Doxorubicin-associated cardiotoxicity is an alarming concern as it limits the use of anticancer drugs in a clinical setting. The objective of this study was to test the therapeutic role of lactoferrin in preventing doxorubicin-induced cardiotoxicity and thus address this severe side effect of chemotherapy.
MethodsWistar rats received vehicle or DOX (2.5 mg/kg, i.p.) three times a week for two weeks and lactoferrin (50 mg/kg, i.p.) for 15 days (i.e., from day 1 to day 15 along with DOX). Changes in body weight, heart weight, and cardiotoxicity markers (blood pressure, LDH, CK-MB, and cardiac troponin I) were evaluated. Furthermore, all the animals were evaluated for the modulation of inflammatory markers (BNP, IL-6, CRP, and TNF-α), histopathological changes in heart tissue, and oxidant and antioxidant status.
ResultsDoxorubicin significantly (p<0.01) reduced body weight and increased the levels of cardiotoxic biomarkers (blood pressure, LDH, CK-MB, BNP, cardiac troponin I, IL-6, CRP, and TNF-α), whereas these effects were reversed in lactoferrin-treated animals. Histopathologically, heart tissue in the doxorubicin-treated group was found to contain muddled myocardial fibers, dilated blood capillaries, and necrosis in muscle fibers; however, these abnormalities were restored to normal levels in the lactoferrin therapy group. Finally, lactoferrin significantly (p<0.05) increased the levels of antioxidative enzymes (glutathione and SOD) and decreased the levels of oxidants (NO) compared with those in the untreated cardiotoxicity group.
ConclusionThe present study revealed the immunomodulatory, antioxidative potential of lactoferrin in restoring normal heart tissue, thereby mitigating the effects of doxorubicin-induced cardiotoxicity.
A cardiotoxicidade associada à doxorrubicina é uma preocupação alarmante por limitar o uso de fármacos antineoplásicos no ambiente clínico. O objetivo deste estudo foi testar o papel terapêutico da lactoferrina na prevenção da cardiotoxicidade induzida pela doxorrubicina para tratar este grave efeito secundário da quimioterapia.
MétodosRatos Wistar receberam veículo ou DOX (2,5mg/kg, i.p.) três vezes por semana durante duas semanas e lactoferrina (50mg/kg, i.p.) durante 15 dias (i.e., do dia 1 ao dia 15 juntamente com DOX). Foram avaliadas as alterações do peso corporal, do peso do coração e dos marcadores de cardiotoxicidade (pressão arterial, LDH, CK-MB e troponina I cardíaca). Além disso, todos os animais foram avaliados quanto à modulação de marcadores inflamatórios (BNP, IL-6, PCR e TNF-α), alterações histopatológicas no tecido cardíaco e estado oxidante e antioxidante.
ResultadosA doxorrubicina reduziu significativamente (p<0,01) o peso corporal e aumentou os níveis de biomarcadores cardiotóxicos (pressão arterial, LDH, CK-MB, BNP, troponina I cardíaca, IL-6, PCR e TNF-α), enquanto que estes efeitos foram revertidos nos animais tratados com lactoferrina. Histopatologicamente, verificou-se que o tecido cardíaco continha fibras miocárdicas confusas, capilares sanguíneos dilatados e necrose nas fibras musculares no grupo tratado com doxorrubicina, mas foi restaurado aos níveis normais no grupo de terapia com lactoferrina. Finalmente, a lactoferrina aumentou significativamente (p<0,05) os níveis de enzimas antioxidantes (glutationa e SOD) e diminuiu os níveis de oxidantes (NO) em comparação com os do grupo de cardiotoxicidade não tratado.
ConclusãoO presente estudo revelou o potencial imunomodulador e antioxidante da lactoferrina na restauração do tecido cardíaco normal, atenuando assim os efeitos da cardiotoxicidade induzida pela doxorrubicina.
