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Modulation by Melatonin of the Cardiotoxic and Antitumor Activities of Adriamycin

Kim, Chunghui*; Kim, Nari*; Joo, Hyun*; Youm, Jae Boum*; Park, Won Sun*; Cuong, Dang Van*; Park, Young Shik*; Kim, Euiyong*; Min, Churl-Ki; Han, Jin*

Journal of Cardiovascular Pharmacology: August 2005 - Volume 46 - Issue 2 - p 200-210
doi: 10.1097/01.fjc.0000171750.97822.a2
Original Article

In this study, we investigated the effects of melatonin on adriamycin-induced cardiotoxicity both in vivo in rats and in vitro, and on the antitumor activities of adriamycin on MDA-231 and NCI breast cancer cells. Rats that received a single intraperitoneal injection of 25 mg/kg adriamycin showed a mortality rate of 86%, which was reduced to 20% by melatonin treatment (10 mg/kg, SC for 6 days). Melatonin attenuated adriamycin-induced body-weight loss, hemodynamic dysfunction, and the morphologic and biochemical alterations caused by adriamycin. Melatonin also reduced adriamycin-induced nuclear DNA fragmentation, as assessed by the comet assay. In addition, the antitumor activity of adriamycin could be maintained using lower doses of this drug in combination with melatonin. Melatonin treatment in the concentration range of 0.1-2.5 mM inhibited the growth of human breast cancer cells. In terms of oncolytic activity, the combination of adriamycin and melatonin improved the antitumor activity of adriamycin, as indicated by an increase in the number of long-term survivors as well as decreases in body-weight losses resulting from adriamycin treatment. These results indicate that melatonin not only protects against adriamycin-induced cardiotoxicity but also enhances its antitumor activity. This combination of melatonin and adriamycin represents a potentially useful regimen for the treatment of human neoplasms because it allows the use of lower doses of adriamycin, thereby avoiding the toxic side effects associated with this drug.

From the *Mitochondrial Signaling Laboratory, Department of Physiology and Biophysics, College of Medicine, Cardiovascular & Metabolic Disease Center, Biohealth Products Research Center, Inje University, Busan, Korea; and †College of Natural Science, Ajou University, Suwon, Korea.

Received for publication December 7, 2004; accepted April 12, 2005.

This work was supported by Grants R05-2003-000-00413-0 and R05-2004-000-00905-0, and by the BioHealth Products Research Center of the Korea Science and Engineering Foundation.

The first two authors contributed equally to this work.

Chunghui Kim's present address is Department of Animal Science & Biotechnology, Jinju National University, Jinju, 660-758, Korea.

Reprints: Jin Han, MD, PhD, Mitochondrial Signaling Laboratory, Department of Physiology and Biophysics, College of Medicine, Biohealth Products Research Center, 2020 Cardiovascular Institute, Inje University 633-165 Gaegeum-Dong, Busanjin-Ku, Busan 614-735, Korea (e-mail: phyhanj@ijnc.inje.ac.kr).

Adriamycin, which is an anthracyclin antibiotic, is effective against a wide range of human neoplasms, such as acute leukemia, lymphoma, and a number of solid tumors.1 However, clinical applications of adriamycin have been restricted by its toxicity, especially its cardiotoxic side effects, which lead to cardiomyopathy and heart failure.2

Although the pathogenesis of adriamycin-induced cardiotoxicity remains unclear, oxygen free radical production and lipid peroxidation are considered to be the 2 most important factors.1,3 The underlying mechanism involves 1-electron reduction of adriamycin for the generation of the semiquinone radical, which reduces oxygen, thereby producing superoxide and regenerating adriamycin. The net result of this process is that adriamycin catalyzes the reduction of oxygen by NADPH to form a superoxide radical, which is subsequently reduced to hydrogen peroxide (H2O2) by the antioxidant enzyme superoxide dismutase. In the presence of Fe2+, H2O2 is further reduced to the extremely reactive hydroxyl (OH) radical, which can react with polyunsaturated fatty acids to yield lipid hydroperoxide. Biologic membranes contain large amounts of polyunsaturated fatty acids, which are susceptible to peroxidative attack by oxygen free radicals, resulting in lipid peroxidation. Of the various organs in which peroxidative tissue damage has been documented, the heart is known to be the most vulnerable to attack by free radicals produced following adriamycin administration.4

