Reduction of doxorubicin-induced cytotoxicity and mitochondrial damage by betanin in rat isolated cardiomyocytes and mitochondria

Abstract

Doxorubicin (DOX) is an anticancer drug which is used for treatment of several types of cancers. But the clinical use of doxorubicin is limited because of its cardiotoxicity and cardiomyopathy. Mitochondrial-dependent oxidative stress and cardiac inflammation appear to be involved in doxorubicin-induced cardiotoxicity. Betanin as a bioactive compound in Beetroot (Beta vulgaris L.) displays anti-radical, antioxidant gene regulatory and cardioprotective activities. In this current study, we investigated the protective effect of betanin on doxorubicin-induced cytotoxicity and mitochondrial-dependent oxidative stress in isolated cardiomyocytes and mitochondria. Isolated cardiomyocytes and mitochondria were treated with three concentrations of betanin (1, 5 and 10 µM) and doxorubicin (3.5 µM) for 6 h. The parameters of cellular and mitochondrial toxicity were analyzed using biochemical and flow cytometric methods. Our results showed a significant toxicity in isolated cardiomyocytes and mitochondria in presence of doxorubicin which was related to reactive oxygen species (ROS) formation, increase in malondialdehyde (MDA), increase in oxidation of GSH to GSSG, lysosomal/mitochondrial damages and mitochondrial swelling. While betanin pretreatment reverted doxorubicin-induced cytotoxicity and oxidative stress in isolated cardiomyocytes and mitochondria. These results suggest that betanin elicited a typical protective effect on doxorubicin-induced cytotoxicity and oxidative stress. It is possible that betanin could be used as a useful adjuvant in combination with doxorubicin chemotherapy for reduction of cardiotoxicity and cardiomyopathy.

Keywords

Beetroot chemotherapy protection cardiomyocyte cardiotoxicity

Introduction

Doxorubicin (DOX) as an anticancer antibiotic belonging to the anthracycline family was discovered in the late 1960s.¹ Doxorubicin is widely used in clinic for treatment of several types of tumors, such as solid tumors, soft-tissue sarcomas, different forms of lymphomas and leukemia.² Despite the beneficial effectiveness of this drug in the treatment of cancers, a few years later after its discovery, severe cardiotoxic side effects such as cardiomyopathy and cardiac toxicity have been reported in patients.³ In addition, the use of doxorubicin is another important reason of dilated cardiomyopathy that creates congestive heart failure.⁴ Therefore, Doxorubicin-induced cardiotoxicity leads to limiting its clinical application. Despite above major side effects induced by doxorubicin, now this drug is widely used in clinic and is still one of the strongest anti-tumor drug available for clinical use.⁵

The mechanism of action of doxorubicin-induced cardiotoxicity is controversial, and there are several hypotheses.⁶ At first, it was accepted that doxorubicin-induced cardiotoxicity is entirely independent from its anti-tumor effect.⁷ Therefore, the majority of studies focused on doxorubicin -induced mitochondrial dysfunction and cardiotoxicity.⁸ Doxorubicin accumulates in mitochondria and can bind to mitochondrial DNA and other biomolecules such as cardiolipin and the mitochondrial abundant phospholipids.⁶ Accumulation of doxorubicin in mitochondria can create intramitochondrial ROS and reactive nitrogen species (RNS) formation mainly through nonenzymatic mechanisms, leading to activation of PARP-dependent cell death pathways.⁶ Doxorubicin-induced mitochondrial DNA oxidation is a major contributor of heart failure development and cardiomyopathy.⁹ Other major mechanisms of doxorubicin-induced ROS production are the redox cycling-dependent formation of superoxide anion, alteration in iron metabolism and formation of hydroxyl radicals through Haber-Weiss reaction.¹⁰ Subsequently enhanced ROS formation causes activation of proinflammatory transcription factor nuclear factor kappa B (NF-κB), inducible nitric oxide synthase and finally cell death in cardiomyocytes.¹¹ Therefore, mitochondrial dysfunction, a decrease in the activity of endogenous antioxidants and an increase of free radicals in the cardiomyocytes play important roles in doxorubicin-induced cardiotoxicity.¹² Therefore, a number of compounds with potential cardioprotective activity, including natural compounds with ROS scavenger properties and mitochondrial protective effect can probably play a promising role in reducing the cardiotoxicity caused by doxorubicin.

