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South African Journal of Botany 81 (2012) 1–7 www.elsevier.com/locate/sajb

Aqueous extract of edulis leaves: Antioxidant activity and cytotoxicity to human breast cancer MCF-7 cell line ⁎ A.J.S. Carvalho a, T. Ishikawa b, C.M.C.P. Gouvêa a,

a Laboratório de Cultura de Células, Instituto de Ciências da Natureza, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas 37130-000, MG, b Laboratório de Plantas Medicinais e Fitoterápicos, Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, Alfenas 37130-000, MG, Brazil

Received 30 January 2012; received in revised form 29 February 2012; accepted 28 March 2012 Available online 21 April 2012

Abstract

The aqueous extract of Plinia edulis leaves (AEP) was evaluated for its in vitro antioxidant potential and cytotoxicity to MCF-7 cells, a human breast adenocarcinoma cell-line that express the estrogen receptor α (ER+). AEP exhibited total antioxidant capacity (324.20±3.14 mg/g) and strong scavenging activity on DPPH free radical, with maximum effect at 20 μg/mL. AEP and cyclophosphamide (cytotoxic agent) treatment for 24 and 48 h decreased the cell protein content, as compared to control cells. Morphological analysis, after hematoxylin–eosin staining of cells, showed changes such as cell rounding-up, shrinkage, nuclear condensation and reduction of colony and cell diameter (pb0.01), thereby indicating that AEP is cytotoxic to MCF-7 cells. AEP treatment also induced cell death, with a maximum effect at 5 μg/mL, and its mechanism of action seems to include the induction of apoptosis, as a DNA ladder-pattern was obtained in the DNA analysis. Oxidative stress is supposed to play an important role in cancer initiation and progression. Considering that AEP showed to be antioxidant and cytotoxic, the extract seems to be a chemopreventive agent and a good candidate for antineoplastic drug development. © 2012 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords: Anticancer; Cambucá; Cell death; Cell morphology; DNA ladder; extract

1. Introduction (Bines and Eniu, 2008; Anderson, 2010; Brasil, 2011). Tissue invasiveness and metastatic spread of breast cancer cells are Breast cancer is a large problem of public health all around responsible for most of the morbidity and mortality associated the world, and is the second most commonly diagnosed cancer with the disease (Nukumi et al., 2007). type (Anderson, 2010; Jemal et al., 2010). Breast cancer-related Breast cancers exhibit remarkable heterogeneity not only mortality rates continue to increase in Brazil and in other less with respect to estrogen (ER), progesterone (PR), and human developed countries (Bines and Eniu, 2008). Increasing breast epidermal growth factor-2 (HER-2) receptor expression but cancer incidence is associated with risk factor diversity, genetic also with respect to tumor size, grade, and nodal status. Thus, characteristics involved in its etiology, later stage disease breast carcinoma is a mixture of diverse phenotypes, which diagnosis, and lack of adequate adjuvant, systemic treatment raises different treatment needs (Jatoi et al., 2008) and at the present moment there is no cure for metastatic breast cancer (Pagani et al., 2010), whereby the necessary search for new drugs to treat and control this disease is much needed. ⁎ Corresponding author. Tel.: +55 35 32991419; fax: +55 35 32991067. Cancer initiation and progression and a number of human E-mail address: [email protected] (C.M.C.P. Gouvêa). diseases including cardiovascular, metabolic, inflammatory, and