Assuming that oxygen radical-induced lipid peroxidation of membrane lipids is indeed a major cause of adriamycin-induced cardiotoxicity, it seems reasonable to speculate that antioxidants would prevent this toxicity. Indeed, several antioxidants have been reported to reduce or delay adriamycin-induced cardiotoxicity in experimental animals.5-7

Melatonin is produced and secreted by the pineal gland in almost all species of animal and is best known for its ability to modulate circadian rhythms.8 There are numerous reports dealing with the effects of melatonin, such as the inhibition of cancer growth,9 stimulation of the immune system,10 and oxygen free radical scavenging.11 The antioxidant activity of melatonin has been ascribed to 2 different mechanisms: (1) melatonin reduces oxidative stress by stimulating antioxidant enzymes12,13; and (2) melatonin directly scavenges OH radicals.14 It has been demonstrated that melatonin protects cells, tissues, and organs from oxidative damage that is induced by a variety of free radical-generating agents and processes.13,15 Melatonin acts effectively as an antioxidant in protecting membrane lipids, nuclear DNA, and proteins from oxidative damage both in vivo and in vitro.16,17

In this study, we investigated whether melatonin could attenuate adriamycin-induced cardiotoxicity in rats both in vivo and in vitro. We examined adriamycin-induced cardiotoxicity using various criteria, such as mortality, body-weight loss, hemodynamic dysfunction, morphologic alterations, nuclear DNA fragmentation as a measure of DNA damage, the level of malondialdehyde (MDA) as an index of lipid peroxidation, and lactate dehydrogenase (LDH) release and the level of serum creatine kinase as indices of irreversible cell damage. We also investigated whether the protective effects of melatonin are mediated by alterations in the above criteria. In addition, it was of interest to examine the effects of melatonin on the antitumor effects of adriamycin. In this respect, we considered the direct antitumor effects of melatonin, which might influence the effects of adriamycin in antagonistic, additive, or synergistic ways. Thus, we determined the antitumor effects of melatonin on human breast cancer cells and examined whether the antitumor efficacy of adriamycin could be maintained at lower doses when used in combination with melatonin.

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MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (175-240 g) were used in these experiments. The rats were fed on standard rat chow and housed in plastic cages. Food and water were provided ad libitum. All of the experiments in this study were performed in accordance with the Guidelines for Animal Research of the National Institutes of Health and were approved by the local Committee on Animal Research.

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Chemicals

Adriamycin was dissolved in normal saline immediately before use in each experiment. Melatonin was dissolved in dimethylsulfoxide (DMSO) and diluted as required in normal saline. The DMSO concentration never exceeded 1%. The chemicals were freshly prepared immediately before use. All the other chemicals used in this study, unless specified otherwise, were purchased from Sigma Chemical Co (St Louis, MO).

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Dose-Response Kinetics of Adriamycin-Induced Mortality

Four groups of rats were used to evaluate the dose-response kinetics of adriamycin toxicity. The percentage of animals that survived in each group was plotted against time (days) after adriamycin injection. Each animal received a single IP injection of adriamycin. Group 1 was the control (1% DMSO) (n = 25); group 2 received 5 mg/kg adriamycin (n = 25); group 3 received 15 mg/kg adriamycin (n = 26); and group 4 received 25 mg/kg adriamycin (n = 23). To assess the effect of melatonin on adriamycin-induced cardiac damage, the rat hearts were used in the assay described below.

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Modulation of Adriamycin-Induced Lethality

The mortality rate for adriamycin was studied after the administration of melatonin. One group of rats (n = 33) served as the control group (1% DMSO). The second group of rats (n = 28) received adriamycin alone (25 mg/kg IP). The third group (n = 31) was injected with melatonin (10 mg/kg SC) 1 hour before receiving adriamycin (25 mg/kg IP) and every 12 hours thereafter for 6 days. The fourth group (n = 24) received only melatonin (10 mg/kg SC) every 12 hours for 6 days. To assess the effect of melatonin on adriamycin-induced cardiac damage, rat hearts were used in the assay described below.