Chemotherapy has been considered the gold standard treatment for most cancers, including both solid tumors and hematologic diseases, for decades.1 However, chemotherapy has several severe side effects that affect patients’ overall health even after completing the treatment schedule. One such chemotherapeutic drug in use is doxorubicin (DOX), an anthracycline derivative and a well-established antineoplastic agent used in a variety of cancers, albeit toxic to cardiomyocytes.2 DOX can cause congestive heart failure when the cumulative dosage exceeds 400–700 mg/m2 in adults and 300 mg/m2 in children; this is a major disadvantage of DOX-based chemotherapy.3 Multiple mechanisms are involved in DOX-induced cardiotoxicity, including increased apoptosis and oxidative stress, which stimulate lipid peroxidation, inflammation, loss of mitochondrial function and integrity, as well as trigger the renin–angiotensin system.4
Owing to the significance and efficacy of DOX as a cancer therapy, various approaches have been explored to avert its toxic effects, including adjuvant candidates with antagonistic properties against DOX-induced cardiotoxicity, herbal compounds, nanoparticle-based therapeutic interventions, and iron-chelating agents.5–7 However, there are no effective treatments for DOX-induced cardiotoxicity, which has prompted the exploration of new agents to counteract its toxic effects.
Antimicrobial peptides (AMPs) are being studied for their potential against various cancers due to their broad-spectrum properties, including immunomodulatory, antioxidative, anticancer, and antimicrobial effects.8 AMPs are short cationic peptides produced by organisms ranging from prokaryotes to eukaryotes and are a key component of the host's innate immune response. Lactoferrin, a cationic antimicrobial peptide with a molecular weight of 80 kDa, has various biological functions. Lactoferrin is found in exocrine secretions such as saliva, milk, tears, and bronchial mucus, as well as in the secondary granules of neutrophils.9 Few studies have described the protective role of lactoferrin against various types of cardiotoxicity and cancer. Mladenka and colleagues reported on the efficacy of lactoferrin against catecholamine induced cardiotoxicity.10 Administration of lactoferrin alleviated myocardial ischemia–reperfusion injury in rats,11 reduced nicotine- and azithromycin-induced cardiotoxicity in rats,12,13 and decreased pathological cardiac hypertrophy in mice.14 In a previous report, reactive oxygen species production was reduced by lactoferrin treatment, increasing the capacity of the body to fight free radicals in dexamethasone-induced hypertension.15 The findings from the abovementioned reports prompted us to explore the therapeutic role of lactoferrin in preventing DOX-induced cardiotoxicity. Therefore, this study aimed to investigate the potential therapeutic role of lactoferrin in treating DOX-induced cardiotoxicity in albino rats.
ObjectivesThe objective of this study was to test the therapeutic role of lactoferrin in preventing doxorubicin-induced cardiotoxicity to address this severe side effect of chemotherapy.
Material and methodsAnimalsMale Wistar rats (weighing 200–250 g, aged 8–10 weeks) were obtained from the KIET School of Pharmacy in Ghaziabad, Uttar Pradesh, India, and kept under standard environmental conditions (temperature 22±2°C; relative humidity 70%; and 12:12 h light/dark cycles) in the transient facility. The animals were given free access to a standard pellet diet (Ashirwad Industries Ltd., Ropar, Punjab, India) and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC/KSOP/E/20/03) of the KIET School of Pharmacy, Ghaziabad, Uttar Pradesh. For experimental purposes, bovine lactoferrin (protein purity 95%; Sigma–Aldrich, India) was used. Lactoferrin was injected by dissolving lactoferrin in phosphate-buffered saline (PBS).