0254-6299/$ -see front matter © 2012 SAAB. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.sajb.2012.03.010 2 A.J.S. Carvalho et al. / South African Journal of Botany 81 (2012) 1–7 neurodegenerative diseases are related to reactive oxygen the aluminum chelating method (Ebrahimzadeh et al., 2008), species (ROS) and reactive nitrogen species (RNS), which using quercetin as standard. once accumulated inside the cell, can attack proteins, lipids, and DNA, causing a state of oxidative stress (Halliwell, 2007; 2.4. Antioxidant assays Vurusaner et al., 2012). Living organisms are equipped with an antioxidant defense system that regulates the toxic impact of The total antioxidant capacity was determined (Prieto et al., ROS and RNS. However, a disturbance in the balance between 1999), and the results were expressed as mg equivalents of the production of ROS or RNS and antioxidant defenses may ascorbic acid/g of extract. The hydrogen atom or electron lead to cell molecule or tissue injury (Gouvêa, 2004; Halliwell, donation ability of AEP was measured from the bleaching of 2007). There is strong evidence that the antioxidants prevent purple colored methanol solution of DPPH according to Yen carcinogenesis, and natural products have proven to be an and Wu (1999). L-Ascorbic acid was used as the reference important source of new and effective antioxidant and standard. anticancer agents (Milaeva, 2011). Secondary plant metabo- lites, such as phenolic compounds, have been found to be 2.5. Cell line and culture strong antioxidants, which can scavenge or suppress ROS and RNS formation by inhibiting some enzymes or chelating trace Human breast cancer MCF-7 cell line (ER+) was purchased metals involved in free radical production, and up-regulate or from Rio de Janeiro Cell Bank (BCRJ 0162) and cultured in protect antioxidant defense, thereby preventing carcinogenesis RPMI 1640 medium, supplemented with 20% (v/v) inactivated (Gouvêa, 2004; Halliwell, 2007; Huang et al., 2010). fetal bovine serum, 100 U/mL penicillin and 100 μg/mL Plinia edulis (Vell.) Sobral, , popularly known as streptomycin. Cells were maintained in a humidified atmosphere “cambucá”, is an arboreous species with edible fruits, which with 5% CO2 at 37 °C. The medium was replaced every 2 days grows naturally in Brazilian Atlantic Rain Forest (Lorenzi et al., and cells were sub-cultured every 5 days, after 0.25% trypsin– 2006). In folk medicine, P. edulis is used to treat stomach EDTA solution treatment. Cell viability was assessed, before problems, sore throat, and diabetes and as tonic (Nascente, beginning each experiment, by the Trypan blue-dye exclusion 2008). This plant is a source of flavonoids, tannins, saponins method and 2×104 viable cells/mL were used in all experiments. and terpenoids (Ishikawa et al., 2008b), and the aqueous Before treatment, viable cells were cultivated for 24 h to reach ethanol extract of P. edulis leaves is antiulcerogenic (Ishikawa exponential growth. et al., 2008a). The aim of the present work was to evaluate the aqueous 2.6. Sulforhodamine B (SRB) uptake assay extract of P. edulis (AEP) in vitro antioxidant activity and its cytotoxicity to MCF-7 cells that express the estrogen receptor α The effect of different concentrations of AEP on cell protein (ER+), a model for the human in situ breast carcinoma. content was determined by the sulforhodamine B (SRB) colorimetric assay (Vichai and Kirtikara, 2006). Briefly, cells 2. Materials and methods were seeded onto 96-well plates and treated with different concentrations of AEP (0.1, 0.2, 0.5, 1.0, 1.5, 2.5, 5.0, 10, 25, μ 2.1. Plant material 50 and 100.0 g/mL) for 24 and 48 h and fixed with 10% trichloroacetic acid (w/v) for 30 min at 4 °C. The plate contents Leaves of P. edulis (Vell.) Sobral, Myrtaceae were collected were carefully removed and each well was washed with in Trindade (Paraty, Rio de Janeiro, Brazil) in the morning distilled water. The plate was then dried for 24 h and stained during flowering, identified by Dr. Lúcia Rossi, and voucher with 0.4% SRB (w/v) in 1% acetic acid (v/v) for 30 min, after specimens (SP 356.472) were deposited at the Herbarium of the which excess SRB was removed, and the wells were washed 4 Instituto de Botânica de São Paulo, Brazil. times with 1% acetic acid (v/v). The bound SRB was dissolved by adding 100 μLof10μM Tris, pH 10.5 for 10 min, and absorbance read at 510 nm. Treated-cells with culture medium 2.2. Extract preparation were the negative control, and with 550 μg/mL cyclophospha- mide, the positive. Leaves were dried at 40–45 °C and pulverized and the aqueous extract (AEP) was obtained by decoction of 10 g leaves 2.7. Cell morphological analysis in 100 mL deionized water, at 90 °C for 30 min (Farmacopéia, 1959), next lyophilized and stored in a desiccator until use. For Cells cultured on coverslips were treated with various use, AEP was dissolved in deionized water. concentrations of AEP (0.1, 0.2, 0.5, 1.0, 1.5 5.0 μg/mL) for 24 and 48 h. After treatment, cells were fixed with 70% acetone 2.3. Determination of total phenolic and flavonoid content for 15 min, washed with PBS, and stained with hematoxylin– eosin. Slides were mounted in Entellan, and observed by light The total phenolic compounds, present in AEP, were microscopy. Cell digital images were acquired using an Olym- determined using Folin–Ciocalteau's method (Rai et al., 2006) pus BX52 microscope and Motic Images Plus 2.0 software. and gallic acid as standard. Total flavonoids were estimated by Fifteen random fields were analyzed per treatment, to describe A.J.S. Carvalho et al. / South African Journal of Botany 81 (2012) 1–7 3