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Modulation of Adriamycin-Induced Body-Weight Changes

The following groups of rats were used to evaluate the body-weight changes and cardiotoxicity of adriamycin: control (n = 12); adriamycin alone (n = 11); melatonin alone (n = 8); and adriamycin plus melatonin (n = 9). In the adriamycin group, 15 mg/kg adriamycin was injected IP as a single dose, whereas in the adriamycin plus melatonin group, the rats were treated with melatonin (10 mg/kg SC) for 6 consecutive days, starting 2 days before adriamycin injection. To observe the effects of melatonin on normal rats, melatonin (10 mg/kg SC) was administered daily to the melatonin-alone group for 5 consecutive days. Body-weight measurements were performed every other day. The numbers of deaths were recorded daily. Finally, the effect of melatonin on adriamycin-induced cardiac damage was assayed as described below.

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Hemodynamics, Heart Weights, and Levels of Cholesterol, Glucose, Insulin, and Creatine Kinase

Using a 2F micromanometer-tipped catheter (Model SPR-407; Millar Instrument, Houston, TX) that was inserted through the right carotid artery, we measured the mean LV systolic and diastolic pressures of 5 consecutive pressure cycles. The maximal rates of left ventricular pressure rise (+dP/dt) and decrease (−dP/dt) were measured. The heart was excised rapidly and subjected to retrograde perfusion with a Langendorff apparatus. At the completion of the heart rate (HR) measurements, the heart was rapidly divided into the right and left atria, and right and left ventricles, and each tissue was weighed individually. To evaluate pulmonary edema, the lungs were also weighed. To determine the confounding roles of cholesterol, glucose, insulin, and creatine kinase, we assayed blood samples from the aorta. The plasma cholesterol levels were determined enzymatically using kits from Hitachi 7150 (Hitachi, Japan), and expressed as milligram per deciliter of serum. The plasma insulin concentrations were measured by collecting 4-mL blood samples in test tubes that contained 2% ethylenediaminetetraacetic acid (80 μL/ml of blood). The blood samples were immediately centrifuged at 3000 × g for 10 minutes, and the plasma samples were stored at −70°C until further analysis. Insulin was measured by the ultrasensitive rat enzyme immunoassay (Mercodia, Uppsala, Sweden). Creatine kinase was determined using kits purchased from Cal-Tech Diagnostics (Chino, CA).

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Electron Microscopy

The following groups of rats were used to evaluate the ultrastructural alterations to the heart that resulted from adriamycin treatment: control (n = 4); adriamycin alone (n = 5); adriamycin plus melatonin (n = 5); and melatonin alone (n = 4). In the adriamycin group, a single dose of 15 mg/kg adriamycin was injected IP, whereas in the adriamycin plus melatonin group, the rats were treated with melatonin (10 mg/kg SC) for 6 consecutive days, starting 2 days before adriamycin injection. Six days after adriamycin injection, the hearts were isolated, as described above, and fixed by perfusion with 0.1 M phosphate buffer that contained 1.7% glutaraldehyde and 1.6% paraformaldehyde through the aorta for 10 minutes at a pressure of 120 mm Hg. The hearts were then excised and stored in a fixative. Random samples (n = 11) of heart tissues were postfixed in Epon 812. Ultrathin sections (50 to 70 nm thick) were stained with uranyl acetate and lead nitrate. The ultrastructure of the cardiac muscles was examined at magnification powers of ×5000 and ×15,000 in the electron microscope (Jeol 1200 EX II; Jeol Ltd, Tokyo, Japan) and analyzed using Image Pro-plus software (Media Cybernetics Inc, Silver Spring, MD).

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Preparation of Single Ventricular Myocytes and Heart Slices

Single ventricular myocytes were isolated from rat hearts by an enzymatic dissociation procedure, as described previously.18 The isolated ventricular cells were stored in KB solution at 4°C and used for measurements of cell length, width, and area as well as for the comet assay described below.

