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C-Phycocyanin Ameliorates Doxorubicin-Induced Oxidative Stress and Apoptosis in Adult Rat Cardiomyocytes

Khan, Mahmood PhD*†; Varadharaj, Saradhadevi PhD*; Shobha, Jagdish C MD; Naidu, Madireddi U MD; Parinandi, Narasimham L PhD*; Kutala, Vijay Kumar PhD*†; Kuppusamy, Periannan PhD*

Journal of Cardiovascular Pharmacology: January 2006 - Volume 47 - Issue 1 - p 9-20
doi: 10.1097/01.fjc.0000191520.48404.27
Original Article

Doxorubicin (DOX), a potent antineoplastic agent, poses limitations for its therapeutic use due to the associated risk of developing cardiomyopathy and congestive heart failure. The cardiotoxicity of doxorubicin is associated with oxidative stress and apoptosis. We have recently shown that Spirulina, a blue-green alga with potent antioxidant properties, offered significant protection against doxorubicin-induced cardiotoxicity in mice. The aim of the present study was to establish the possible protective role of C-phycocyanin, one of the active ingredients of Spirulina, against doxorubicin-induced oxidative stress and apoptosis. The study was carried out using cardiomyocytes isolated from adult rat hearts. Doxorubicin significantly enhanced the formation of reactive oxygen species (ROS) in cells as measured by the 2′,7′-dichlorodihydrofluorescein diacetate and dihydroethidium fluorescence. The doxorubicin-induced reactive oxygen species formation was significantly attenuated in cells pretreated with C-phycocyanin. It was further observed that the doxorubicin-induced DNA fragmentation and apoptosis, as assayed by TUNEL assay and flow cytometry coupled with BrdU-FITC/propidium iodide staining, were markedly attenuated by C-phycocyanin. C-phycocyanin also significantly attenuated the doxorubicin-induced increase in the expression of Bax protein, release of cytochrome c, and increase in the activity of caspase-3 in cells. In summary, C-phycocyanin ameliorated doxorubicin-induced oxidative stress and apoptosis in cardiomyocytes. This study further supports the crucial role of the antioxidant nature of C-phycocyanin in its cardioprotection against doxorubicin-induced oxidative stress and apoptosis.

From the *Center for Biomedical EPR Spectroscopy and Imaging, Davis Heart and Lung Research Institute, Department of Internal Medicine, The Ohio State University, Columbus, OH; and †Department of Clinical Pharmacology and Therapeutics, Nizam's Institute of Medical Sciences, Hyderabad, India.

Received for publication June 25, 2005; accepted October 5, 2005.

We acknowledge the financial support from NIH Grant CA 102264.

Reprints: Periannan Kuppusamy, Davis Heart and Lung Research Institute, The Ohio State University, 420 West 12th Ave, Room 114, Columbus, OH 43210 (e-mail: kuppusamy.1@osu.edu).

Doxorubicin (DOX) is a potent antineoplastic agent used to manage a variety of human malignancies.1 However, the clinical use of DOX is limited due to its dose-dependent cardiotoxicity leading to congestive heart failure.2,3 Some of the proposed mechanisms of the cardiotoxicity of DOX include release of vasoactive amines,4 changes in adrenergic function,5 mitochondrial impairment,6 free radical formation,7,8 depletion of myocardial antioxidants, and lipid peroxidation. The probable mechanism of DOX-induced mitochondrial impairment involves the activation of DOX to a semiquinone intermediate, which undergoes redox-cycling in mitochondrial complex I to generate reactive oxygen species (ROS). Subsequently, ROS can cause oxidative damage to the cardiac mitochondria and myocytes and ultimately lead to apoptosis and cell death.7,9

Oxidative stress such as exposure to hydrogen peroxide causes apoptosis in a variety of cell systems including cardiomyocytes.9-13 The signal transduction pathway leading to DOX-induced apoptosis may involve p53,14 nuclear factor κB,13 p38 MAP kinase,15 C-Jun-N-terminal kinase,16 and/or the mitochondrial death pathway.17 Other pathways involving PI3 kinase/Akt18 and Fas/FasL19 were also suggested. Apoptosis in the myocardium ultimately leads to cardiomyopathy and congestive heart failure.20 The DOX-induced apoptosis was reported to be mediated by the formation of oxygen free radicals21 as this process was inhibited by various ROS scavengers, such as Ebselen (a mimetic of glutathione peroxidase, GPX),9 and by the overexpression of antioxidant enzymes.22-24

