INTRODUCTION:

Doxorubicin (DOX) a secondary metabolite of Streptomyces peucetius, related to anthracyclines. DOX is reduced intracellularly to doxorubicinol (active metabolite), in addition to a semiquinone radical by several intracellular oxidoreductases. Re-oxidation of semiquionone leads to the production of reactive oxygen species (ROS).

There are many risk factor for DOX cardiotoxicity including the cumulative dose, schedule of administration, prior irradiation, combination therapy, age (old or age <4 years), ethnicity, hypertension, female gender, CVD, chromosomal deformities and liver disease^(1,2,3).

Several mechanisms are indicated in DOX induced cardiotoxicity, e.g. cardiomyopathy is largely associated with increasing oxidative stress. Other important mechanisms are apoptosis, extracellular remodeling and deterioration of myofibrillar and intracellular dysregulation of calcium level. Furthermore, the cardiotoxicity associated with DOX involved also changes in endothelin-1 levels⁽⁴⁾.

Metformin (Met) is a biguanide (1,1-dimethylbiguanide) commonly used in type 2 diabetes mellitus (T2DM) patients. Met inhibits the mitochondrial respiratory chain complex 1 in a variety of tissues such as hepatocytes, pancreatic beta cells, endothelial cells, skeletal muscle, and neurons⁽⁵⁾. This inhibition caused a transient reduction in energy status cells, which changes a balance between the production of ATP and its consumption, causing increases of intracellular AMP to ATP ratio that lead to the activation of AMP activated protein kinase (AMPK)⁽⁶⁾.

Metformin decreases the production of ROS in cultured endothelial cells, and in animal model studies of heart failure⁽⁷⁾, also have myocardial protection from oxidative stress induced by H₂O₂ or TNFα⁽⁸⁾. These effects occur by its activation of AMPK. Additionally, AMPK have an important role in maintaining cellular or whole body energy homeostasis, so the reduced ATP during ischemia was significantly decreased by Met. The AMPK also demonstrated significant roles in the genotoxic stress response and apoptosis regulation⁽⁹⁾.

Several studies demonstrated that Met increases NO production⁽¹⁰⁾. This result indicated that its protective effect was mediated largely by the production of NO during ischemia since the beneficial effect of Met was eliminated by administration of the nitric oxide synthase inhibitor⁽¹¹⁾.

Aim of study:

This study aimed to evaluate the cardioprotective effect of Met against DOX-induced acute and chronic cardiotoxicity in an attempt to help improve survival of patients receiving DOX therapy by antagonized the oxidative stress and the inflammatory mediators.

MATERIALS AND METHODS:

Animals:

Thirty-six white male rabbits were used in this study purchased from Baghdad University- College of Veterinary Medicine – animal house. These male rabbits were kept in large cages with free water and food. These cages placed in controlled temperature room in a same 12:12 light-dark cycle. The weight of the rabbits varied between 0.6–2kg. These rabbits allowed 12 days acclimatization period before using in experimental study.

Study design:

The thirty-six male rabbits divided randomly into six groups, each group comprising of six rabbits.

1 Control group received single dose of 2ml saline intraperitoneally.

2 Met group received 300mg/kg/day, daily for 14 days orally.

3 Chronic DOX induction group received 4mg/kg, twice a week for two weeks intraperitoneally.

4 Acute DOX induction group received 16mg/kg single dose intraperitoneally.

5 Met + chronic DOX induction group received DOX 4mg/kg, twice a week for two weeks intraperitoneally and Met 300mg/kg/day, daily for 14 days orally, starting three days prior to DOX therapy.

6 Met + acute DOX induction group received doxorubicin 16mg/kg single dose intraperitoneally and Met 300mg/kg/day, daily for 14 days orally, starting three days prior to DOX therapy.