Study designThe animals were randomly assigned to four experimental groups, each consisting of six animals. Group I: Animals were injected with normal saline (0.5 ml/kg, i.p.) daily for two weeks and served as controls. Group II: Animals were injected with DOX (2.5 mg/kg, i.p.) three times a week for two weeks and served as toxic controls. Group III: Animals were injected with lactoferrin (50 mg/kg, i.p.) for fifteen days along with DOX treatment, i.e., one hour after the DOX injection. Group IV: Animals were injected with lactoferrin (50 mg/kg, i.p.) for fifteen consecutive days (Figure 1). The experimental animals were weighed at the beginning and at the end of the study period. After fifteen days of treatment, the animals were starved for 12 hours, and 500 μL of blood was withdrawn from the retro-orbital plexus of each rat for serum isolation. After blood sample collection, the animals were euthanized by cervical dislocation, and cardiac tissues were collected. In the present study, the dose and route of lactoferrin administration were selected on the basis of previous studies.10,12,16
Estimation of hemodynamic changes
On the final day of the study, the hemodynamic profile, such as the systolic and diastolic blood pressure and heart rate, of each animal was monitored via a noninvasive blood pressure measuring apparatus (AD Instruments Pvt. Ltd.). Each rat was restrained for 10 min for 15 days prior to the estimation of the hemodynamic parameters.
Estimation of heart-to-body weight ratioAt the end of the study, heart tissue was removed and washed with normal saline. Then the tissue was dried with the help of tissue paper. Dried heart tissue was weighed, and the heart weight-to-body weight ratio was calculated by dividing the heart weight by the body weight of the rat to normalize the heart-to-body weight ratio.17
Estimation of cardiotoxicity and inflammation markersThe activity of creatine kinase-myocardial band (CK-MB) and lactate dehydrogenase (LDH) in the serum was estimated using an ELISA kit (Q-Line Biotech Pvt. Ltd., India) according to the given protocol. Inflammatory markers, including C-reactive protein (CRP), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α), as well as cardiotoxicity markers such as brain natriuretic peptide (BNP) and cardiac troponin I (cTnI), were assessed in rat serum samples via an ELISA kit (ELABSCIENCES, US) according to the manufacturer's protocol.
Assessment of oxidant/antioxidant levelsBriefly, heart tissue homogenates were prepared in 0.1 M potassium phosphate buffer (pH 7.4) for assessment of glutathione (GSH) and nitrite levels. On the other hand, the homogenates were centrifuged at 10000×g for 30 min, and the supernatant was collected for further determination of the superoxide dismutase (SOD) level.
Superoxide dismutase activityThe SOD level was estimated as previously described by Kono et al. (1978).18 For this purpose, 0.1 ml of sample homogenate was taken with 2 ml of nitroblue tetrazolium (NBT) salt. Thereafter, 0.5 ml of hydroxylamine hydrochloride was added to start the reaction. A spectrophotometer was used to observe the change in absorbance at 560 nm.
Nitrite (NO) estimationNO generation was estimated via the use of Griess reagent as described previously. This assay is based on the tendency of NO to be converted into nitrate and nitrite under physiological circumstances. The NO level in the tissue homogenate was determined with slight modifications to the method of Green et al. (1982).19 The reaction mixture was prepared at a 1:4:5 ratio (tissue homogenate:distilled water:Griess reagent) and then incubated at room temperature for 10 min in the dark. The reaction mixture was observed at 546 nm using spectroscopy.
GSH estimationThe tissue homogenates were prepared for estimation of GSH levels via the method described by Moron et al. (1979).20 Briefly, the homogenate (100 μl) was precipitated with 25% TCA and centrifuged at 1500×g for 10 min. Thereafter, the supernatant was separated and mixed with a phosphate buffer solution and DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)). Furthermore, the yellow color of the solution was detected at 412 nm.
Histopathological studiesFor hematoxylin and eosin staining, the obtained heart tissues were preserved in a 10% formaldehyde solution prepared in PBS (pH 7.4). The tissues were briefly dehydrated using increasing concentrations of alcohol, cleared in xylene, and embedded in paraffin wax. Using a microtome, tissue sections with a thickness of 0.5 μm were cut, expanded in a hot water bath, and placed on glass slides coated with albumin.
Statistical analysisThe values are presented as the mean±standard error of the mean and were analyzed using one-way ANOVA followed by Tukey's multiple comparison test with SPSS 14 software. p<0.05 was considered significant.
ResultsEffect of lactoferrin on hemodynamic parametersSystolic blood pressure (SBP) (F=36.23, df=3 and 20) and diastolic blood pressure (DBP) (F=2.10, df=3 and 20) were significantly greater (p<0.05), whereas HR (F=25.57, df=3 and 20) was significantly lower (p<0.001) in DOX-treated rats than in control rats. Compared with DOX treatment, lactoferrin treatment restored the hemodynamic parameters to normal levels (Table 1).