Betanin (betanidin-5-O-b-glucoside) as a major phytochemical agent found in Beetroot (Beta vulgaris L.) displays the antioxidant potential and anti-inflammatory effects.¹³ Because of the resistance to gastrointestinal digestion, Betanin is absorbed by the epithelial cells and reaches the plasma.¹⁴ This betanin shows free-radical scavenger potential through electron or hydrogen donation.¹⁵ Also, betanin suppresses the pro-inflammatory NF-κB pathways and simultaneously induces the transcription of antioxidant genes through the nuclear factor erythroid-2-related factor 2.¹⁶ It has been reported that betanin can reduce low-density lipoprotein (LDL) and lipid membranes peroxidation, modulate gene expression and ROS formation in order to decrease inflammatory cytokines and enhancing antioxidant enzyme activities.¹⁵ As a supportive therapy, betanin shows potential to attenuate the pathophysiological effects caused by the inflammation and oxidative stress that lead to cardiovascular disease (CVD).¹⁷ Due to the promising effect of betanin on ROS scavenger, antioxidant, anti-inflammatory and cardioprotection, it deserves to be more studied. Therefore, here we aim to evaluate the effect of betanin against doxorubicin-induced toxicity in isolated cardiomyocytes and mitochondria obtained from rat heart.

Materials and methods

Chemicals and reagents

Collagenase (Type II), Penicillin and Streptomycin Solution, Fetal Bovine Serum (FBS), 199 Medium, Creatine, 2′,7′-Dichlorofuorescin Diacetate (DCFH-DA), Carnitine, Potassium chloride, Taurine, N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), Hank’s Trypan blue, Rhodamine123, Balanced Salt Solution (HBSS), Bovine Serum Albumin (BSA), Dimethyl sulfoxide (DMSO), -Amino-2-hydroxymethyl-propane-1,3-diol (TRIS), D-mannitol, Sucrose, 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2 Monopotassium phosphate, 3-morpholinopropane-1-sulfonic acid (MOPS), Sodium succinate, Rotenone, Magnesium chloride and Acridine Orange were purchased from Sigma (St. Louis, MO USA). Xylazine and Ketamine were obtained from CEVA Santé Animale (Naaldwijk, the Netherlands). Doxorubicin with CAS number 25316-40-9 and a purity of about 99%, was gifted from Dr. Abidi Pharmaceuticals Co. Iran. It was freshly prepared before use and dissolved in 0.05% DMSO. Also, betanin with CAS number 0007659952 and a purity of about 99%, were purchased from Sigma (St. Louis, MO USA) and dissolved in 0.05% DMSO.

Animals

Adult male Wistar rats (8- to 9-week-old) weighting 180–220 g were obtained from the experimental animal center at Baqiyatallah University of Medical Sciences, Tehran, Iran. The Committee of Experimental Animals of Shahid Beheshti University of Medical Sciences approved this study. All animals were cared and experimented on in accordance with the recommendations in the Guide for the Use and Care of Laboratory Animals approved by the Ethics Committee at the Shahid Beheshti University of Medical Sciences, Tehran, Iran. The approval code is IR.SBMU.RETECH.REC.1399.725. In the current study, isolated cardiomyocytes (10⁶ cells/ml) and mitochondria (1000 µg/ml) obtained from five animals were used.

Cardiomyocyte isolation

Cardiomyocytes were isolated from adult male Wistar rats. Rats were intraperitoneally anesthetized with combination of ketamine (50 mg/kg) and xylazine (10 mg/kg). After rats appeared calm, hearts were quickly excised, and directly perfused on a modified Langendorff perfusion apparatus with Powell medium (containing: 110 mM NaCl, 1.2 MgSO₄ 7H₂O, 2.5 mM KCl, 1.2 mM KH₂PO₄, 25 mM HEPES and 10 mM D (+)-Glucose monohydrate in ultra-pure sterile water with pH = 74 adjusted with 2 M NaOH) for 5 min. Powell medium were replaced by enzyme mixtures containing 30 µM CaCl₂ and 5 mg/ml collagenase, in warm Powell medium for 25 min. Then, hearts were dissected mechanically to small pieces and shaken in flasks with enzyme mixture for another 10 min. The collagenase digested ventricle suspensions were then filtered through a sieve (180 µm) to remove remnants of connective tissues. After rinsing with Powell medium, cell suspensions were placed on top of BSA solution where non-cardiomyocytes were removed by a density gradient. Isolated cardiomyocytes were suspended in CCT medium containing taurine, creatine and carnitine in medium 199 supplemented with antibiotics (100 µg/ml penicillin and 100 µg/ml streptomycin) at 37°C under a 5% CO₂−95% air atmosphere. One hour after plating, the CCT medium was changed to remove unattached dead.¹⁸ The isolated cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and 10 µM betanin group for 6 h.

Measurement of cell viability

After the cardiomyocytes were seeded and allowed to attach, the MTT assay was used to evaluate the effects of doxorubicin and betanin on cell viability. Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 96 well plate (10⁴ cells per well) at 37°C for 6 h. 25 µl of 0.5 mg/mL MTT was added to each well. After 2 h of incubation the formazan crystals were dissolved in 100 µL DMSO and the absorbance was measured at 570 nm.¹⁹ The viability was represented as the percentage absorbance compared with untreated control group.