Cultured cardiomyocytes pretreated with antioxidants such as Trolox, vitamin E, Carvedilol, or Probucol followed by DOX exposure, showed decreased oxidative stress, cardiomyocyte damage, and apoptosis.25-29 An increase in the expression of superoxide dismutase (SOD), catalase, and glutathione S-transferase (GST) in the heart of transgenic mice has been shown to protect against DOX-induced cardiotoxicity.22,24,30 In isolated cardiomyocytes, iron chelators attenuated the DOX-induced apoptosis, but did not protect the cells from oncosis induced by high dose of DOX.21,31 Recently, we have demonstrated that the use of antioxidants containing herbal extracts namely, Gingko biloba and CardiPro significantly protected mice from DOX-induced cardiotoxicity.32,33

C-phycocyanin is one of the major biliproteins present in blue-green algae such as Spirulina (Arthospira) accounting for 20% of algal dry weight.34 It contains a tetrapyrrole chromophore known as phycocyanobilin (PCB), which is covalently attached to the apoprotein. PCB, for the most part, accounts for the biologic activity of C-phycocyanin.35 The extracts of Spirulina and isolated/purified C-phycocyanin have been reported to possess marked antioxidant and radical scavenging properties.36-38 C-phycocyanin has been shown to scavenge peroxyl,38 hydroxyl39 and superoxide37 radicals, and peroxynitrite.40 C-phycocyanin effectively inhibits carbon tetrachloride-induced lipid peroxidation in rat livers, prevents hepatotoxicity,41 and exhibits anti-inflammatory properties37 by inhibiting cyclooxygenase-2.42 It has also been shown to protect against oxalate-mediated renal cell injury43 and kainic acid-induced neural damage in rat hippocampus.44 In our recent study, we have demonstrated that pretreatment of mice with Spirulina preparation significantly decreased the DOX-induced cardiotoxicity.45 However, the exact mechanism and component present in the Spirulina responsible for the protection against DOX-induced cardiotoxicity have not been identified. The aim of the present study was to determine the effect of C-phycocyanin on DOX-induced oxidative stress and apoptosis in isolated cardiomyocytes. The results revealed that C-phycocyanin significantly attenuated the DOX-induced oxidative stress and apoptosis in cardiomyocytes and this effect is attributed to the antioxidant activity of the biliprotein.

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MATERIALS AND METHODS

Reagents

Collagenase type 2 (Worthington Biochemical Corporation, Lakewood, NJ), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), dihydroethidium (DHE), and C-phycocyanin were purchased from Sigma (St. Louis, MO). Laminin was purchased from Upstate Biotechnology (Lake Placid, NY). Caspase-3 colorimetric assay kit, enhanced DNA ladder assay kit, and Apo BrdU in situ DNA fragmentation assay kit were purchased from Biovision, CA. Medium 199, fetal bovine serum, penicillin, streptomycin, Tris-acetate-EDTA (TAE) buffer, and agarose were obtained from Life Technologies (Carlsbad, CA). Spirulina was obtained in the form of spray-dried powder from New Ambadi Estates, India. It had the following compositions: proteins (65.38%), phycocyanin (15.37%), minerals (7.95%), total carotenoids (0.43%), β-carotene (0.167%), and total pheophorbide (0.02%). Spirulina and C-Phycocyanin were freshly prepared in sterile distilled water and filtered using a 1-μm filter (Millipore) prior to use.