Effect of lactoferrin on hemodynamic and serum CK-MB, and LDH parameters in doxorubicin-induced cardiotoxicity in rats.
| Groups | Parameters | ||||
|---|---|---|---|---|---|
| SBP (mm Hg) | DBP (mm Hg) | HR (BPM) | CK-MB (IU/L) | LDH (IU/L) | |
| Control | 108.7±3.12 | 82.46±6.13 | 373.5±7.25 | 192±15.64 | 458±48 |
| DOX | 158.4±5.34# | 98.57±4.65# | 228.6±12.4## | 798±29.48## | 1486±72## |
| DOX+lactoferrin | 116.5±4.28* | 88.68±5.57* | 348.4±18.2** | 353±18.82** | 654±38** |
| Lactoferrin | 110.6±2.06 | 82.76±4.23 | 358.2±12.6 | 245±24.27 | 525±32 |
Data expressed as mean±SEM (n=6). Statistical significance was determined by using one-way ANOVA followed by Tukey's multiple comparison test. CK-MB: creatine kinase-MB; DBP: diastolic blood pressure; DOX: doxorubicin; HR: heart rate; LDH: lactate dehydrogenase; SBP: systolic blood pressure.
Significant reductions in body weight (F=6.60, df=3 and 20; p<0.05) and the heart weight-to-body weight ratio (F=2.75, df=3 and 20; p<0.05) were observed in DOX-treated rats compared with those in the control group. Moreover, lactoferrin significantly increased both body weight and the heart weight-to-body weight ratio compared with those of DOX-treated rats (Figure 2).
(A) Effects of lactoferrin on body weight. The data are expressed as the means±SEMs (n=6) and were analyzed via one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05 vs the control group; #p<0.05 vs the DOX group. (B) Effects of lactoferrin on the heart-to-body weight ratio. The data are expressed as the means±SEMs (n=6) and were analyzed via one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05 vs the control group; #p<0.05 vs the DOX group.
In DOX-treated rats, significant (p<0.001) changes in the levels of CK-MB (F=147.7, df=3 and 20) and LDH (F=91.47, df=3 and 20) were observed compared with those in the control group. Compared with DOX treatment, lactoferrin treatment significantly restored these levels to control values (Table 1).
Furthermore, the serum levels of BNP (F=82.48; df=3 and 20) and cTnI (F=1599; df=3 and 20) were significantly (p<0.001) greater in DOX-treated rats than in control rats. Conversely, lactoferrin therapy significantly improved the serum levels of cTnI and BNP compared with those of the control group, although they were not restored to normal levels (Table 2).
Effect of lactoferrin on serum cTnI, BNP, CRP, IL-6, and TNF-α parameters in doxorubicin-induced cardiotoxicity in rats.
| Groups | Parameters | ||||
|---|---|---|---|---|---|
| cTnI (pg/ml) | BNP (pg/ml) | CRP (ng/ml) | IL-6 (pg/ml) | TNF-α (pg/ml) | |
| Control | 0.828±0.035 | 53.33±2.29 | 9.55±0.18 | 8.81±0.12 | 100.78±1.09 |
| DOX | 3.656±0.037# | 90.33±1.54# | 90.18±1.08# | 43.75±0.83# | 182.08±0.80# |
| DOX+lactoferrin | 2.576±0.026* | 64.16±1.81* | 55.20±1.15* | 34.96±0.75* | 163.83±0.81* |
| Lactoferrin | 0.850±0.037 | 57.33±1.58 | 11.39±0.81 | 11.34±0.35 | 105.01±0.80 |
Data expressed as mean±SEM (n=6). Statistical significance was determined by using one-way ANOVA followed by Tukey's multiple comparison test. BNP: brain natriuretic peptide; CRP: C-reactive protein; cTnI: cardiac troponin I; DOX: doxorubicin; IL-6: interleukin-6; TNF-α: tumor necrosis factor alpha.