Intracellular ROS measurement

Intracellular ROS accumulation was examined using the fluorescence probe 2′-7′dichlorofluorescin diacetate (DCFH-DA). Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 24 well plate (10⁵ cells per well) at 37°C for 6 h. After incubation, the cells were washed twice with PBS and incubated with 5 µM DCFH-DA dissolved in CCT medium for 15 min in a dark chamber. Then the fluorescence intensity of DCF was detected by a Cyflow Space-Partec flowcytometry.²⁰

Measurement of mitochondrial damages

Mitochondrial membrane potential collapse was examined using the fluorescence probe rhodamine 123. Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 24 well plate (10⁵ cells per well) at 37°C for 6 h. After incubation, the cells were washed twice with PBS and incubated with 1 µM rhodamine 123 dissolved in CCT medium for 15 min in a dark chamber. Then the fluorescence intensity of rhodamine 123 was detected by a Cyflow Space-Partec flowcytometry.²¹

Measurement of lysosomal damages

Lysosomal membrane integrity was measured using the fluorescence probe acridine orange (AO). Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 24 well plate (10⁵ cells per well) at 37°C for 6 h. After incubation, the cells were washed twice with PBS and incubated with 5 µM acridine orange dissolved in CCT medium for 15 min in a dark chamber. Then the fluorescence intensity of acridine orange was detected by a Cyflow Space-Partec flowcytometry.¹⁹

Measurement of the reduced (GSH) and oxidized (GSSG) glutathione levels

The levels of glutathione (GSH) and glutathione disulfide (GSSG) were measured by Hissin and Hilf method.²² Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 24 well plate (10⁵ cells per well) at 37°C for 6 h. After incubation, the cells were washed twice with PBS and mechanically lysed using a glass homogenizer in phosphate buffer (0.1 M with pH 7.4), then the cells were centrifugated at 8,000 ×g at 4°C for 10 min. Then 100 µl supernatant was blended with 3 ml reaction solution (500 mM TRIS-HCl and 10 mM DTNB with pH = 8.0) for measurement of GSH. The optical density was measured at 412 nm, after 15 min of incubation at 25°C. Similarly, 100 µl of supernatant was blended to 3 ml of reaction solution (150 μM NADPH, 500 mM TRIS–HCl buffer, 1 mM EDTA, glutathione reductase, 10 mM DTNB and3 mM MgCl₂) for measurement of GSSG. The optical density was measured at 412 nm, after 15 min of incubation at 25°C.

Measurement of malondialdehyde

As an indicator of lipid peroxidation, malondialdehyde (MDA) levels was measured. Briefly, cardiomyocytes were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin (IC50 6 h) group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 24 well plate (10⁵ cells per well) at 37°C for 6 h. After incubation, the cells were washed twice with PBS and mechanically lysed in 1 ml 0.1% (w/v) trichloroacetic acid (TCA) by glassy homogenizer and centrifugated at 10,000 ×g for 10 min. The obtained supernatant was combined with 4 ml of 20% TCA and 0.5% TBA. The mixture was boiled for 15 min at 90°C. After cooling on ice and centrifugation at 1000 ×g for 10 min, the absorbance was measured at 532 nm.²³

Mitochondria isolation

Mitochondria were isolated from the rat heart as previously described by Gostimskaya and Galkin.²⁴ Briefly, after deep anesthesia the animals with combination of ketamine and xylazine, the heart was cleared from blood vessels, chopped and homogenized with a glass homogenizer in isolation buffer (10 mM Tris-HCl (pH 7.4), 75 mM sucrose, 1 mM EDTA, 225 mM mannitol at 4°C). After centrifugation of the homogenate at 1000 ×g for 10 min, the pellet was removed and supernatant was collected. The mitochondria in the supernatant were sedimented at 10000 ×g for 10 min at 4°C. The protein content in mitochondria was measured using the Bradford assay. Bovine serum albumin (BSA) is used as a protein concentration standard.

Measurement of mitochondrial function

Mitochondrial Function was measured by the activity of succinate dehydrogenase (complex II). Briefly, isolated mitochondria were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 96 well plate (100 µg/ml) at 37°C for 1 h. Then 25 µl of 0.4% MTT was added to each well and incubated for 30 min at 37°C. The formazan crystals were dissolved in DMSO and the optical density was detected at 570 nm.²⁵ The succinate dehydrogenase was represented as the percentage absorbance compared with untreated control group.

Measurement of mitochondrial swelling

Mitochondrial swelling was estimated by examining changes in light scattering as monitored spectrophotometrically at 540 nm.²⁶ Briefly, isolated mitochondria were randomized into six experimental groups: 0.05% DMSO as the control group, 3.5 µM doxorubicin group, 3.5 µM doxorubicin + 1 µM betanin group, 3.5 µM doxorubicin + 5 µM betanin group, 3.5 µM doxorubicin + 10 µM betanin group and betanin group in a 96 well plate (100 µg/ml) at 37°C for 1 h. After 1 h, the absorbance at 540 nm was monitored during 60 min using a spectrophotometer.

Statistics

The results are presented as mean ± SD from three repeated experiments. Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA). Data were subjected to one- and two-way ANOVA followed by post hoc Tukey and Bonferroni test respectively. A value of <0.05 was considered to indicate a significant difference.