Thin (0.4- to 0.5-mm-thick) slices of rat heart were prepared as described previously,3 and the sections were stored in ice-cold modified Cross-Taggart medium that contained 130 mM NaCl, 10 mM KCl, 1.5 mM CaCl2, 5 mM glucose, and 20 mM Tris-HCl (adjusted to pH 7.4 with NaOH). They were used for measurements of LDH release and lipid peroxidation described below.

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Measurement of LDH Release and Lipid Peroxidation

For the measurement of LDH release, the heart slices were centrifuged at 1000 rpm for 5 minutes. The pellets were discarded, and the supernatants were saved. LDH activity was determined in the supernatants and incubation medium using the LDH Assay Kit (Asan Pharmaceutical Co, Ltd, Seoul, Korea). The final values are expressed as percentages of the control value.

Lipid peroxidation was estimated by measuring the content of malondialdehyde (MDA) according to the method of Okawa et al.19 The final values are expressed as percentages of the control value.

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Comet Assay

The comet assay was performed as described by Sestili and Cantoni,20 with slight modifications. The assay works on the principle that strand breakage of the supercoiled duplex DNA leads to the reduction of the size of the large molecule, and these strands can be stretched out by electrophoresis. Also, under highly alkaline conditions there is denaturation, unwinding of the duplex DNA, and expression of alkali-labile sites as single strand breaks. Comets form as the broken ends of the negatively charged DNA molecule become free to migrate in the electric field toward the anode. Two principles in the formation of the comet are that (1) DNA migration is a function of both size and the number of broken ends of the DNA, and that (2) tail length increases with damage initially and then reaches a maximum that is dependent on the electrophoretic conditions, not the size of fragments. In each experiment, the DNA migration distance was calculated for at least 50 randomly selected cells. For each nucleus in the cell, the total length of the image (including the nucleus and tail) and the diameter of the nucleus were measured in micrometers. The extent of DNA damage was assessed from the extent of DNA migration (derived by subtracting the diameter of the nucleus from the total length of the image).

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Colony-Forming Assay

A clonogenic assay was used to measure the ability of cells to form colonies. MDA-231 and NCI cells were cultured in 5% CO2 at 37°C in DMEM that was supplemented with 10% FBS. The cells (n = 500) were seeded on 100-mm2 tissue culture dishes, following treatment with melatonin and/or adriamycin. The controls consisted of untreated cells. The cells were exposed continuously for 7 days to the indicated concentrations of melatonin and/or adriamycin; colonies were then stained with crystal violet, and colonies that contained >50 cells were counted. Each point represents the mean value ± SEM for 6 plates. The dose-response relationships for MDA-231 or NCI cell growth inhibition by adriamycin or melatonin were fitted to the Hill equation using the least-squares method:

where [Drug] is the concentration of adriamycin or melatonin, Ki is the concentration that gave half-maximal inhibition by adriamycin or melatonin, and n is the Hill coefficient.

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Statistical Analysis

All the values are expressed as mean ± SEM. The differences between the mean values were analyzed by the Student t test, Dunnett procedure after analysis of variance or χ2 analysis, ANOVA, and Kaplan-Meier (Mantel-Cox) methods. A P < 0.05 value was considered to be statistically significant.

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RESULTS

Survival Study

Single-dose adriamycin treatment was dose-dependently lethal for rats (Fig. 1A). The following survival rates were noted: 100% for group 1 (control) and group 2 (5 mg/kg adriamycin); 85% for group 3 (15 mg/kg adriamycin); and 14% for group 4 (25 mg/kg adriamycin). Figure 1B shows how the survival rate from IP administration of 25 mg/kg adriamycin was modified by SC injection of 10 mg/kg melatonin for 7 days. Melatonin treatment significantly attenuated adriamycin-induced mortality from 86% to 20% (P < 0.05). On the other hand, the administration of melatonin alone according to this regimen did not kill any of the rats.

FIGURE 1

FIGURE 1

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Body Weight Changes

Adriamycin injection significantly decreased (−16% of the initial weight) the rat body weights by day 7 (Fig. 1C; P < 0.05 versus control). In contrast, the body weights of the rats that were treated with adriamycin plus melatonin decreased by only 5%, which indicates that treatment with melatonin ameliorates adriamycin-induced body weight loss (P < 0.05 versus adriamycin alone). However, melatonin alone did not produce any significant difference in body weight, as compared with the control group.