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Isolation of Cardiomyocytes

Adult rat ventricular cardiomyocytes were isolated from male Sprague-Dawley rats (280-350 g, b.w.) as previously described,46 but with some modifications. Briefly, isolated hearts were perfused with the Krebs Henseleit (KH) buffer containing 117 mM NaCl, 4.7 mM KCl, 1.5 mM NaH2PO4, 20 mM Hepes, 8 mM NaHCO3, 16.6 mM glucose, 3 mM MgCl2, 1 mM CaCl2, 5 mM sodium pyruvate, 12 mM taurine, 10 mM creatine, and 1 μM insulin. The hearts were then perfused with calcium-free KH buffer containing 0.1% collagenase and 0.1% bovine serum albumin (BSA) for 30 minutes. The hearts were minced, filtered through 2 layers of cheese cloth, and centrifuged. The cell pellet was then washed 3 times with the KH buffer containing increasing concentrations of BSA, to separate the dead round cells from viable rod-shaped cardiomyocytes. This procedure yielded >95% viable cardiomyocytes. The myocytes were then plated onto sterile culture dishes or coverslips treated with laminin (50 μg/mL). Before the experiments, the cells were maintained in M199 medium containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated overnight at 37°C in an atmosphere of 5% CO2. The protocols were approved by the Institutional Animal Care and Use Committee at The Ohio State University, and conformed to the Guide for the Care and Use of Laboratory Animals (NIH publication No.86-23, revised 1985).

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Cell Culture and Treatment

Adult rat cardiomyocytes (1 × 105 cells/dish) were plated in 100-mm sterile dishes coated with laminin and maintained in M199 medium containing 10% FBS (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated in a humidified atmosphere containing 5% CO2. For all the experiments, cells were plated 2 days preceding treatment with DOX and harvested at chosen time periods. After treatment, the cells were washed with PBS, scraped into PBS and centrifuged at 3000×g for 15 minutes. The final cell pellets were used for DNA isolation, caspase-3 assay, and TUNEL assays by fluorescence microscopy and flow cytometry.

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Cell Viability

Cardiomyocytes were incubated for 24 or 48 hours at standard culture conditions to determine the viability following DOX, DOX + C-phycocyanin, or DOX + Spirulina treatments. The number of viable cells in the culture was determined using a Nucleo Counter™ (New Brunswick Scientific, Edison, NJ), an automatic cell counter. This technique uses a fluorescent dye, namely propidium iodide, which binds to cellular nuclei and the resulting counts provide total cells or viable cells, depending on sample preparation.

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Lactate Dehydrogenase Assay

Cytotoxicity was determined by measuring the amount of lactate dehydrogenase (LDH) released into the cell culture medium using an optimized LDH procedure according to the manufacturer's instructions (Sigma). Cardiomyocytes were exposed to DOX (1 μM) for 24 hours following pretreatment, without and with C-phycocyanin (10 μM) or Spirulina (50 μg/mL) for 1 hour in serum-free medium M199. After incubation, an aliquot of the medium was taken and assayed for LDH activity. The rate of NADH oxidation was determined by measuring the decrease in absorbance at 340 nm at 25°C using a Varian (model Cary 50) spectrophotometer.

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Measurement of Oxidative Stress

The level of DOX-induced intracellular oxidant production in cardiomyocytes was measured using H2DCF-DA and DHE fluorescence. For DCF fluorescence, cells were plated on laminin-coated circular coverslips in 6-well plates (1 coverslip/well) containing serum-free medium and were preincubated with 10 μM of H2DCF-DA for 30 minutes and then washed twice with PBS. Cells were then incubated with DOX (10 μM) and with/without Spirulina (50 μg/mL) or C-phycocyanin (10 μM) for 4 hours. Because H2DCF-DA and DHE probes are not stable in cells for longer incubation times, we instead used higher concentration of DOX (10 μM) and incubated for 4 hours. Both Spirulina and C-phycocyanin were added 1 hour prior to DOX treatment. H2DCF-DA, a nonfluorescent probe is oxidized by peroxides to give DCF, which emits green fluorescence. The DCF fluorescence was measured by a Nikon fluorescence microscope equipped with a fluorescein isothiocyanate filter. For DHE studies, cells grown on circular cover slips in 6-well plates containing serum-free medium were incubated for 1 hour with 10 μM DHE, Spirulina or C-phycocyanin before adding DOX. After further incubation for 4 hours, the cells were washed several times with PBS and were viewed immediately with a Nikon TE 300 microscope using a rhodamine filter. High-resolution images were obtained using a constant exposure time. A single scan of a new field was used to limit any change in intensity caused by overexposure. The fluorescence intensity was quantified using MetaMorph software. The mean intensity of the fluorescence in a low-power field was used for comparison between the treatment groups.