Results

Cytoprotective effect of betanin on doxorubicin-induced cytotoxicity in isolated cardiomyocytes

Addition of doxorubicin (3.5 µM) to isolated cardiomyocytes resulted in a decrease in the number of cells as shown by MTT assay at 6 h (Figure 1(A)). As seen in Figure 1(B), pretreatment of isolated cardiomyocytes with 10 µM betanin, 30 min before doxorubicin addition prevented cytotoxicity caused by the drug. The obtained results showed that 10 µM betanin significantly (p < 0.001) prevented of doxorubicin-induced cytotoxicity.

Figure 1.

Effects of betanin treatment on doxorubicin-induced cytotoxicity (A). DOX, doxorubicin; Data presented are the mean ± SD (n = 3). ***p < 0.001 compared with the group with no pretreatment; ^###p < 0.001 compared with the group with doxorubicin. (B) Effects of betanin treatment on doxorubicin-induced lipid peroxidation. Data presented are the mean ± SD (n = 3). ***p < 0.001 compared with the group with no treatment; ^###p < 0.001 compared with the group with doxorubicin.

Protective effect of betanin on doxorubicin-induced oxidative stress in isolated cardiomyocytes

The MDA amounts in isolated cardiomyocytes after the treatment with doxorubicin and betanin was shown in Figure 1(B). The MDA amounts increased rapidly 6 h after the treatment with 3.5 µM doxorubicin when compared with untreated control group (p < 0.001). While these data revealed that pretreatment of isolated cardiomyocytes with 10 µM betanin, 30 min before doxorubicin addition, significantly (p < 0.001) decreased the MDA level in the culture medium. That means betanin could inhibit the lipid peroxidation and subsequent cardiomyocytes membrane damage under doxorubicin-induced oxidative stress.

Protective effect of betanin on doxorubicin-induced GSG depletion in isolated cardiomyocytes

Altered intracellular redox status (GSH/GSSG) has a pivotal role in doxorubicin-induced cell death in the cardiomyocytes. To determine whether the intracellular redox status (GSH/GSSG) contributes to the protective effect of betanin on doxorubicin-induced cytotoxicity, we measured GSH and GSSG levels in isolated cardiomyocytes. As shown in Table 1, doxorubicin exposure at 3.5 µM resulted in increased oxidation of GSH to GSSG, and the GSH/GSSG ratio fell significantly (p < 0.01) after doxorubicin addition. Pretreatment with 10 µM betanin 30 min before doxorubicin addition, prevented GSH reduction and GSSG elevation, leading to almost complete recovery of GSH/GSSG ratio.

Table 1.

Effects of betanin pretreatment on doxorubicin-induced GSH depletion in cardiomyocytes.

Groups	GSH (µM)	GSSG(µM)	GSH/GSSG ratio
Groups	6 h	6 h	6 h
Control	28 ± 1.3	11 ± 1.2	2.5 ± 0.02
DOX (3.5 µM)	17 ± 1.4^a	22 ± 1.3^a	0.7 ± 0.02^a
DOX (3.5 µM) + betanin (1 µM)	18 ± 1.3	21 ± 1.6	0.85 ± .03
DOX (3.5 µM) + betanin (5 µM)	18 ± 1.7	21 ± 1.5	0.85 ± 0.01
DOX (3.5 µM) + betanin (10 µM)	24 ± 1.1^b	9 ± 1.7^b	2.6 ± 0.04^b
Betanin (10 µM)	29 ± 1.1	9 ± 1.1	3.2 ± 0.02

^a Shows significant difference (p < 0.01) with control.

^b Shows significant difference (p < 0.01) with doxorubicin group.

Protective effect of betanin on doxorubicin-induced ROS formation in isolated cardiomyocytes

A significant increase of ROS was seen when isolated cardiomyocytes were treated with 3.5 µM doxorubicin (Figure 2), which could suggest the occurrence of oxidative stress after doxorubicin addition to cardiomyocytes. Pretreatment with 5 and 10 µM betanin 30 min before doxorubicin addition reduced the level of ROS formation caused by the drug. Betanin (5 and 10 µM) effectively reduced the levels of ROS formed after doxorubicin. Butylated hydroxytoluene (BHT) as a standard antioxidant was able to reduce ROS levels in isolated cardiomyocytes after doxorubicin addition.

Figure 2.

Effects of betanin treatment on doxorubicin-induced ROS Formation in cardiomyocytes. Representative fluorescence intensity of ROS staining with DCF-DA. Cardiomyocytes with DCF-DA fluorescence indicate elevated intracellular ROS. As shown the fluorescence intensity is increased after exposure with doxorubicin. The peak is translocated to the right compared to control. While treatment of betanin (5 and 10 µM) with doxorubicin showed that the fluorescence intensity is decreased and the peaks are translocated to the left.