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Hemodynamics, Heart Weight, and Levels of Cholesterol, Glucose, and Insulin

Hemodynamic changes were observed in all the surviving rats 6 days after treatment with adriamycin (Table 1). Adriamycin caused significant alterations in the hemodynamic indices (heart rate, LVSEP, +dP/dt, and −dP/dt). The cardiac contractile parameters of LVEDP, +dP/dt, and −dP/dt in the adriamycin-treated group were decreased significantly. LVEDP, which is a ventricular diastolic property, and the lung-weight to body-weight ratios and the cholesterol levels were increased. However, the insulin levels decreased in the adriamycin group. Melatonin treatment improved all of the adriamycin-impaired cardiac function parameters. The addition of melatonin to the adriamycin group also reduced the serum cholesterol level. No differences were observed in the left-ventricular-weight/body-weight ratios, the right-ventricular-weight/body-weight ratios, and the serum glucose levels among the 4 groups.

TABLE 1

TABLE 1

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Morphologic Analysis

Cardiac muscle cells, which are irregularly shaped cylinders, comprise the Z lines with diads, sarcomeres, elliptical nucleus, myofibrils, and round or elongated mitochondria (Fig. 2A). The morphologic changes seen in the adriamycin group were typical of adriamycin-induced cardiomyopathy in that they showed mitochondrial swelling, vacuolization of the cytoplasm, thinning of the Z lines, and loss of myofibrils (Fig. 2B). The ultrastructures of the hearts from the melatonin plus adriamycin group were indistinguishable from those of the control group, ie, regular myofibrillar arrangements and preserved mitochondria; this was not the case for the length of the Z line (Fig. 2C). Melatonin reduced the adriamycin-induced structural changes including the length of the sarcomere (Fig. 2D), Z line lengths (Fig. 2E), and mitochondrial areas (Fig. 2F). To characterize the morphologic alterations at the cellular level, we isolated rat ventricular myocytes from different treated groups (Table 2). The cell length and area of the adriamycin-treated myocytes were significantly smaller than those of the control group and melatonin-alone group. The combination of melatonin and adriamycin restored the cell length and area values.

TABLE 2

TABLE 2

FIGURE 2

FIGURE 2

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Changes in the Levels of MDA, LDH Release, and Serum Creatine Kinase

In Vitro Study

The effect of adriamycin on lipid peroxidation was examined over the concentration range 100-500 μM in rat heart slices that were incubated for 6 hours at 37°C. Adriamycin doses of >200 μM caused significant increases in the levels of MDA (Fig. 3A) and LDH release (Fig. 3E), and the effect was proportional to the concentration of adriamycin. We observed that lipid peroxidation (Fig. 3B) and LDH release (Fig. 3F) increased linearly with incubation time when 300 μM adriamycin was used. The incubation time of 4 hours was selected for the subsequent experiments because this was deemed to be the optimal time for the determination of lipid peroxidation.

FIGURE 3

FIGURE 3

Melatonin inhibited adriamycin-induced lipid peroxidation (Fig. 3C) and LDH release (Fig. 3G) in a concentration-dependent manner. The minimal effective concentration of melatonin for the inhibition of lipid peroxidation and LDH release induced by 300 μM adriamycin was 1 mM.

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In Vivo Study

Figures 3D and 3H show the effect of melatonin on adriamycin-induced lipid peroxidation and LDH release in vivo. Significantly elevated levels of lipid peroxidation and LDH release were observed in rats that received adriamycin alone (15 mg/kg IP) when compared with the control (both P < 0.05). When the adriamycin-injected animals were pretreated with melatonin (10 mg/kg SC for 5 days), the levels of lipid peroxidation and LDH release were not different from those of the control. Melatonin alone did not change the levels of lipid peroxidation and LDH release. In addition, adriamycin increased the level of serum creatine kinase as compared with the control group. The addition of melatonin to the adriamycin group significantly decreased the level of serum creatine kinase (Fig. 4; P < 0.05).