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DNA Laddering

The extent of DNA fragmentation in cardiomyocytes treated with DOX (1 μM) and with/without C-phycocyanin or Spirulina pretreatment was determined using an enhanced apoptotic DNA ladder detection kit (BioVision, Mountain View, CA). Cells were washed with PBS, lysed using tris-acetate EDTA (TAE) lysis buffer, and DNA was isolated according to the manufacturer's protocol. The final DNA pellet was washed with 70% ethanol and air-dried for 10 minutes. The dried DNA pellet was then reconstituted in 20 μL of DNA suspension buffer, loaded onto a 1.8% agarose gel in TAE buffer, and electrophoresed at a constant voltage of 50 V for 2 hours in TAE buffer until the yellow dye reached the leading edge of the gel. The gel was then stained with gentle shaking for 30 minutes. The DNA ladder was visualized under UV light in a trans-illuminator and the image was captured using Alpha software (Innotech Corp, San Leandro, CA).

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TUNEL Assay

Following fixation of cells with 1% paraformaldehyde for 5 minutes, the cells were washed once with PBS and centrifuged at 3000×g for 5 minutes. The cells were then washed with 70% ethanol and subjected to TUNEL assay by using bromodeoxyuridine triphosphate nucleotides (BrdUTP) according to the manufacturer's protocol. The BrdUTP is readily incorporated into DNA strand breaks and is identified by a fluorescence-labeled anti-BrdU monoclonal antibody, which gives green staining for apoptotic cells over an orange-red propidium iodide (PI) counterstaining. Cells were briefly washed with 1 mL of the wash buffer provided in the kit and labeled with DNA labeling solution for 60 minutes at 37°C by shaking the cells every 15 minutes to resuspend. Cells were then centrifuged and resuspended in the antiBrdU-FITC antibody for 30 minutes in dark and then stained with PI for 30 minutes. The cells were mounted on a glass slide for examination under the Zeiss confocal microscope. The labeled cells were used within 3 hours of staining for flow cytometry.

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Western Blot Analysis

The total cell lysate was used to detect the expression of Bcl-2 and Bax protein. After treatment, the lysates were prepared by using cell lysis buffer containing 10 μg aprotinin, and 2 μg leupeptin in a protease inhibitor cocktail. The cell lysates were sonicated (5 seconds for 3 times) on ice and centrifuged at 5000×g for 10 minutes at 4°C. To obtain the mitochondrial-free cytosolic fraction for detecting the release of cytochrome c, the cells were suspended in ice-cold isotonic buffer containing 25 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 17 μg/mL PMSF, 8 μg/mL aprotinin, 2 μg/mL leupeptin, and 5 μg/mL pepstatin, at pH 7.4. The cells were lysed by two successive sonications on ice for 10 seconds each and the cell lysate was centrifuged at 10,000×g for 10 minutes to separate the mitochondria. The supernatant was further centrifuged at 75,000×g for 1 hour using a Beckman Coulter ultracentrifuge. The protein concentration was determined with a BCA protein assay kit (Pierce Chemical Co, Rockford, IL) using BSA as standard. Thirty micrograms of protein were resolved in 12% SDS-polyacrylamide gel along with SeeBlue Plus 2 protein ladder (Invitrogen, Mountain View, CA), transferred onto a polyvinylidene diflouride membrane, blocked with 5% milk PBS-T (0.1% Tween-20) pH 7.4 for 1 hour, and incubated with the primary antibody as indicated at 1:1000 dilution with 3% milk PBS-T overnight at 4°C. The membranes were then washed 3 times at 15-minute intervals and incubated with mouse IgG peroxidase conjugate at 1:5000 dilution for about 1 hour at room temperature. Protein bands were detected using ECLTM Western blot detection reagents (Amersham Biosciences, Piscataway, NJ) and Biomax-Kodak imaging film. The image was scanned and the intensities of the bands were digitized using Un-Scan-it™ (Version 5.1, Silk Scientific, Orem, UT) and the pixel values for Bcl-2, Bax, and cytochrome c were corrected using β-actin as a loading control.