Additionally, the levels of proinflammatory cytokines (TNF-α [F=2276; df=3 and 5] and IL-6 [F=1179; df=3 and 5]) and CRP (F=1653; df=3 and 5) significantly (p<0.001) increased in the serum after DOX treatment. These levels were restored to normal compared with those in DOX-treated rats (Table 2).
Effect of lactoferrin on oxidative stress parametersCompared with those in the control group, nitric oxide (NO) levels were increased in the DOX-induced cardiotoxicity group. In contrast, lactoferrin resulted in a significant (p<0.05) reduction in NO (F=134000; df=3 and 20) levels, which was attributed to its antioxidative role in the stress environment.21 In addition, the levels of the antioxidant enzymes SOD (F=3828; df=3 and 20) and GSH (F=1393; df=3 and 20) were significantly (p<0.05) lower in the DOX group than in the control group. Lactoferrin therapy reversed the changes in the levels of NO, SOD, and GSH toward those of the control (Figure 3).
Effects of lactoferrin on GSH, SOD and NO levels. The results are expressed as the mean±SEM (n=6) and were analyzed via one-way ANOVA followed by Tukey's multiple comparison test. *p<0.05 vs the control group; #p<0.05 vs the DOX group. GSH: glutathione; NO: nitric oxide; SOD: superoxide dismutase.
Histopathological studies of the DOX-induced cardiotoxicity group revealed specific pathological changes in cardiac tissue morphology, such as disorganization of myocardial fibers, distortion of blood capillaries, necrosis of muscle fibers, and vacuolization of the cytoplasm, compared with the organized fibrillar structure observed in regular cardiomyocytes of the control tissue. Lactoferrin results in the restoration of the disorganization of myocardial fibers, leading to mild to moderate lesions, a low occurrence of degeneration, and the absence of necrosis (Figure 4).
Histopathological analysis of heart tissue from all the experimental groups via H&E staining. (A) Control group: normal histoarchitecture with organized cardiac fibers; (B) DOX group: dilated capillaries along with disorganized cardiac fibers; (C) lactoferrin-treated DOX group: restored cardiac myocytes with less distortion and disorganization; (D) lactoferrin group: normal histoarchitecture. The tissue sections were visualized at 40× under a light microscope. Scale: 100 μm. *p<0.001 vs the LF+DOX group.
In this study, the role of lactoferrin in preventing doxorubicin-induced cardiotoxicity was evaluated using a well-established animal model. Body weight and the heart-to-body weight ratio are markedly reduced in DOX-treated animals, most likely because DOX causes local tissue necrosis in the heart.1 These changes were reversed by lactoferrin treatment compared to those in DOX-treated rats. In this study, DOX-induced cardiotoxicity was manifested by elevated levels of BP, CK-MB, LDH, and cTnI. Previous findings also suggest that these parameters are increased in DOX-induced cardiotoxicity in rats.4,13,21,22 Furthermore, increased inflammation and oxidative stress may also be involved in DOX-induced cardiotoxicity.14
The release of intracellular enzymes, including CK-MB and LDH, into the serum by DOX caused cellular distension of the subsarcolemmal bulbs, which damaged the cardiomyocytes.13,24 The findings of this study are consistent with those of previous studies.13,22–24 Both early and late myocardial damage manifest as elevated blood levels of CK-MB and LDH.25 Lactoferrin treatment reduces the release of serum CK-MB and LDH. These findings demonstrate that lactoferrin can mitigate the cardiac damage caused by DOX. Moreover, the administration of lactoferrin mitigated cardiac contractility impairment in isolated stunned rat hearts, isoproterenol-induced myocardial infarction, and nicotine- and azithromycin-induced cardiac injury; additionally, lactoferrin ameliorated pathological cardiac hypertrophy.10–14 In these studies, lactoferrin was shown to have cardioprotective effects through different mechanisms. However, the administration of lactoferrin failed to provide significant protection against isoproterenol-induced cardiotoxic injury because its hydrophilic nature limits intracellular penetration, and the complexity of catecholamine cardiotoxicity.10