Induction of cardiotoxicity:

Induction of cardiotoxicity in rabbits carried out by injection of DOX intraperitoneally in a dose of 16mg /kg as a single dose for acute induction and 4mg/kg, twice a week for two weeks for chronic cardiotoxicity induction⁽¹²⁾.

Sample collection and preparation:

At the end of therapy, the 5 ml blood withdrawal from each rabbit by heart puncture, the blood sample were put in plain gel tube, then centrifugation at 3000x for 15 minutes for serum preparation which stored at -80°C for ELISA analysis. Rabbits then sacrificed by using di-ethyl ether for anesthesia, and the heart removed immediately and washed by distilled water. Small portion of the heart tissue was kept in a sterile tube containing normal saline which at -80ºC, to be used for molecular tests (DNA extraction and RT- PCR), and the other portion kept in 10% buffered neutral formalin to prepare paraffin embedded blocks for histopathological assessment.

Statistical analysis:

Statistical analysis was done by using SPSS version 16.0. The results were expressed as mean ± standard deviation (SD). To compare the results among groups, multiple analyses of variance (One-way ANOVA test) were used, followed by a post hoc Tukey test. Statistically significant differences were considered when the p<0.05 for data.

RESULTS:

Effect of metformin on matrix metalloproteinase 2:

The descriptive statistics for DNA load of MMP2 concentration which is represented as mean ± SD was significantly elevated in both acute and chronic DOX induction group (2190±667.57, 8338.34±348.18 copies/gm respectively) in comparison with the control and Met group (419.43 ±118.95, 759.5±289.45 copies/ gm respectively; P>0.05). The MMP2 DNA load concentration was significantly reduced in both Met + acute DOX induction and Met +chronic DOX induction group in comparison with the acute DOX induction and chronic DOX induction group (198±54.46, 708±96.26 copies/gm respectively; P> 0.05). The MMP2 DNA load concentration was non-significantly differences in both Met+acute DOX induction and Met + chronic DOX induction group in compare with the control and Met group as shown in (table-1).

Table-1: Effect of metformin on the MMP2 DNA load concentration in both acute and chronic doxorubicin cardiotoxicity

Group	MMP2(copies/gm)
Control	419.43 ±118.95 *#
Metformin	759.5±289.45 *#
Acute doxorubicin induction	2190±667.57
Metformin +Acute doxorubicin induction	198±54.46 *
Chronic doxorubicin induction	8338.34±348.18
Metformin +Chronic doxorubicin induction	708±96.26 #

Each value expressed as mean ±SD. The statistical analysis done by using one-way ANOVA followed by Tukey test.

*Significant difference (p<0.05) when compared among acute doxorubicin induction with control, Metformin and Metformin + Acute doxorubicin induction groups.

# Significant difference (p<0.05) when compared among chronic doxorubicin induction with control, Metformin and Metformin + Chronic doxorubicin induction groups.

Effect of metformin on TNFα:

The descriptive statistics for TNF α concentration which is represented as mean ± SD was significantly elevated in both acute and chronic DOX induction group (126.00 ± 3.61, 111.32 ± 8.48pg/ml respectively) in comparison with the control and Met group (30.53 ± 17.94, 29.16 ± 18.41pg/ml respectively; P>0.05). The serum TNF- α concentration was significantly reduced in both Met +acute DOX induction and Met +chronic DOX induction group in compare with the acute doxorubicin induction and chronic DOX induction group (50.52 ± 15.28, 42.18±36.5pg/ml respectively; P>0.05). Serum TNF-α concentration was non-significantly differences in both Met + acute DOX induction and Met + chronic DOX induction group in compare with the control and Met group, as shown in (table-2).

Table-2: Effect of metformin on serum TNF α level in both acute and chronic Doxorubicin cardiotoxicity

Group	TNF α(pg/ml) Mean ± SD
Control	30.53 ± 17.94*#
Metformin	29.16 ± 18.41*#
Acute doxorubicin induction	126.00 ± 3.61
Metformin +Acute doxorubicin induction	50.52 ± 15.28 *
Chronic doxorubicin induction	111.32 ± 8.48
Metformin +Chronic doxorubicin induction	42.18 ± 36.53#

Each value expressed as mean ±SD. The statistical analysis done by using one-way ANOVA followed by Tukey test.