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Caspase-3 Assay

Caspase-3 activity was assayed using Caspase-3/CPP-32 colorimetric assay kit (Biovision, Mountain View, CA). The assay is based on spectrophotometric analysis of the chromophore p-nitroanilide (pNA) after cleavage from the labeled substrate DEVD-pNA. The magnitude of the increase in the CPP-32 activity was determined by comparing the absorbance of the apoptotic sample with the level of an untreated control. Cardiomyocytes were treated with DOX and Spirulina or C-phycocyanin as mentioned before. The cells were lysed with ice-cold lysis buffer for 10 minutes and centrifuged at 10,000×g for 1 minute. The caspase-3 activity in the supernatant was assayed spectrophotometrically by measuring the released pNA at 405 nm according to the manufacturer's protocol. The activity of caspase-3 was expressed per microgram protein.

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Effect of C-Phycocyanin on the Antitumor Activity of Doxorubicin

Human ovarian cancer cells were used to check the interference of C-phycocyanin on the antitumor activity of DOX. The ovarian cancer cells were seeded into 96-well plates at a starting density of 2.5 × 104 cells/well and cultured overnight in a phenol red-free RPMI-1640 medium containing 5% fetal-calf serum at 37°C humidified with 5% CO2. The following day, doxorubicin (10 μM) and/or C-phycocyanin (10 μM) or Spirulina (50 μg/mL) were added to the medium. Twenty-four hours later, 0.5% MTT was added, incubated for 3 hours, then the medium was removed. The water-insoluble blue formazan dye formed was solubilized in DMSO, and the absorbance was measured using a 96-well plate ELISA reader (Beckmann Coulter, AD 340) at 550 nm. All experiments were run in at least 4 parallels and repeated 3 times.

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Statistical Analysis

All values are expressed as Mean ± SD of 3 to 5 independent experiments. For comparison between the groups, ANOVA and Student's t test were used. Differences between groups were considered to be significant at P < 0.05.

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RESULTS

Ventricular cardiomyocytes isolated from adult rat hearts were treated with DOX in presence of C-phycocyanin or Spirulina and incubated for 24 hours or 48 hours. Cell viability was assessed using an automatic cell counter as described in the Methods section. The results, shown in Figure 1, indicated that DOX (1 μM) caused a significant reduction in the number of viable cells, when the cells were exposed to DOX for 24 or 48 hours. Pretreatment with C-phycocyanin (10 μM) or Spirulina (50 μg/mL) significantly prevented the reduction in cell viability induced by DOX indicating that both agents had a cytoprotective role against DOX-induced cell death.

FIGURE 1

FIGURE 1

Lactate dehydrogenase release was measured in the cell culture supernatant as a marker of cellular injury. There was a significant increase in LDH levels (P < 0.001) in cells treated with DOX (1 μM) for 24 hours (Fig. 2). The increase in LDH release was significantly reduced in cells pretreated with C-phycocyanin or Spirulina.

FIGURE 2

FIGURE 2

Experiments were performed to determine whether pretreatment of cells with C-phycocyanin or Spirulina could attenuate the DOX-induced oxidative stress in cardiomyocytes. Cells were pretreated with Spirulina or C-phycocyanin for 1 hour and then treated with DOX in the presence of redox-sensitive fluorescent probes, H2DCF-DA and DHE for 4 hours. Representative sets of fluorescence micrographs are shown in Figures 3 and 4. The untreated (control) cells showed a low baseline level of fluorescence. Cells exposed to DOX showed a significant increase in fluorescence, indicating DOX-induced oxidative stress (Figs. 3A and 4A). However, pretreatment of the cells with C-phycocyanin or Spirulina showed a significant reduction in the DOX-induced fluorescence. Figures 3B and 4B show the mean fluorescence intensity in control (untreated) cells. In cells treated with DOX, the fluorescence intensity of DCF increased by 3.2-fold and HE increased by 4.2-fold as compared with untreated controls (P < 0.001). Both C-phycocyanin and Spirulina pretreatment significantly attenuated the DOX-induced oxidative stress to the level that was exhibited by the untreated cells.