FIGURE 4

FIGURE 4

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DNA Damage Determined by the Comet Assay

The cardiotoxicity of adriamycin was further examined in rat ventricular myocytes using the comet (single-cell gel electrophoresis) assay. Cardiac myocytes that were treated with 30 μg/mL adriamycin showed the comet-like morphology (Fig. 5A, middle 3 panels), in contrast to the spherical shape of the control (Fig. 5A, left panel). Melatonin (1 mM) inhibited the DNA damage induced by adriamycin (Fig. 5A, right panel). The control, adriamycin-treated, and adriamycin- plus melatonin-treated cells exhibited various DNA migration distances in several different experiments; the data were pooled and are expressed graphically in Figure 5B. The distribution of the control cells with respect to DNA migration distance was similar in all the experimental series. The DNA migration distance increased markedly in adriamycin-treated cells, and this effect was prevented by melatonin treatment.

FIGURE 5

FIGURE 5

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The Effect of Melatonin on the Antitumor Activity of Adriamycin

An initial series of experiments was performed to determine the effect of different concentrations of adriamycin, within the range of 0.01 to 1 μg/mL, on in vitro cultured human breast cancer cell lines MDA-231 and NCI. Adriamycin inhibited the growth of both cell lines. Figure 6A shows the dose-response relationships for MDA-231 cell growth inhibition by adriamycin in the absence and presence of 1 mM melatonin. The adriamycin concentration at half-maximal inhibition (Ki) and the Hill coefficient were 0.17 ± 0.03 μg/mL and 0.99 ± 0.18, respectively, in the absence of melatonin and 0.10 ± 0.01 μg/mL and 0.99 ± 0.11, respectively, in the presence of melatonin (P < 0.05). For the NCI cells (Fig. 6B), treatment of cells with 1 mM melatonin changed the Ki of adriamycin (0.06 ± 0.01 μg/mL in the absence of melatonin and 0.03 ± 0.01 μg/mL in the presence of melatonin; P < 0.05). No significant changes in the Hill coefficient were observed in the absence (0.98 ± 0.16) and presence (0.98 ± 0.14) of melatonin. These data indicate that melatonin increases the efficacy of adriamycin.

FIGURE 6

FIGURE 6

At concentrations of 0.5-10 mM, melatonin induced significant reductions in cell growth. The melatonin concentration at half-maximal inhibition and the Hill coefficient were 1.07 ± 0.06 mM and 0.99 ± 0.13, respectively, for the MDA-231 cells (Fig. 6C) and 0.95 ± 0.12 mM and 0.99 ± 0.16, respectively, for the NCI cells (Fig. 6D). The melatonin concentration at half-maximal inhibition in the presence of adriamycin was significantly less than that in the absence of adriamycin (0.18 ± 0.02 mM in the presence of 0.17 μg/mL adriamycin for the MDA-231 cells, Fig. 6C; and 0.48 ± 0.09 mM in the presence of 0.06 μg/mL adriamycin for the NCI cells, Fig. 6D). No significant changes in the Hill coefficient were observed in the absence or presence of adriamycin (0.99 ± 0.15 in the presence of 0.17 μg/mL adriamycin for the MDA-231 cells and 0.98 ± 0.14 in the presence of 0.06 μg/mL adriamycin for the NCI cells).

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DISCUSSION

Previous studies have shown that adriamycin induces cardiac dysfunction and causes severe cell damage.1-3 Adriamycin cardiotoxicity is mainly the result of the formation of reactive oxygen species (ROS).4 Although several ROS scavengers have been tested, they do not completely eliminate adriamycin cardiotoxicity because the problems of high-density lipoprotein lowering, unchanged mortality rate and weight loss, and potentiation of hematotoxicity persist, as well as the fact that these scavengers interfere with the antitumor properties of adriamycin.21,22 Recently, it has been reported that melatonin is a potent antioxidant and ROS scavenger.11-14 On the basis of previous findings, it seems reasonable to assume that melatonin represents a valid candidate for reducing adriamycin-induced cardiotoxicity. Melatonin, besides being an efficient ROS scavenger, is a lipophilic compound that readily crosses cell membranes and distributes itself within the subcellular compartments, where the antioxidant protection is required. Furthermore, it is well tolerated and safe after oral administration.23 In this study, melatonin was effective in attenuating adriamycin-induced cardiotoxicity in rats. It is interesting to note that melatonin itself inhibited the growth of human cancer cells. In addition, the concomitant administration of melatonin improved the antitumor action of adriamycin. The functionally refractory nature of adriamycin-induced cardiomyopathy and the heart failure observed in humans have also been observed in rats.24,25 It seems likely that rats simulate most of the adriamycin-induced clinical and hemodynamic changes. Thus, in this study, we chose the rat as the animal model of adriamycin-induced cardiomyopathy.

