Melissa officinalis Protects against Doxorubicin

RESEARCH ARTICLE

Melissa officinalis Protects against Doxorubicin-Induced Cardiotoxicity in Rats and Potentiates Its Anticancer Activity on MCF-7 Cells Alaaeldin Ahmed Hamza1*, Mahguob Mohamed Ahmed2, Hanan Mohamed Elwey3, Amr Amin4,5*

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1 Hormone Evaluation Department, National Organization for Drug Control and Research (NODCAR), Giza, Egypt, 2 Molecular Drug Evaluation Department, NODCAR, Giza, Egypt, 3 Analytical Chemistry Department, NODCAR, Giza, Egypt, 4 Biology Department, UAE University, Al-Ain, UAE, 5 Zoology Department, Cairo University, Giza, Egypt * [email protected] (AAH); [email protected] (AA)

OPEN ACCESS Citation: Hamza AA, Ahmed MM, Elwey HM, Amin A (2016) Melissa officinalis Protects against Doxorubicin-Induced Cardiotoxicity in Rats and Potentiates Its Anticancer Activity on MCF-7 Cells. PLoS ONE 11(11): e0167049. doi:10.1371/journal. pone.0167049 Editor: Aamir Ahmad, University of South Alabama Mitchell Cancer Institute, UNITED STATES Received: May 19, 2016 Accepted: November 8, 2016 Published: November 23, 2016 Copyright: © 2016 Hamza et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract Cardiotoxicity is a limiting factor of doxorubicin (DOX)-based anticancer therapy. Due to its beneficial effects, we investigated whether standardized extract of Melissa officinalis (MO) can attenuate doxorubicin-induced cardiotoxicity and can potentiate the efficacy of DOX against human breast cancer cells. MO was administered orally to male albino rats once daily for 10 consecutive days at doses of 250, 500 and 750 mg/kg b.wt. DOX (15 mg/kg b. wt. i.p.) was administered on the 8th day. MO protected against DOX-induced leakage of cardiac enzymes and histopathological changes. MO ameliorated DOX-induced oxidative stress as evidenced by decreasing lipid peroxidation, protein oxidation and total oxidant capacity depletion and by increasing antioxidant capacity. Additionally, MO pretreatment inhibited inflammatory responses to DOX by decreasing the expressions of nuclear factor kappa-B, tumor necrosis factor-alpha and cyclooxygenase-2 and the activity of myeloperoxidase. MO ameliorated DOX-induced apoptotic tissue damage in heart of rats. In vitro study showed that MO augmented the anticancer efficacy of DOX in human breast cancer cells (MCF-7) and potentiated oxidative damage and apoptosis. Thus, combination of DOX and MO may prove future cancer treatment protocols safer and more efficient.

Data Availability Statement: All relevant data are within the paper. Funding: This work was supported by the Terry Fox Foundation, grant# 21S088. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: ABTS, 2,2-azino-bis(3ethylbenzothiazoline-6-sulfonate); AST, Aspartate

Introduction Doxorubicin (DOX), an anthracycline antibiotic, is regularly used to treat various malignancies, including solid tumors, lymphoma and leukemia. Its association with severe forms of cardiomyopathy and/or congestive heart failure in cancer patients greatly limits DOX application [1–2]. Cellular damage induced by DOX is mediated by the oxidative damage of cardiomyocytes, plasma membranes and the consequent death of those cells by apoptosis [2–3]. Synthetic agents (such as antioxidants and metal chelators) have been investigated with some degree of

PLOS ONE | DOI:10.1371/journal.pone.0167049 November 23, 2016

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Melissa officinalis Protects against Cardiotoxicity & Possesses Anti Breast Cancer Activity

aminotransferase; MDA, malondialdehyde; CAT, catalase; CK, Creatine kinase; CK-MB, CK isoenzyme-MB; COX-2, Cyclooxygenase-2; DOX, Doxorubicin; DPPH, 1,1-diphenyl-2-picrylhydrazyl; MPO, Myeloperoxidase; NF-kB, nuclear factorkappa B; NO, nitric oxide; P.carbonyl, protein carbonyl; SOD, superoxide dismutase; TNF-α, Tumor necrosis alpha.

success [3–4]. While some of those agents such as vitamin E failed to inhibit DOX cardiotoxicity [5] others, such as deferasirox (an iron chelator) increased its toxicity [6]. Cardiotoxicity associated with DOX treatment has been successfully prevented by different medicinal plants [7–9]. Thus, it is well justified to explore more plant derived-natural compounds that prevent the cardiotoxicity of DOX and enhance its chemotherapeutic efficacy. Among these, Melissa officinalis L (MO), Lemon Balm, (Lamiaceae family) is one of the most used medicinal plants in Europe and the Mediterranean region. Normally, herbal tea of MO is used for its aromatic, digestive and antispasmodic properties and to reduce gastrointestinal disorders and sleep disturbance [10–11]. Moreover, MO has been reported to show potent anti-tumor effects in a variety of human cancer cell lines [12–14] and to induce apoptosis in colon carcinoma cells through formation of reactive oxygen species (ROS) [1]. Caffeic acid, protocatechuic acid, rosmarinic acid, ferulic acid, and syringic acid have been reported as the most abundant phenolic compounds in MO [15]. Other phenolic compounds have also been characterized from this plant including triterpene acids and terpenes (ursolic and oleanolic acids and luteolin) [10–11]. The present study was carried out to investigate the protective effect of MO against DOXinduced cardiotoxicity in rats and to elucidate its antitumor effect alone or in combination with DOX on breast cancer cell line (MCF-7). The interest to use the estrogen receptor-positive MCF-7 cell line as a model to evaluate the DOX/MO combinatory anticancer effect stemmed from the evidence that although DOX is among the most active chemotherapeutic drugs for the treatment of breast cancer; it has several limitations particularly in estrogen dependent breast cancer [16]. MCF-7 cell line, thus, serves as an excellent in vitro model for studying the mechanisms of chemo resistance as it relates to susceptibility to apoptosis [17]. Markers of oxidative stress, inflammation and apoptosis were also assessed to unravel possible mechanism/s of action of MO.