Protective effect of betanin on doxorubicin-induced mitochondrial damages in isolated cardiomyocytes

A significant increase of fluorescence intensity of rhodamine 123 as indicator of the mitochondrial permeability transition and mitochondrial damages was observed when isolated cardiomyocytes were treated with 3.5 µM doxorubicin (Figure 3), which could show the mitochondria damages after doxorubicin addition to cardiomyocytes. Pretreatment with 5 and 10 µM betanin 30 min before doxorubicin addition reduced the fluorescence intensity of rhodamine 123 as indicator of the mitochondrial damages caused by the drug. Betanin (5 and 10 µM) effectively reduced the mitochondrial damages after doxorubicin. Cyclosporine A as a mitochondrial permeability transition (MPT) pore sealing agents was able to reduce mitochondrial damages in isolated cardiomyocytes after doxorubicin addition.

Figure 3.

Effects of betanin treatment on doxorubicin-induced loss of mitochondrial ΔΨm in cardiomyocytes. (A) Representative fluorescence intensity of rhodamine 123 staining. (B) Quantitative analysis of rhodamine 123 staining. DOX, doxorubicin. Data presented are the mean ± SD (n = 3). ***p < 0.001 compared with the group with no treatment; ^###p < 0.001 compared with the group with doxorubicin.

Protective effect of betanin on doxorubicin-induced lysosomal damages in isolated cardiomyocytes

A significant increase of fluorescence intensity of acridine orange as indicator of the lysosomal membrane permeabilization was observed when isolated cardiomyocytes were treated with 3.5 µM doxorubicin (Figure 4), which could show the lysosomal damages after doxorubicin addition to cardiomyocytes. Pretreatment with 5 and 10 µM betanin 30 min before doxorubicin addition reduced the fluorescence intensity of acridine orange as indicator of lysosomal membrane disintegrity caused by the drug. Betanin (5 and 10 µM) effectively reduced the lysosomal membrane disintegrity after doxorubicin. Chloroquine as a lysosomotropic agent was able to reduce the lysosomal membrane disintegrity in isolated cardiomyocytes after doxorubicin addition.

Figure 4.

Effects of betanin treatment on doxorubicin-induced loss of lysosomal membrane destabilization in cardiomyocytes. (A) Representative fluorescence intensity of acridine orange staining. (B) Quantitative analysis of acridine orange staining. DOX, doxorubicin. Data presented are the mean ± SD (n = 3). ***p < 0.001 compared with the group with no treatment; ^##p < 0.01, ^###p < 0.001 compared with the group with doxorubicin.

Succinate dehydrogenase activity in isolated mitochondria

Addition of doxorubicin (3.5 µM) to isolated rat heart mitochondria resulted in a decrease in the succinate dehydrogenase activity as shown by MTT assay at 60 min. As seen in Figure 5(A), pretreatment of isolated rat heart mitochondria with 5 and 10 µM betanin, 30 min before doxorubicin addition prevented mitochondrial dysfunction caused by the drug. This observation confirmed that the used concentration (5 and 10 µM) of betanin significantly (p < 0.001) prevented of doxorubicin-induced mitochondrial dysfunction.

Figure 5.

Effect of doxorubicin on succinate dehydrogenase activity and protective effect of betanin in isolated mitochondria (A). Mitochondrial SDH activity was evaluated by MTT assay. Presented data showed doxorubicin (3.5 µM) significantly decreased SDH activity compared to control group. Also, data showed betanin significantly increased SDH activity compared to treated groups with doxorubicin (3.5 µM). Effect of doxorubicin on mitochondrial swelling and protective effect of betanin in isolated mitochondria (B). Mitochondrial swelling was evaluated by monitoring absorbance at 540 nm. Presented data showed doxorubicin (3.5 µM) significantly induced mitochondrial swelling compared to control group. Also, data showed betanin significantly inhibited mitochondrial swelling compared to treated groups with doxorubicin (3.5 µM). SDH, Succinate Dehydrogenase Activity; DOX, doxorubicin. Data presented are the mean ± SD (n = 3). ***p < 0.001 compared with the group with no pretreatment; ^###p < 0.001 compared with the group with doxorubicin.

Mitochondrial swelling in isolated mitochondria

The decreased absorbance at 540 nm, as a criteria of mitochondrial membrane permeability and the mitochondrial swelling in isolated rat heart mitochondria was evaluated after addition of doxorubicin and betanin. Doxorubicin addition (3.5 µM) significantly caused mitochondrial swelling in treated isolated rat heart mitochondria as compared with untreated control group (p < 0.001). While pretreatment with 5 and 10 μM betanin significantly (p < 0.001) decreased mitochondrial swelling as compared with doxorubicin treated group (p < 0.001) (Figure 5(B)).