Since the first report on adriamycin-induced cardiomyopathy,26 several mechanisms have been proposed to explain the development of this disease.4,27-41 It is generally accepted that mechanisms involving ROS are the main contributors to adriamycin-mediated cardiotoxicity.35,36 In this study, increased ROS formation caused by adriamycin was evidenced by an increase in tissue MDA formation, which is a breakdown product of lipid peroxidation (Fig. 3A-D). The measurement of thiobarbituric acid-reactive substances (TBARs) is a widely accepted and sensitive method for monitoring oxidative stress in tissues. These results are consistent with those of previous studies conducted by other investigators that suggest that adriamycin-induced cardiotoxicity is associated with ROS generation and lipid peroxidation.4,38,42 The results of the comet assay reported in the present study (Fig. 5) clearly demonstrate adriamycin-induced DNA damage of cardiac myocytes. Because the comet assay is a highly sensitive system for detecting DNA damage, these results are indicative of adriamycin-induced apoptosis. Furthermore, the levels of LDH release and serum CK levels were used to evaluate adriamycin-induced necrosis because adriamycin causes peroxidation and disruption of the cardiac myocyte membrane. Adriamycin treatment resulted in increased LDH release (Fig. 3E-H) and serum CK level (Fig. 4), as compared with the control group. These results support the findings of recent studies that oxidative stress and lipid peroxidation may result in membrane and macromolecule damage, both of which induce cell apoptosis and necrosis.43

In this study, electron microscopic evidence of myocardial damage was observed in all animals that were treated with adriamycin (Fig. 2). The morphologic alterations characteristic of adriamycin-induced cardiotoxicity include the loss and disorganization of myofibrils, dilation of the sarcoplasmic reticulum, cytoplasmic vacuolization, mitochondrial degeneration and swelling, and increased number of lysosomes.21,22 Of these, the loss of myofibrils and vacuolization of the cardiac myocytes are known to be the 2 most important ultrastructural changes; they were observed consistently in the myocardium of rats exposed to adriamycin (Fig. 2B). These changes were associated with adriamycin-induced ROS production and lipid peroxidation, which activate lysosomal enzymes in the heart and induce cell inflammation and necrosis.43 As the mitochondria swelled (Fig. 2B,F), they appeared to leak intramembranous proteolytic enzymes into the cytosol, thereby cleaving nearby myofilament proteins. In addition, adriamycin shortened the length of the sarcomere (Fig. 2D) and Z line (Fig. 2E) of cardiac myocytes, consistent with the results observed on the cellular level (Table 2) that suggest the presence of contracted cardiac myocytes. It seems likely that this persistent contraction of myocytes is caused by an adriamycin-induced decrease in mitochondrial Ca2+ loading capacity.44

In this study, the mean body weight and mortality rates, as well as the hemodynamic data, were used to demonstrate the presence of adriamycin-induced cardiomyopathy. The mean body weight of the adriamycin-treated rats was reduced significantly from day 3 to the end of the experiment (day 7), which is consistent with the results of a previous study.45 Interestingly, the decrease in body weight correlated with an increase in mortality (Fig. 1). The adriamycin-induced hemodynamic changes in the rats (Table 1) were similar to those observed in humans,46 in that they included poor performance of the left ventricle (LVSEP, LVEDP, and ±dp/dt) and reduced heart rate. These functional changes could lead to pulmonary congestion, which is indicated by an increase in the lung to body-weight ratio (Table 1). Thus, congestive heart failure is also likely to be a characteristic sign of adriamycin-induced cardiotoxicity. It is interesting to note that adriamycin caused an increase in the cholesterol level and a decrease in the insulin level. These data suggest that adriamycin-induced adverse effects, such as hypercholesterolemia and hypoinsulinemia, which are also deleterious for cardiac function, are secondary to the other clinical side effects of adriamycin.