3. Results and discussion

3.1. Phenolic compound determination

AEP presented 54.34±0.02 mg/g total phenols and 35.16± 0.01 mg/g flavonoids, a higher content of flavonoids than the hydroalcoholic extract from Plinia cauliflora leaves (1 mg/g; Souza-Moreira et al., 2011), and also that reported by other previously examined sources of antioxidants (Kim et al., 2011; Santos Filho et al., 2011). Phenolic compounds from medicinal and dietary are Fig. 1. DPPH scavenging activity. AEP, aqueous extract of Plinia edulis leaves; bioactive and play an important role in prevention of cancer. AA, ascorbic acid. The results are mean±SE of three independent experiments They have a complementary and overlapping mode of action, performed in triplicate. Different letters indicate significant differences (pb0.05) including antioxidant activity, scavenging free radicals, and μ by the Tukey test. The maximum effect was obtained with 20 g/mL AEP. modulation of carcinogen metabolism that alter important cellular and molecular mechanisms related to carcinogenesis, a multistep cell morphology. Treated-cells with culture medium were the process involving the transformation, survival, proliferation, in- negative control, and with 550 μg/mL cyclophosphamide, the vasion, angiogenesis, and metastasis of the tumor cells (Huang et positive. Cell and colony diameters were determined by mea- al., 2010). Epidemiological studies confirmed that phenolics, suring 150 cells and 20 colonies per treatment (Campanella et al., namely flavonoids, appear to be beneficial compounds in various 2012). stages of carcinogenesis (Clere et al., 2011; Fresco et al., 2010). In fact, little is known about the chemical composition of the genus Plinia. Research is currently in progress in our laboratory 2.8. Cell death assay to better identify the AEP constituents.

Cell death was estimated using the Fast green-dye exclusion 3.2. Antioxidant activity method, according to Weisenthal et al. (1983),withmodifica- tions. The rationale of this method is based on the principle that The total antioxidant capacity of AEP was 324.20±3.14 mg/g. viable cells are able to exclude the dye while dead cells lose this The extract also showed strong DPPH scavenging activity (Fig. 1), ability. Cells were treated with several concentrations of AEP which was concentration-dependent, showing the maximum effect μ (0.1, 0.2, 0.5, 1.0 and 5.0 g/mL) for 24 h, whereupon cells were with 20 μg/mL AEP, an activity similar to ascorbic acid. – stained with 2% Fast green, followed by hematoxylin eosin, and The results of the present work indicate that AEP acts as an slides were mounted in Entellan. With this procedure, viable cells antioxidant and it could be attributed to the extract constituents. (i.e., cells excluding Fast green) were stained reddish-pink, while Phenolic compounds are regarded to be the most important dead cells (cells unable to exclude Fast green) were stained green. antioxidant constituents of plants. In general, the phenolic OH To achieve the percentage of dead cells, a total of 600 cells/ is considered a scavenger of free radicals with consequently treatment was analyzed using an Olympus BX52 microscope and antioxidant activity. Flavonoids may also contribute to AEP Motic Images Plus 2.0 software. Treated-cells with culture me- antioxidant activity, due to its hydrogen-donating ability (Rice- dium were the negative control, and with 550 μg/mL cyclo- phosphamide, the positive.

2.9. DNA isolation and electrophoresis

MCF-7 cell genomic DNA was isolated using the SDS/ Proteinase K/RNase A extraction method (De Siervi et al., 2002). Samples were electrophoresed in 0.8% (w/v) agarose gel and DNA was visualized by ethidium bromide staining. The presence of apoptosis was indicated by the appearance of a ladder of oligonucleosomal DNA fragments on the agarose gel.

2.10. Statistical analysis Fig. 2. MCF-7 cell protein content decreasing (%), estimated by the sulforhodamine B assay, after treatment with cyclophosphamide (CP, 550 μg/mL) and different – μ Obtained data were compared by one-way analysis of concentrations of aqueous extract of Plinia edulis leaves (AEP 0.1 5 g/mL) for 24 b and 48 h. The results are expressed as mean±SEM of three independent experiments. variance (ANOVA) followed by the Tukey test, when p 0.05. Different letters indicate significant differences (pb0.001) by the Tukey test. Protein Data are shown as the mean±SEM of three independent content was calculated relative to the negative control and 5 μg/mL AEP produced experiments. the maximum effect. 4 A.J.S. Carvalho et al. / South African Journal of Botany 81 (2012) 1–7 A.J.S. Carvalho et al. / South African Journal of Botany 81 (2012) 1–7 5

Fig. 4. MCF-7 cell diameter (μm) after treatment with cyclophosphamide (CP, Fig. 5. MCF-7 cell death (%) after treatment with cyclophosphamide (CP, μ – μ 550 μg/mL) and different concentrations (0.1–5 μg/mL) of aqueous extract of 550 g/mL) and different concentrations (0.1 5 g/mL) of aqueous extract of Plinia edulis leaves (AEP) for 24 or 48 h, compared to the negative control cells Plinia edulis leaves (AEP) for 24 h, compared to the negative control cells (NC). Results are expressed as mean±SEM of three independent experiments. (NC), estimated by the Fast green color dye exclusion. Results are expressed as Different letters indicate significant differences (pb0.01) by the Tukey test. mean±SEM of three independent experiments. Different letters indicate b Note cell-diameter reduction after treatment, in comparison to control cells, significant differences (p 0.01) by the Tukey test. Inset: appearance of cells – thereby indicating AEP cytotoxicity. after fast green-hematoxylin eosin staining; 1, green dead cell; 2, reddish-pink living cell. AEP treatment kills MCF-7 cells in a concentration-dependent manner.