Discussion

Doxorubicin as a potent anticancer and the first-choice drug is used for many cancers such as the leukemia, lymphoma, breast, colon and liver cancer.²⁷ However, its use is decreased due to toxicity in several organs such the heart, skeleton, kidney, brain, muscle, liver and lung.²⁷ Among the above toxicities, the cardiotoxicity and cardiomyopathy are the most feared and serious side effect of this anticancer.²⁸ Despite the apparent toxicity of this drug due to its good effectiveness it is still widely used in the clinic. Since the main mechanism of action of doxorubicin is based on the inhibition of the enzyme topoisomerase II, causing DNA damage and induction of apoptosis and this mechanism is independent of the mechanism of its toxicity and oxidative stress.²⁹ Therefore, finding a solution to reduce the toxicity of doxorubicin during treatment can help to increase the effectiveness of the drug and reduce its toxic effects, especially cardiac toxicity. Therefore, this study aimed to provide a solution to reduce the toxicity of doxorubicin based on its mechanism of action through the mitochondrial-dependent oxidative stress at the cellular and mitochondrial level.

Doxorubicin-related cytotoxicity is often irreversible.³ Doxorubicin accumulates in mitochondria and binds to the phospholipid cardiolipin in the inner mitochondrial membrane. This binding leads to inhibition of complex I and complex II and disruption of the electron transport chain and inducing ROS generation in mitochondria.³⁰ Also, free radicals might be generated by other doxorubicin-mediated mechanisms, for example a quinone segment in the structure of doxorubicin is reduced by mitochondrial complex I into a reactive semiquinone which transfers an electron to a molecule oxygen and produces the superoxide anion free radical. Then, the produced semiquinone free radical is oxidized and rebounds to the quinone form in a sequence of reactions which is known as the redox cycling of doxorubicin.³¹ Other mechanisms such as the alteration in Iron homeostasis,³² induction of mtDNA damage through binding to endothelial nitric oxide synthase (eNOS) and disruption of Ca²⁺ homeostasis is involved in doxorubicin-related cytotoxicity.³³ The above mechanisms directly and indirectly lead to ROS and peroxynitrite (ONOO−) formation, mitochondrial membrane permeability, mitochondria dysfunction, cytochrome c and leading to apoptosis or necrosis.³⁴ The increasing levels of TNFα and NADPH oxidase mediated by doxorubicin can also generate the excessive oxidative stress and lead to redox modifications of macromolecules.³⁵ Previous studies have been reported that the antioxidative defense of cells reduces after exposure to doxorubicin.^12,34 Our studies on isolated cardiomyocytes and mitochondria showed that doxorubicin-induced cytotoxicity is exerted by oxidative stress, depletion of antioxidant defenses, production of reactive oxygen radical, and organelle damage such as lysosome, and especially mitochondria. Our data in the current study, are consistent with previously published studies. As shown in our results and other studies, mitochondria play a main role in doxorubicin-induced cytotoxicity and cardiotoxicity,³⁴ Therefore, protection of mitochondria and reduction of harmful factors to them can be an important strategy in reducing cytotoxicity and cardiac toxicity induced by doxorubicin.

Scientific documents have demonstrated the protective effect of the plant-derived antioxidant natural products on mitochondrial damage, oxidative stress and cardiovascular disease (CVD).³⁶ Several mechanisms such as the maintaining a redox state in the cells due to their scavenging ROS abilities, modulating the expression of genes involved in intracellular defense, binding to receptor sites as antagonists and competing for active sites in enzymes in various subcellular structures are suggested.³⁷ One of the plant-derived antioxidant natural products is betanin which has been proven that it’s antioxidant and anti-inflammatory activities can reduce the cardiovascular disorders.¹⁷ Previous studies have reported the free-radical scavenger activity of betanin.¹⁷ In the current study we showed that betanin significantly decreased ROS formation and oxidative stress induced by doxorubicin. The high level of ROS inside and outside mitochondria after exposure with doxorubicin leads to damaged mitochondrial components, opening of MPT pores, mitochondrial depolarization, high amplitude mitochondrial swelling and outer membrane rupture.³⁸ In the current study, increased mitochondrial depolarization and mitochondrial swelling were found in the doxorubicin group compared to control group, demonstrating mitochondrial toxicity of doxorubicin in isolated cardiomyocytes and mitochondria. These toxicity parameters were reversed in betanin groups, consistent with a protective effect of betanin on mitochondria.^39,40 This has demonstrated that mitochondrial dysfunction damages lysosomal structure in a ROS-dependent manner⁴¹ and that loss of Transient Receptor Potential Mucolipin (TRPML1) as a pore channel in lysosome induces the accumulation of ROS, which in turn cause loss of mitochondrial membrane potential and fragmentation of mitochondria.⁴² It has been reported that doxorubicin accumulates in lysosomes as a hydrophobic weak base anticancer drug and may be associated with lysosomal damage.⁴³ Our findings in this study showed that betanin reduced doxorubicin-induced lysosomal damage in isolated cardiomyocytes.

