PT, and activation of anti-oxidant enzymes,45-48 all of which are consistent with doxorubicin induction of apoptosis.49-51

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TVP1022 Does Not Cause Proliferation and Does Not Interfere With the Anti-cancer Activity of Doxorubicin

Because our aim is to develop TVP1022 as a potential cardioprotective drug for anthracycline-treated cancer patients, it was important to determine whether TVP1022 causes proliferation of human cancer cells and/or interferes with the anti-cancer activity of doxorubicin. To our satisfaction, TVP1022 neither caused proliferation of the human cancer cell lines HeLa and MDA-231 nor diminished the anti-cancer efficacy of doxorubicin. The clinical implication of these findings are (once established in vivo) that TVP1022 can be safely coadministered with doxorubicin to cancer patients.

In summary, we have shown that TVP1022 and propargylamine attenuated doxorubicin and serum starvation-induced apoptosis in NRVM. TVP1022 neither caused proliferation of human cancer cell lines nor interfered with the anti-cancer efficacy of doxorubicin in vitro. On the basis of these encouraging findings, we are currently developing TVP1022 as a cardioprotective drug in doxorubicin and other anthracyclines-treated patients as well as in myocardial ischemic events.

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Keywords:

TVP1022; propargylamine; doxorubicin; serum starvation; cardiotoxicity; ventricular cardiomyocytes; apoptosis; caspase 3

© 2008 Lippincott Williams & Wilkins, Inc.