FIGURE 3

FIGURE 3

FIGURE 4

FIGURE 4

To assess the occurrence of apoptosis in culture, fragmented DNA in the nucleus was labeled with an antiBrdU-FITC conjugate by TUNEL assay and visualized by confocal microscopy and flow cytometry. Figure 5 shows a typical TUNEL assay where the apoptotic nuclei were stained green with BrdUTP-FITC. All nuclei, apoptotic and non-apoptotic, were counterstained with PI and appeared red. In the control cells, all the nuclei had a normal oblong appearance and were stained red. Exposure to DOX resulted in nuclear fragmentation showing green staining. While no apoptosis was observed in control (untreated) cells, significant apoptosis was observed in cells treated with DOX. Figure 6 shows the flow cytometric analysis of labeled cells. In DOX-treated cells, the presence of TUNEL-positive apoptotic cells was observed with higher fluorescence intensity as compared with non-apoptotic cells. Pretreatment with Spirulina or C-phycocyanin considerably reduced the number of apoptotic cells compared with DOX treatment alone. Quantitative assessment of nuclear fragmentation and apoptosis was done using flow cytometry. DOX-treated cells showed a significant amount of apoptosis as compared with untreated cells (Fig. 6B). The DOX-induced apoptosis was significantly attenuated in cells pretreated with C-phycocyanin or Spirulina. The DNA fragmentation was further confirmed by agarose gel electrophoresis. The presence of DNA laddering, indicative of apoptosis, was observed in cardiomyocytes after 24 hours of DOX treatment (Fig. 7). Pretreatment of cardiomyocytes with C-phycocyanin or Spirulina completely inhibited the DOX-induced DNA fragmentation (Fig. 7).

FIGURE 5

FIGURE 5

FIGURE 6

FIGURE 6

FIGURE 7

FIGURE 7

We further examined the effect of C-phycocyanin on expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins in DOX-treated cardiomyocytes. As shown in Figure 8A, DOX enhanced the expression of Bax, whereas cells pretreated with C-phycocyanin inhibited the DOX-induced expression of Bax. On the other hand, Bcl-2 expression was low in DOX-treated cells, but treatment with C-phycocyanin enhanced the expression of Bcl-2 (Fig. 8B). The Bcl-2 to Bax ratio was significantly increased in cells treated with C-phycocyanin plus DOX (Bcl-2/Bax = 0.96) as compared with DOX alone (Bcl2/Bax = 0.4).

FIGURE 8

FIGURE 8

Apoptosis is reported to be accompanied by an efflux of cytochrome c from the mitochondria into the cytosolic compartment. Hence, an immunoblot analysis was performed to detect cytosolic cytochrome c release from mitochondria. As shown in Figure 9, treatment of cardiomyocytes with 1 μM DOX for 24 hours resulted in the release of cytochrome c from mitochondria. Pretreatment of C-phycocyanin or Spirulina totally inhibited the DOX-induced cytochrome c release from mitochondria.

FIGURE 9

FIGURE 9

To investigate the DOX-induced apoptosis through the activation of caspase-3, the activity of caspase-3 was determined (Fig. 10). Caspase-3 activity was increased 4-fold following the treatment of cells with 1 μM of DOX. Both C-phycocyanin and Spirulina pretreatments significantly (P < 0.001) inhibited the DOX-induced activation of caspase-3 in cardiomyocytes.

FIGURE 10

FIGURE 10

To evaluate whether C-phycocyanin or Spirulina could modify the chemotherapeutic efficacy of DOX, we investigated the effect of C-phycocyanin and Spirulina on the DOX-induced cell killing in human ovarian cancer cells in vitro. Results, shown in Figure 11, demonstrate that co-administration of DOX with C-phycocyanin or Spirulina had no significant effect on DOX-induced cell death. These results suggest that the antitumor activity of DOX was not compromised by C-phycocyanin or Spirulina.