* Significant difference (p<0.05) when compared among acute doxorubicin induction with control, Metformin and Metformin + Acute doxorubicin induction groups.

# Significant difference (p<0.05) when compared among chronic doxorubicin induction with control, Metformin and Metformin + Chronic doxorubicin induction groups.

Effect of metformin on inducible nitric oxide synthase:

The descriptive statistics for DNA load of iNOS concentration which is represented as mean ± SD was significantly elevated in both acute and chronic doxorubicin induction group (1266.66±292.68, 4471.16±851.16 copies/gm respectively) in comparison with the control and Met group (75±17.60, 100±16.73 copies/gm respectively; P >0.05). The DNA load of iNOS concentration was significantly reduced in both Met+acute DOX induction and Met +chronic DOX induction group in compare with the acute DOX induction and chronic DOX induction group (166.66±77.63, 300±89.44 copies/gm respectively; P>0.05). The iNOS concentration was non-significantly differences in both Met+acute doxorubicin induction and Met +chronic DOX induction group in compare with the control and Met group. as shown in (table-3).

Table-3: Effect of metformin on DNA load of iNOS level in both acute and chronic doxorubicin cardiotoxicity

Group	iNOS(copies/gm)
Control	75 ± 17.60 *#
Metformin	100 ±16.73 *#
Acute doxorubicin induction	1266.66 ±292.68
Metformin +Acute doxorubicin induction	166. 66 ±77.63 *
Chronic doxorubicin induction	4471.16 ±851.16
Metformin +Chronic doxorubicin induction	300 ± 89.44 #

Each value expressed as mean ±SD. The statistical analysis done by using one-way ANOVA followed by Tukey test.

* Significant difference (p<0.05) when compared among acute doxorubicin induction with control, Metformin and Metformin + Acute doxorubicin induction groups.

# Significant difference (p<0.05) when compared among chronic doxorubicin induction with control, Metformin and Metformin + Chronic doxorubicin induction groups.

Histological change of cardiac tissues by Haematoxylin and Eosin stain:

Metformin improved the histological change of cardiac tissues produced by acute and chronic DOX induction toxicity. The result for all groups illustrated below:

A- Control group:

The cardiac myocyte is normal showed centrally located nuclei with scanty collagen fibers between cardiac cells, normal myofibrillar structure with striation as shown in figure (A).

B- Met group:

The cardiac myocyte is normal showed centrally located nuclei with scanty collagen fibers between cardiac cells, normal myofibrillar structure with striation as shown in figure (B).

C- Chronic DOX induction group:

The cardiac tissue in this group showed swelling of cardiac myocyte, nuclear change (some of nuclei undergo pyknosis, other nuclei are lost called karyolysis) that lead to cell necrosis or apoptosis, cytoplasmic vacuoles, eosinophilic infiltration, oedema, increase collagen fibers between the cardiac cells and loss of striation as shown in figure (C).

D- Acute DOX induction group:

E- Chronic DOX induction +Met group:

The cardiac tissue in this group showed mild necrosis, cytoplasmic vacuoles, mild oedema, mild collagen fibers between the cardiac cells and myofibrillar structure with striation relatively well preserved in compare with chronic doxorubicin group as shown in figure (E).

F- Acute DOX induction + Met group:

The cardiac tissue in this group showed mild necrosis, cytoplasmic vacuoles, mild oedema and myofibrillar structure with striation relatively well preserved in compare with acute doxorubicin induction group as shown in figure (F).

Fig-A: Section of rabbit cardiac tissue of control group showing normal myocardial muscle fibers, no inflammation or cardiac damage (H&E, 40 X).