Furthermore, the widely used biomarkers for assessing anthracycline-induced cardiotoxicity are cTnI and BNP. These proteins are released when the heart muscle is damaged, such as during cardiomyopathy, ventricular wall distention, or cardiac tissue injury.26 In contrast to the findings of Mladenka et al.,10 the results of this study revealed that lactoferrin decreased the release of cTnI and BNP from cardiomyocytes. Heart tissue inflammation has been reported in animals exposed to DOX.27 The progression of cardiomyocyte injury and cellular loss caused by inflammation and cell death results in ventricular wall weakening and impaired systolic function.27 This study demonstrated a significant increase in the levels of proinflammatory cytokines (CRP, IL-6, and TNF-α) in DOX-treated animals, which is well supported by a previous report providing compelling evidence that DOX triggers a cascade of inflammatory responses within the myocardium via the release of several proinflammatory cytokines, such as IL-6 and TNF-α.28
Multiple reports have suggested that lactoferrin plays a pivotal role in modulating the immune response and controlling the levels of proinflammatory markers.29
C-reactive protein is another important key marker of inflammation,30 atherothrombosis,31 endothelial dysfunction, and cardiovascular diseases.32 Lactoferrin resulted in a significant reduction in CRP levels, highlighting the role of lactoferrin in cardioprotection. The efficacy of lactoferrin against stunned myocardium revealed its cardioprotective potency.33 NO, an oxidant, plays an essential role in the development of cardiac toxicity, such as cardiac failure, ischemia/perfusion injury, and cardiomyopathy. It is an important marker of myocardial contraction.34 In this study, NO levels were significantly greater in the DOX-induced cardiotoxicity group than in the control group. In contrast, lactoferrin results in a significant reduction in NO levels, which is attributed to its antioxidative role in the stress environment. GSH and SOD are crucial intracellular antioxidant enzymes that protect macromolecules and membrane lipids from damage when they are present in sufficient quantities. GSH levels decreased as a result of the interaction of DOX with the biomembrane, which promoted oxidizing activity.4,10,13 The findings of this study show that the administration of DOX triggers a significant reduction in GSH in cardiac cells. They also suggest that lactoferrin acts as a cardioprotective agent by combating pronounced oxidative stress. Our results are in line with previous findings suggesting that the antioxidant effect of lactoferrin contributes to its cardioprotective effect and that the antioxidant effect of lactoferrin is mediated through its iron chelating property.10,13,14
Compared with animals treated with DOX, lactoferrin caused the myocardial fibers to reorganize, resulting in mild to moderate lesions, a low incidence of degeneration, and no necrosis. Previous research has shown that lactoferrin may have a cardioprotective effect by reducing autophagy, mitochondrial dynamics, and apoptosis.14,35 In line with other studies, the cardioprotective effect of lactoferrin is mediated by the suppression of oxidative stress, inflammatory markers, and cardiotoxicity markers, viz. LDH, CK-MB, and cTnI.
ConclusionIn conclusion, it has been hypothesized that lactoferrin might play a beneficial role in combating/modulating the increased production of proinflammatory cytokines, which in turn helps to lower the oxidative stress prevailing in the heart due to DOX metabolism. Lactoferrin could be administered clinically as an adjunct along with DOX to reduce its cardiotoxicity on the basis of available evidence, viz. antioxidant, anti-inflammatory, and immunomodulatory activities.
Authors’ contributionsVinay Kumar: supervision, review and editing; Virendra Pratap Singh Rathor: investigation and formal analysis; Paritosh Kunwar Singh: conceptualization and data curation; Mohamad Taleuzzaman: writing – original draft and methodology.
Ethical statementThe experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC/KSOP/E/20/03) of the KIET School of Pharmacy, Ghaziabad (UP).
FundingThe research work was not supported by any external funding agency.
Conflict of interestThe authors declare that they have no conflicts of interest.
The authors are thankful to the KIET School of Pharmacy, Ghaziabad (UP), for providing the facilities to carry out the experimental work in the laboratory.