FIGURE 11

FIGURE 11

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DISCUSSION

In the present study, DOX-treated cells showed a significant level of ROS generation revealed by an increase in the DCF and HE fluorescence intensities. Our results are consistent with the results reported by others, who demonstrated the generation of superoxide47 and hydrogen peroxide9,24,47,48 in cardiomyocytes treated with DOX. Some studies suggested superoxide as the major source for oxidative stress,7,49 whereas others suggested H2O2 as more probable oxidant species in inducing oxidative stress under DOX treatment.9,13,23,48,50 The overexpression of MnSOD has been shown to be cardioprotective in DOX-treated animals, suggesting the primary role of superoxide in causing DOX-induced cardiotoxicity.22 Pretreatment of cardiomyocytes with C-phycocyanin or Spirulina preparation significantly attenuated the DOX-induced DCF and HE fluorescence, suggesting that they both possess ROS-scavenging properties. Recently, using an in vitro electron paramagnetic resonance (EPR) spin-trapping technique, we demonstrated that both C-phycocyanin and Spirulina posses significant superoxide scavenging activity.45 It has also been shown by others that C-phycocyanin scavenges superoxide, peroxyl, and peroxynitrite.37-40 A recent study has shown that C-phycocyanin, extracted from blue-green alga, Aphanizomenon flos-aquae, possesses antioxidant activity.51

Oxidative stress that results from exposure to H2O2 and reactive oxygen radicals causes apoptosis in several cell systems, including cardiomyocytes.9-12,14 Cardiomyocytes exposed to DOX undergo apoptosis and this effect is mainly due to the formation of oxygen free radicals.9,21,24,28 The inhibition of apoptosis by antioxidants such as FeTBAP, Trolox®, Carvedilol, vitamin E, Ebselen (GPX mimetic), PBN (spin-trap), and by the overexpression of antioxidant enzymes, including SOD, catalase, and GST further supports the involvement of oxidative stress in DOX-induced apoptosis.24-27,46,52 It was reported that the iron chelator, dexrazoxane abrogates DOX-induced apoptosis and this was attributed to the inhibition of DOX-induced hydroxyl radical formation.21,31 In the present study, pretreatment of isolated cardiomyocytes with Spirulina or C-phycocyanin significantly ameliorated the DOX-induced oxidative stress, inhibited the apoptotic pathway, prevented DNA fragmentation, and decreased the number of apoptotic cells, suggesting that these protective effects could be attributed to C-phycocyanin.

In the present study, the occurrence of apoptosis in cardiomyocytes was confirmed by independent methods: TUNEL assay, DNA laddering, Bax and Bcl-2 protein expression, cytochrome c release, and caspase-3 activity. Flow cytometric and confocal microscopy studies showed a significant increase in the number of apoptotic cells following DOX treatment. Bcl-2 family proteins are key mediators of apoptosis. In DOX-treated cells, Bcl-2 was severely degraded suggesting Bcl-2 degradation may contribute to apoptosis.53 H2O2 exposure promotes translocation of the proapoptotic proteins such as Bax and Bad to the mitochondria, and a subsequent release of cytochrome c in cytosol from the mitochondria leading to apoptosis.24 In the present study, DOX-treated cardiomyocytes showed increased Bax-protein expression and cytochrome c release in cytosol. DOX-treated cardiomyocytes also showed decreased Bcl-2 protein expression. Our results are in agreement with the results published previously showing an increase in Bax and a decrease in Bcl-2 protein expression in cardiomyocytes following DOX treatment.47,54 Caspase-3 activity was increased by several-fold in DOX-treated cardiomyocytes. Our results suggest that DOX-induced apoptosis occurs, in part, via the activation of caspase-3. These findings are in agreement with the earlier reported studies, demonstrating the DOX-induced caspase-3 activation.9,47,48 H9c2 cardiac muscle cells exposed to DOX have also been reported to undergo apoptosis and it is suggested that this is due to induction of Bax and caspase-3.55 Recent studies have proposed several mechanisms implicating DOX-induced cardiomyocyte apoptosis, including a mitochondrial-dependent pathway, an extrinsic receptor-mediated pathway, and an endoplasmic reticulum pathway.56-59 The redox-cycling of DOX has been widely implicated in DOX-induced cardiomyocyte apoptosis by direct initiation of a mitochondrial-dependent apoptotic pathway.9,48 In the mitochondrial pathway, mitochondrial dysfunction due to DOX-induced oxidative stress can cause the release of cytochrome c, leading to the formation of an apoptosome complex, which activates caspase-3, a key apoptotic cell death proteosome.9,48,49,60,61