In summary, we showed that betanin has protective effect against doxorubicin-induced cytotoxicity, oxidative stress and mitochondrial/lysosomal damages in isolated cardiomyocytes and mitochondria. The obtained results of the current study are consistent with previous studies for inhibition and reduction of cardiotoxicity and cytotoxicity induced by doxorubicin though antioxidant action.^44–47 We also showed betanin’s antioxidant activities, protection of mitochondria and lysosome, are likely the mechanism responsible for its protection against doxorubicin. In the light of these findings, we concluded that betanin exerted protective role in DOX-induced toxicity in isolated cardiomyocytes and mitochondria, and it would be valuable to examine in vivo effects and its potential in clinical uses.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Shahid Beheshti University of Medical Sciences, Deputy of Research with ethics code IR.SBMU.RETECH.REC.1399.725.

ORCID iD

A Salimi

References

Zhao

Zhang

. Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes. Sci Rep 2017; 7: 1–11.

Rivankar

. An overview of doxorubicin formulations in cancer therapy. J Cancer Res Therap 2014; 10: 853.

Chatterjee

Zhang

Honbo

, et al. Doxorubicin cardiomyopathy. Cardiology 2010; 115: 155–162.

Haq

Legha

Choksi

, et al. Doxorubicin-induced congestive heart failure in adults. Cancer 1985; 56: 1361–1365.

Baxter-Holland

Dass

. Doxorubicin, mesenchymal stem cell toxicity and antitumour activity: implications for clinical use. J Pharm Pharmacol 2018; 70: 320–327.

Varga

Ferdinandy

Liaudet

, et al. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am J Physiol-Heart C 2015; 309: H1453–H1467.

Wenningmann

Knapp

Ande

, et al. Insights into doxorubicin-induced cardiotoxicity: molecular mechanisms, preventive strategies, and early monitoring. Mol Pharmacol 2019; 96: 219–232.

Osataphan

Phrommintikul

Chattipakorn

, et al. Effects of doxorubicin-induced cardiotoxicity on cardiac mitochondrial dynamics and mitochondrial function: insights for future interventions. J Cell Mol Med 2020; 24(12): 6534–6557.

Lauritzen

Kleppa

Aronsen

, et al. Impaired dynamics and function of mitochondria caused by mtDNA toxicity leads to heart failure. Am J Physiol Heart Circ Physiol 2015; 309: H434–H449.

10.

Kalyanaraman

. Teaching the basics of the mechanism of doxorubicin-induced cardiotoxicity: have we been barking up the wrong tree? Redox Biol 2020; 29: 101394.

11.

Mukhopadhyay

Rajesh

Bátkai

, et al. Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol Heart Circ Physiol 2009; 296: H1466–H1483.

12.

Cappetta

De Angelis

Sapio

, et al. Oxidative stress and cellular response to doxorubicin: a common factor in the complex milieu of anthracycline cardiotoxicity. Oxid Med Cell Longev 2017; 2017: 1521020.

13.

Baião

DDS

De Freitas

Gomes

, et al. Polyphenols from root, tubercles and grains cropped in Brazil: chemical and nutritional characterization and their effects on human health and diseases. Nutrients 2017; 9: 1044.

14.

Tesoriere

Allegra

Butera

, et al. Absorption, excretion, and distribution of dietary antioxidant betalains in LDLs: potential health effects of betalains in humans. Am J Clin Nutr 2004; 80: 941–945.

15.

Esatbeyoglu

Wagner

Motafakkerazad

, et al. Free radical scavenging and antioxidant activity of betanin: electron spin resonance spectroscopy studies and studies in cultured cells. Food Chem Toxicol 2014; 73: 119–126.

16.

Krajka-Kuźniak

Paluszczak

Szaefer

, et al. Betanin, a beetroot component, induces nuclear factor erythroid-2-related factor 2-mediated expression of detoxifying/antioxidant enzymes in human liver cell lines. Br J Nutr 2013; 110: 2138–21349.

17.

Silva

DVTd

Baião

DdS

Ferreira

, et al. Betanin as a multipath oxidative stress and inflammation modulator: a beetroot pigment with protective effects on cardiovascular disease pathogenesis. Crit Rev Food Sci Nutr 2020: 1–16. Online ahead of print.

18.

Nippert

Schreckenberg

Schlüter

K-D

. Isolation and cultivation of adult rat cardiomyocytes. JoVE (J Vis Exp). 2017; 128: e56634.

19.

Ahangari

Khezri

Jahedsani

, et al. Ellagic acid alleviates clozapine-induced oxidative stress and mitochondrial dysfunction in cardiomyocytes. Drug Chem Toxicol 2020: 1–9. Online ahead of print.

20.

Eruslanov

Kusmartsev

. Identification of ROS using oxidized DCFDA and flow-cytometry. In: Armstrong

(ed) Advanced protocols in oxidative stress II. Berlin: Springer, 2010, pp. 57–72.

21.

Juan

Cavazzoni

Saez

, et al. A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry. Cytometry A 1994; 15: 335–342.

22.

Hissin

Hilf

. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 1976; 74: 214–226.

23.