Table 1 MCF-7 cell colony diameter (μm). Treatment Incubation time (h) significant (pN0.05) hindrance was achieved by increasing AEP 24 48 concentration or incubation time. Therefore, it is possible to NC 12.72±1.39a 31.48±1.83a sustain the positive cytotoxic role of AEP in the inhibition of CP 8.65±0.64b 18.53±1.94b MCF-7 cell growth and spread, thus affecting cell viability. AEP0.1 12.05±1.74a 16.30±1.44b The most readily observed effect, following cell exposure to NC — negative control cells, CP — 550 μg/mL cyclophosphamide-treated toxicants, is morphological alteration in the cell-layer and/or cells, and AEP0.1 — 0.1 μg/mL aqueous extract of Plinia edulis leaves-treated cell-shape in monolayer cultures (Ekwall et al., 1990). AEP and cells. Results are expressed as mean±SEM of three independent experiments. CP treatment induced alterations in cell morphology. Untreated b Different letters in the same column indicate significant differences (p 0.01) by control cells grow as colonies, presented irregular morphology, the Tukey test. and were homogeneously stained, with the cytoplasm less stained than the nucleus and the nucleolus plainly visible. On Evans et al., 1996). The interplay between free radicals and the other hand, when exposed to AEP cells shrank, became antioxidants is important in maintaining health and preventing rounded and individually separated, with wrinkled cytoplasmic the onset and progression of several pathologies, including cancer membrane and nuclear condensation, indicating cytoskeleton (Gouvêa, 2004; Halliwell, 2007; Huang et al., 2010). disruption (Fig. 3). Cell shrinkage was further confirmed by the determination of cell and colony diameter that were signifi- cantly (pb0.01) decreased in comparison to control cells 3.3. Determination of cytotoxicity (Fig. 4 and Table 1). AEP seemed to be more effective than CP, even at lower concentrations (Fig. 4). Cell colonies were visible We evaluated the cytotoxicity of AEP using the SRB assay only with 0.1 μg/mL AEP treatment; upon higher AEP (Fig. 2). SRB, in moderated acid conditions, binds stoichiomet- concentrations only individually separated cells were seen. rically to basic protein amino acids so that the decrease in OD A cytotoxic compound induces short-term loss of cell observed with AEP treatment could be correlated to cell number, viability either by triggering cell-death or decreasing cell sur- thus being an indicator of cell growth (Vichai and Kirtikara, vival (Sumantran, 2011), so we next investigated the AEP 2006) or the extract could affect the protein turnover, affecting effect on cell death. AEP and CP treatment induced significant cell viability. The results depicted in Fig. 2 show that the extract (pb0.01) cell death, in a concentration-dependent manner with reduced cell protein content in a concentration-dependent man- maximumeffectat5μg/mL (Fig. 5). ner. Cell treatment with 0.1 μg/mL AEP for 48 h or with 0.2 to DNA fragmentation analysis (Fig. 6) indicates that CP and 2.5 μg/mL for 24 and 48 h reduced protein content by 80% and AEP induced apoptosis, as a DNA ladder pattern was obtained. the maximum effect was obtained with 5 μg/mL AEP. No further Laddering-DNA is one of the best-studied biochemical

Fig. 3. Morphology of MCF-7 cells stained with hematoxylin and eosin, after 24 and 48 h incubation. Cont, control cells; CP, cells treated with 550 μg/mL cyclophosphamide; AEP0.1, cells treated with 0.1 μg/mL aqueous extract of Plinia edulis leaves; AEP5, cells treated with 5 μg/mL aqueous extract of P. edulis leaves. All the pictures are typical of three independent experiments, each carried out under identical conditions. Bar=5 μm. N — nucleolus; R — rounding; NC — nuclear condensation; W — wrinkled cytoplasmic membranes. 6 A.J.S. Carvalho et al. / South African Journal of Botany 81 (2012) 1–7

Acknowledgments

Financial assistance from FAPEMIG (grant APQ-1305/06) and the fellowship from PET-MEC-SESU (CMCPG) are grate- fully acknowledged.

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