Pretreatment of cells with C-phycocyanin or Spirulina significantly inhibited the DOX-induced apoptosis. DOX-induced increase in TUNEL positive cells, DNA fragmentation was inhibited by C-phycocyanin and Spirulina. The increase in caspase-3 activity and cytosolic cytochrome c from mitochondria by DOX was significantly attenuated by C-phycocyanin and Spirulina. DOX-induced increase in Bax protein expression was inhibited by C-phycocyanin. The DOX-induced alteration in Bax protein, caspase-3 activity, and cytosolic cytochrome c levels were restored to normal levels with C-phycocyanin. Furthermore, a significant increase in the Bcl-2 to Bax ratio is likely to be one of the factors responsible for the inhibitory effect of C-phycocyanin on DOX-induced apoptosis. This indicates that C-phycocyanin inhibits the DOX-induced apoptosis by inhibiting Bax expression, cytosolic cytochrome c, and caspase-3 activity. The antiapoptotic effect of C-phycocyanin can be attributed to a free radical scavenging mechanism. This finding is consistent with the previous reports on the protection of DOX-induced apoptosis by antioxidants namely, FeTBAP,62 Trolox®,28 Probucol29 PBN,9 Carvedilol,47 and with the overexpression of the genes of antioxidant enzymes in transgenic mice, including SOD,23 thioredoxin,63 and GST.24

Although the results obtained from this study suggest C-phycocyanin protection of cardiomyocytes from DOX-induced apoptosis through the inhibition of oxidative stress, other possible mechanism cannot be excluded. The signal transduction pathway connecting oxidant exposure to apoptosis may involve p38 MAPK,14 JNK,64 and PI3-Kinase/Akt.65 H2O2-induced activation of NF-κB during DOX-induced apoptosis has been reported.13 A link between oxidative stress, JNK activation, and apoptotic cell death involving the mitochondrial death pathway have been demonstrated in cardiomyocytes.16,17 It has been recently shown that the MAP kinase family plays a role in DOX-induced apoptosis and is known to be activated in presence of ROS.15,66,67 p38 MAPK was activated in daunomycin-induced apoptosis in cardiomyocytes and the inhibition of p38 MAPK, significantly reduced the amount of apoptosis,15 suggesting the involvement of p38 MAPK in DOX-induced apoptosis. The involvement of the transcription factor p53 on DOX-induced cardiotoxicity was also reported.68 It is likely that the C-phycocyanin might alter the oxidative stress-activated signaling cascade thereby inhibiting the apoptosis induced by DOX. This requires further investigation.

In summary, our study demonstrated that C-phycocyanin and Spirulina significantly attenuated the DOX-induced increase in ROS, reduced the number of apoptotic cells, and inhibited the Bax protein expression and caspase-3 activity. The anti-apoptotic activity of C-phycocyanin and Spirulina is therefore attributed to its antioxidant activity. These findings support our earlier observation, which demonstrated the cardioprotective role of Spirulina in DOX-induced cardiotoxicity in mice, and further suggested C-phycocyanin as one of the major active constituents of Spirulina that is responsible for its cardioprotective effects. Furthermore, C-phycocyanin does not compromise the antitumor effect of DOX. Perhaps understanding of the exact mechanism(s) of action of C-phycocyanin in modulating DOX-induced apoptotic signaling pathway will be critical in minimizing and/or avoiding cardiotoxicity of DOX in cancer patients with DOX chemotherapy.

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Keywords:

apoptosis; biliprotein; cardiotoxicity; C-phycocyanin; doxorubicin; oxidative stress; Spirulina

© 2006 Lippincott Williams & Wilkins, Inc.