Fig. B Section of rabbit cardiac tissue of Metformin group showing normal myocardial muscle fibers, no inflammation or cardiac damage (H&E, 40 X).

Fig-C Section of rabbit cardiac tissue of chronic doxorubicin induction group showing inflammation, necrosis and myocardial swelling (H&E,40X).

Fig- D Section of rabbit cardiac tissue of acute doxorubicin induction group showing oedema, loss of striation, necrosis and myocardial swelling (H&E,40 X).

Fig-E Section of rabbit cardiac tissue of chronic doxorubicin induction +Metformin group showing mild collagen fiber, mild oedema and mild necrosis (H&E,40 X).

Fig-F Section of rabbit cardiac tissue of acute doxorubicin induction+Metformin group showing mild oedema and mild necrosis (H&E,40X).

DISCUSSION:

Doxorubicin is a secondary metabolite of Streptomyces peucetius and is one of the anthracyclines families, greatly effective anticancer drugs that used to treat many pediatric and adult cancers, e.g. solid tumors, lymphomas, leukemia and breast cancer. Doxorubicin has severe toxicities, but the cardiotoxicity being the most important one. Met has been reported to have cardioprotective effect in addition to reducing basal and postprandial glucose levels, weight loss, and reducing lipid serum levels. Animal model studies of isolated myocardial infarction and heart failure shown that Met increases the tolerance of myocardial cells to ischemia-reperfusion injury, and reduce the development of heart failure after infarction.

Matrix metalloproteinase are proteolytic enzymes which are responsible for the degradation of extracellular matrix components and are very important in the normal tissues remodeling and growth process. Matrix metalloproteinase have been activated in cardiac injury such as ischemia, toxic injury and during increasing the oxidative stress. Several myocardial injury models demonstrated that MMP-2 play an important role in cardiac remodeling and ventricular dilation. The gene expression MMP and its activity have been to increase after chronic DOX therapy⁽¹³⁾. Several studies demonstrated that MMP-2 is increased acutely after DOX therapy in rats and indicated for the first time that activation of MMP-2 is combined for both left ventricular systolic dysfunction and increased myocardial stiffness in rats⁽¹⁴⁾. Esfahanian et al 2012, revealed that MMP-2 level significantly decreased after Met treatment⁽¹⁵⁾. This study showed that MMP-2 level significantly decreased (P<0.05) in both acute DOX induction + Met and chronic DOX induction+ Met groups in comparison with acute and chronic DOX induction groups. This suppressive effect may be related to the activation of AMPK that leading to decreasing the oxidative stress⁽¹⁶⁾.

Tumor necrosis factor α is a proinflammatory cytokine formed by many cells such as activated dendritic cells, macrophages and T cells. TNF-α is involved in many process such as inflammation, differentiation, growth, cellular survival and apoptosis. There is a direct correlation between serum TNF-α level and the severity and progression of heart failure⁽¹⁷⁾.

iNOS was recognized to be induced by inflammatory cytokines, via the NF-KB activation. Gochman et al. confirmed that iNOS expression is contributed in inflammatory disease and its inhibition as a potential therapeutic treatment. The expression of iNOS in cardiovascular system has been proposed to be related to disease states, as that its upregulation and increased activation stimulates the production of iNOS and could contributed to cardiotoxicity. Aldieri et al, revealed that the increase in the NO level after DOX treatment of the cardiac cells was associated with an increase in the gene expressionof iNOS⁽¹⁸⁾.

Abd El-Aziz et al, confirmed that doxorubicin increased the release of proinflammatory cytokines such as TNF α via the activation of NF-κB in the cardiac rats treated with DOX over two weeks⁽¹⁹⁾.

Pecoraro et al, illustrated that DOX treatment is associated with an increase in expression of inflammatory cytokines levels, such as TNF-α and IL-6, in addition to induced iNOS expression and NO release within 24 h from first DOX treatment⁽²⁰⁾.