The present study demonstrates that treatment with melatonin offers complete protection against adriamycin-induced cardiotoxicity, improving both the survival rate and body-weight loss (Fig. 1) while preserving both cardiac function (Table 1) and morphology (Fig. 2 and Table 2) as well as inhibiting the increases in lipid peroxidation (Fig. 3), LDH and CK levels (Figs. 3 and 4), and nuclear DNA damage (Fig. 5). Lipid peroxidation, as measured by MDA, is generally accepted as an indicator of oxidative stress that results from free radical overproduction and reduction in antioxidant reserves. Adriamycin treatment caused a significant increase in myocardial lipid peroxidation (Fig. 3A,B), which was abrogated by melatonin (Fig. 3C,D). This indicates that the antioxidant property of melatonin may be directed against the lipid radical-mediated chain reaction. It is believed that adriamycin-induced radicals and ROS induce membrane injury by lipid peroxidation and disruption. Measurements of LDH release and serum CK are widely accepted and sensitive methods for monitoring severe cell damage (Fig. 3E,F). The protective effects of melatonin against adriamycin-induced membrane damage observed in this study (Fig. 3G,H) are comparable to those observed with other chemoprotectants in rats.47-49 Our findings are also consistent with previous studies demonstrating that melatonin protects against the oxidative damage caused by other ROS-generating agents.50

Nuclear DNA is one of the major biologic targets of adriamycin. ROS are known to induce genotoxic damage (Fig. 5), including single- and double-strand breaks, and play an important role in the pathogenesis of cytotoxicity. Adriamycin-induced free radicals can also induce DNA damage and cardiotoxicity. In the present study, we used the single-cell gel electrophoresis (SCGE) assay, also known as the comet assay, to test the ability of melatonin to prevent DNA breaks induced by adriamycin. The comet assay is a very sensitive method for the detection of DNA damage. The administration of melatonin (1) decreased the extent of DNA damage (DNA migration distance) and (2) increased the number of cells with decreased levels of DNA damage (Fig. 5). These results are of interest in view of the proposed chemopreventive activity of melatonin.

Our results suggest that melatonin may be a useful adjuvant to prevent adriamycin toxicity in cancer chemotherapies. The anticancer activity of adriamycin and lipid peroxidation are reportedly independent properties of adriamycin; and antioxidants, such as vitamin E and vitamin C, have been reported to reduce the lipid peroxidation and cardiotoxic activities of adriamycin.6,38 In this context, it seems unlikely that the ameliorating effect of melatonin on adriamycin toxicity has any affect on the antitumor activity of adriamycin. In this study, we also examined whether the antioxidant effect of melatonin interfered with the antineoplastic effects of adriamycin. Surprisingly, melatonin itself had antiproliferative effects on human breast cancer cells. Furthermore, the direct antitumor effects of melatonin were additive or synergistic with those of adriamycin, which suggests the potential of this drug combination for improving the efficacy of chemotherapy in terms of both survival and quality of life. These results are consistent with previous reports that melatonin does not attenuate the antitumor activity of adriamycin under various in vitro and in vivo conditions.51 Indeed, in a large group of advanced cancer patients who were treated with chemotherapy, melatonin increased the 1-year and 5-year survival rates as well as the overall rate of tumor regression.52

In conclusion, the results of the present study demonstrate that melatonin protects against adriamycin-induced lethality, body-weight loss, hemodynamic changes, lipid peroxidation, severe cell damage, morphologic changes, and DNA damage in rat heart and that it also inhibits the growth of the human cancer cells. These findings support the results of clinical trials that suggest the use of melatonin to prevent adriamycin-induced cardiotoxicity and to enhance the antitumor activity of adriamycin.

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REFERENCES

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Keywords:

melatonin; adriamycin; cardiotoxicity; nuclear DNA fragmentation; antitumor activity

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