Materials and Methods Chemicals All chemicals were of analytical grade and chemicals required for sensitive biochemical assays were obtained from Sigma Chemical Co., St. Louis, MO, USA. Radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (sc-24948) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Poly vinylidene difluoride (PVDF) membrane and blocking reagent were obtained from Roche Diagnostics GmbH (Mannheim, Germany). The primary antibodies used in the western blotting stage were obtained from BioVision (Milpitas, CA, USA) whereas secondary antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RNA easy Mini Kit (Qiagen, Valencia, CA, USA). TM first strand kit for cDNA synthesis and SYBR Green Real-time PCR Master Mix were bought from Applied Biosystems (Foster City, CA, USA). DOX (Adriblastina, 50 mg) was purchased from Pharmacia Italia S.P.A., Italy.

Preparation of plant extract Dried aerial parts (leaves and stems) of MO, grown in May in Syria, where the average temperature is 20˚C and average relative humidity is 60%, were purchased from local (Cairo) herbal store. The plant material was authenticated by Dr. Nael M. Fawzi, The Flora and Taxonomy Department, Agricultural Research Center, Giza, Egypt. Plant was stored in light-protected glass bottles at 4˚C until the extraction step. Air-dried and ground aerial parts of MO (1000g) were extracted in 70% (v/v) ethanol (2000 mL) by maceration for 48 h at 4˚C. The resulting compound was then filter-dried under reduced pressure in a rotary evaporator at 40˚C. This


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crude extract was weighed, dissolved in water for animal study and kept at−20˚C for further analysis. The yield of the MO was 12.5 g per 100 g of used plants.

Ethics statement Animals were cared for in accordance with the standard guidelines (Canadian Council on Animal Care 1993). The protocol was approved by the Ethics Committee of Animal Care and Use at National Organization for Drug Control and Research, Giza (Approval No 181, 1-7-2015).

Experimental animals Adult male Wistar albino rats (Forty eight) were obtained from the animal house of the National Organization for Drug Control and Research (NODCAR). They were maintained on standard pellet diet and tap water ad libitum and were kept in polycarbonate clean cages under a 12 hrs. light/dark cycle and room temperature 22–24˚C. Rats were acclimatized for two week prior to experimental use.

Treatment regime Rats were randomly divided into six groups consisting of eight animals in each group (n = 8) and were subjected to the following treatments: The first group was the control group and received 5ml / kg distilled water through oral gavage for 10 days and injected with single dose of saline (5ml /kg b.wt.) after 7 days of water administration. The second group was the MO group and received 750 mg/kg b.wt. of MO for 10 days. The third group was the DOX-treated group and received a daily dose of distilled water (5ml / kg b.wt.) for 10 days followed by a single intraperitoneal (i.p.) injection of DOX (15 mg/kg) on the 8th day. This DOX dose was selected as it has been used previously to induce acute cardiotoxicity in male albino rats [18]; [19]. Doses of MO were selected based on previously reported pharmacological properties of this plant [20]. DOX solution was freshly prepared in a saline solution. A sample of 1 g MO extract was suspended in 10 ml distilled water. Groups four, five and six received 250, 500 and 750 mg/kg of MO respectively orally and daily for 10 days followed by a single i.p. injection of DOX (15 mg/kg b.wt.) on the 8th day 1 hr after MO treatment. Twenty- four hours after the last MO or vehicle solution administration, blood and heart tissues were collected from all groups and stored at -20˚C for further processing.

Sample preparation Blood was collected from the retro-orbital plexus and the serum was immediately separated by centrifugation in a refrigerated centrifuge (4˚C) at 3000 r.p.m. for 20 minutes. Rats were euthanized by cervical dislocation under diethyl ether anesthesia. The hearts were removed and weighed to calculate the heart to the body weight ratio. Hearts were sliced frontally into two halves (each half includes parts of all chambers of the heart) and for histopathological examination, cardiac tissue samples were immediately fixed in 10% buffered formalin. For biochemical determination, cardiac tissue samples from the other half were homogenized in ice-cold KCl (150 mM). The ratio of tissue weight to homogenization buffer was 1:10. Then, suitable dilutions from that were prepared to determine the levels of oxidative stress biomarkers.

Biochemical assays and histopathology Cardiotoxicity indices. Aspartate aminotransferase (AST), creatine kinase (CK) and creatine kinase-MB (CK-MB) activities were estimated in serum samples using Randox (Randox


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Table 1. Effect of MO treatments on severity of histopathologic lesions in DOX-treated rats. Groups

Disorganization

Focal necrosis

Degeneration

Inflammation

Control

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

DOX

3.00 ± 0.00a

1.83 ± 0.17a

2.17 ± 0.31a

2.00 ± 0.26a

DOX+MO(LD)

1.33 ± 0.21a

0.67 ± 0.21a

0.67 ± 0.21a

1.00 ± 0.36a

a

a

a

0.67 ± 0.21a

a

0.50 ± 0.22a

DOX+MO(MD) DOX+MO(HD)

1.00 ± 0.00

a

0.50 ± 0.22

0.33 ± 0.21

a

0.17 ± 0.17

0.33 ± 0.21 0.00 ± 0.00

Severity of injury is expressed as mean ± SEM of three scores for seven animals in each group. a P 30% severe, a

P