Arai et al, revealed that Met reduce inflammatory responses by suppressing the TNF α production⁽²¹⁾. Kelleni et al, showed that Met significantly decreased the iNOS expressions⁽²²⁾.

This study shown that Met led to significant decrease (P<0.05) in TNFα and iNOS level in Met+ acute DOX and Met + chronic DOX groups when compared with acute and chronic DOX groups. Wulster-Radcliffe et al, showed that APN treatment reduced the production of TNFα and IL6 in lipopolysaccharide stimulated porcine macrophages⁽²³⁾. APN also accumulates in myocardial damaged tissue and protects the myocardium by inhibiting the expression of iNOS and NADPH oxidase and the resulting oxidative stress. So the TNFα and iNOS suppressive effect by Met may be related to activation of APN system⁽²⁴⁾.

The cardiac myocyte in control and Met groups of this study is normal, showed centrally located nuclei, normal myofibrillar structure with striation. While, in acute and chronic DOX induction groups showed swelling of cardiac myocyte, necrosis, cytoplasmic vacuoles, eosinophilic infiltration, edema and loss of striation.

Janeesh et al, showed that DOX have changes on normal morphology of cardiomyocyte including necrosis, myofibrillar loss, vacuolization, and mononuclear cells infiltration due to the action of oxidative stress that considered as an indication for cardiac injury and dysfunction⁽²⁵⁾. Bahadır et al, showed that DOX 15mg/kg (cumulative dose) causing myocyte edema, myocyte vacuolization and loss of myofibrils⁽²⁶⁾.

In this study both acute and chronic DOX + Met groups show mild necrosis, cytoplasmic vacuoles, mild edema and myofibrillar structure with striation relatively well preserved in compare with DOX groups. These finding agreement with Ashour et al, that reported oral therapy of Met (50mg/kg and 500mg/kg) removed histopathological changes produced by DOX 18mg/kg (cumulative dose) so exerting a cardioprotective effect⁽²⁷⁾. Sheta et al, showed that Met treatment reduced DOX induced myocardial alterations, reported by normal appearance of muscle fibers, less widening of interstitial space, moderate interstitial cellular infiltration, no myocytic degeneration and normal nuclei location.

Finally the capability of Met to prevent DOX induced cell death may weaken the effectiveness of the anthracycline as an anticancer agent. However, the use of Met as an anticancer agent itself may improve the responsiveness to antitumor agent and reducing the cancer development risk⁽²⁸⁾. In addition, many groups showed that the co-administration of Met with antitumor agents, including DOX, decreases cancer growth and prevents relapse in many types of cancer. Metformin is able to selectively kill stem cells of cancer, which are resistant to antitumor agent and can act synergistically with DOX to block cancer stem cells in addition to non transformed cells⁽²⁹⁾. Recently, another study suggests that Met can suppress the emergence of the multidrug resistance phenotype in cancer cells. This reflects the ability of Met to decrease the dose of DOX required for several cancers treatment^(30,31). These observations suggest that Met may act as a preventative agent against DOX induced cytotoxicity without any affecting on its anticancer capability.

CONCLUSIONS:

From this study and depending on the result one can conclude that DOX administration caused cardiotoxicity in both acute and chronic induction. Metf co-administration markedly decreases the inflammatory marker TNF-α and iNOS in addition to MMP-2 markers and decreased the size of necrosis and other histopathological changes in myocardial tissue. So the current study showed that Met has a good cardioprotective agent against DOX cardiotoxicity by much mechanism by activation of AMPK and increased APN level and its receptor (adipoR1 and adipoR2).

ACKNOWLEDGMENTS:

The authors would like to thank Mustansiriyah University (www.uomustansiryiah.edu.iq) for their support in the present work.

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Received on 18.05.2019 Modified on 21.06.2019

Research J. Pharm. and Tech. 2019; 12(12): 5815-5821.

DOI: 10.5958/0974-360X.2019.01007.2