Beach

Giroux

. Inhibition of lipid peroxidation promoted by iron (III) and ascorbate. Arch Biochem Biophy 1992; 297: 258–264.

24.

Gostimskaya

Galkin

. Preparation of highly coupled rat heart mitochondria. JoVE (J Vis Exp). 2010: e2202. Online ahead of print.

25.

Zhang

Sun

. Mitochondrial Ca²⁺ retention capacity assay and Ca²⁺-triggered mitochondrial swelling assay. JoVE (J Vis Exp). 2018: e56236. Online ahead of print.

26.

Zhao

Liu

, et al. Vanadium compounds induced mitochondria permeability transition pore (PTP) opening related to oxidative stress. J Inorg Biochem 2010; 104: 371–378.

27.

Pugazhendhi

Edison

TNJI

Velmurugan

, et al. Toxicity of Doxorubicin (Dox) to different experimental organ systems. Life Sci 2018; 200: 26–30.

28.

Oliveira

Melo

Carvalho

, et al. Doxorubicin cardiotoxicity and cardiac function improvement after stem cell therapy diagnosed by strain echocardiography. J Cancer Sci Ther 2013; 5: 052.

29.

Thorn

Oshiro

Marsh

, et al. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011; 21: 440.

30.

Mordente

Meucci

Silvestrini

, et al. Anthracyclines and mitochondria. In: Scatena

Bottoni

Giardina

(eds) Advances in mitochondrial medicine. Springer, 2012, pp. 385–419.

31.

Zhu

Sarkar

Scott

, et al. Doxorubicin redox biology: redox cycling, topoisomerase inhibition, and oxidative stress. React Oxyg Species (Apex) 2016; 1: 189.

32.

Montaigne

Hurt

Neviere

. Mitochondria death/survival signaling pathways in cardiotoxicity induced by anthracyclines and anticancer-targeted therapies. Biochem Res Int 2012; 2012: 951539.

33.

Sawyer

Peng

Chen

, et al.

Mechanisms of anthracycline cardiac injury: can we identify strategies for cardioprotection?

Prog Cardiovasc Dis 2010; 53: 105–113.

34.

Gorini

De Angelis

Berrino

, et al. Chemotherapeutic drugs and mitochondrial dysfunction: focus on doxorubicin, trastuzumab, and sunitinib. Oxid Med Cell Longev 2018; 2018: 7582730.

35.

Gilliam

Ferreira

Bruton

, et al. Doxorubicin acts through tumor necrosis factor receptor subtype 1 to cause dysfunction of murine skeletal muscle. J Appl Physiol 2009; 107: 1935–1942.

36.

Chang

Zhang

, et al. Natural drugs as a treatment strategy for cardiovascular disease through the regulation of oxidative stress. Oxid Med Cell Longev 2020; 2020: 1–20.

37.

Kurutas

. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. Nutr J 2015; 15: 1–22.

38.

Wallace

Sardão

Oliveira

. Mitochondrial determinants of doxorubicin-induced cardiomyopathy. Circ Res 2020; 126: 926–941.

39.

Han

Zhang

Yang

, et al. Betanin attenuates paraquat-induced liver toxicity through a mitochondrial pathway. Food Chem Toxicol 2014; 70: 100–106.

40.

Ahmadian

Khosroushahi

Eghbal

, et al. Betanin reduces organophosphate induced cytotoxicity in primary hepatocyte via an anti-oxidative and mitochondrial dependent pathway. Pestic Biochem Phys 2018; 144: 71–78.

41.

Demers-Lamarche

Guillebaud

Tlili

, et al. Loss of mitochondrial function impairs lysosomes. J Biol Chem 2016; 291: 10263–10276.

42.

Coblentz

St. Croix

Kiselyov

. Loss of TRPML1 promotes production of reactive oxygen species: is oxidative damage a factor in mucolipidosis type IV? Biochem J 2014; 457: 361–368.

43.

Zhitomirsky

Assaraf

. Lysosomal accumulation of anticancer drugs triggers lysosomal exocytosis. Oncotarget 2017; 8: 45117.

44.

Patil

Balaraman

. Effect of green tea extract on doxorubicin induced cardiovascular abnormalities: antioxidant action. Iran J Pharm Res 2011; 10: 89.

45.

Mandziuk

Gieroba

Korga

, et al. The differential effects of green tea on dose-dependent doxorubicin toxicity. Food Nutr Res 2015; 59: 29754.

46.

Alpsoy

Aktas

Uygur

, et al. Antioxidant and anti-apoptotic effects of onion (Allium cepa) extract on doxorubicin-induced cardiotoxicity in rats. J Appl Toxicol 2013; 33: 202–208.

47.

Kaiserová

Šimůnek

van der Vijgh

, et al. Flavonoids as protectors against doxorubicin cardiotoxicity: role of iron chelation, antioxidant activity and inhibition of carbonyl reductase. Biochim Biophys Acta 2007; 1772: 1065–1074.