Study of the antiproliferative potential of seed extracts from Northeastern Brazilian plants

This study assessed the antiproliferative and cytotoxic potential against tumor lines of ethanolic seed extracts of 21 plant species belonging to different families from Northeastern Brazil. In addition, some underlying mechanisms involved in this cytotoxicity were also investigated. Among the 21 extracts tested, the MTT assay after 72 h of incubation demonstrated that only the ethanolic extract obtained from Myracrodruon urundeuva seeds (EEMUS), which has steroids, alkaloids and phenols, showed in vitro cytotoxic activity against human cancer cells, being 2-fold more active on leukemia HL-60 line [IC50 value of 12.5 (9.5-16.7) μg/mL] than on glioblastoma SF-295 [IC50 of 25.1 (17.3-36.3) μg/mL] and Sarcoma 180 cells [IC50 of 38.1 (33.5-43.4) μg/mL]. After 72h exposure, flow cytometric and morphological analyses of HL-60-treated cells showed that EEMUS caused decrease in cell number, volume and viability as well as internucleosomal DNA fragmentation in a dose-dependent way, suggesting that the EEMUS triggers apoptotic pathways of cell death.


INTRODUCTION
There is considerable scientific and commercial interest in discovering new anticancer agents from natural product sources (Kinghorn et al. 2003).The potential of using natural products as anticancer agents was recognized in the 1950s by the U.S. National Cancer Institute (NCI) and since then several studies have given valuable contributions to the discovery of new naturally occurring anticancer agents.In the 1980s, the development of new screening technologies led the research for new anticancer agents in plants and other organisms, focus- Santos et al. 2008).Despite this great biodiversity, Northeastern Brazilian plants are relatively underexploited with regard to discoveries of active biological substances (Luna et al. 2005).
Seeds, especially from legumes, are recognized by their nutritional value, rich in proteins, carbohydrates and oil, though they are not merely a site to accumulate organic materials.They need physical and chemical mechanisms for protection and/or defense for the developing embryo.The compounds involved in chemical defense include lectins, protease and amylase inhibitors, toxins and low molecular mass compounds (secondary metabolites) (Xavier-Filho 1993, Sampaio et al. 1992, Ferreira et al. 2009).According to some reports many seeds and other parts from Northeastern Brazilian plants are exploited in popular medicine and many of these present important pharmacological properties.The ethnomedical data and some pharmacological activities of the studied plants are shown in Table I.Nevertheless, these plants, especially their seeds, are scarcely studied concerning cytotoxicity on tumor cell strains.Thus, the aim of the present study was to assess the antiproliferative potential of ethanolic seed extracts of twenty-one plant species belonging to different families from Northeastern Brazil on tumor cells and study some underlying mechanisms involved in this cytotoxicity.

ANIMALS
Adult Swiss mice (Mus musculus Linnaeus, 1758) were obtained from the animal facilities of the Universidade Federal do Ceará, Fortaleza, Brazil.They were kept in well ventilated cages under standard conditions of light (12 h with alternative day and night cycles) and temperature (27 ± 2 • C) and housed with access to commercial rodent stock diet (Nutrilabor, Campinas, Brazil) the Caatinga forest (dry land vegetation), Araripe National Forest (rain forest), and in the coastal zone.Mature wild seeds (at least 500 g) of each plant species were collected during the dry period (from January 2005 to November 2007), with help of native people.Plants were identified and voucher specimens were deposited at Herbarium Prisco Bezerra -EAC, Universidade Federal do Ceará (Fortaleza, Ceará, Brazil).Table I described all the species studied in this work, their voucher numbers, common names, harvest data and medicinal applications.

PREPARATION OF CRUDE EXTRACTS
Seeds of freshly collected plant material were separated, immediately air dried and finally ground in a laboratory mill (Quimis, Campinas, São Paulo, Brazil) to a moderately-fine powder (mesh size 0.5 mm).Powdered material (500 g) was submitted to extraction with 99% ethanol (1.5 L) at room temperature (25-27 • C) for 3 days and filtered.The residue was re-extracted twice in a similar manner.The extracts were evaporated and bulked under reduced pressure in a rotary evaporator.Crude extracts were stored in a freezer at -20 • C until required.A stock solution containing 10 mg/mL of each crude extract was prepared by suspending 10 mg of extract in 1 mL of sterile dimethylsulphoxide (DMSO, Sigma Aldrich) (Torres et al. 2005, Costa et al. 2008, Buriol et al. 2009, Magalhães et al. 2010), mixed by sonication (Bandelin, model RK-100, Berlin, GER) for 20 min.

CYTOTOXICITY AGAINST HUMAN TUMOR CELL LINES
The antiproliferative potential of the seed extracts was evaluated by the MTT assay (Mosmann 1983) against 4 human tumor cell lines: HL-60 (leukemia), SF-295 (glioblastoma),  and MDA/MB-435 (melanoma), all obtained from the National Cancer Institute (Bethesda, MD, USA).All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/mL penincillin and 100 μg/mL streptomycin, at 37 • C with 5% CO 2 .Tumor cell growth was quantified by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H -tetrazolium bromide (MTT) to a purple formazan product.Briefly, cells were plated in 96-well plates [0.7 × 10 5 cells/well for adherent cells  All cytometry analyses were determined in a Guava EasyCyte Mine (Guava Express Plus software).Five thousand events were evaluated per experiment and cellular debris was omitted from the analysis.Experiments were performed in triplicate using HL-60 cells and analyzed after 72 h of incubation with EEMUS (6.25, 12.5 and 25 μg/mL).

Cell number and membrane integrity
Cell membrane integrity was evaluated by the exclusion of propidium iodide (50 μg/mL, Sigma Aldrich Co. -St.Louis, MO/USA).Briefly, 100 μL of treated and untreated cells were incubated with propidium iodide (50 μg/mL).The cells were then incubated for 5 min.
Fluorescence was measured and cell number and membrane integrity were determined (Darzynkiewicz et al. 1992).

Internucleosomal DNA fragmentation
Internucleosomal DNA fragmentation was evaluated by the incorporation of propidium iodide (50 μg/mL).Briefly, HL-60 cells were treated and then incubated at 25 • C for 30 min, in the dark, in a lysis solution containing 0.1% citrate, 0.1% Triton X-100 and 50 μg/mL propidium iodide.Fluorescence was measured and DNA fragmentation was analyzed according to Nicoletti et al. (1991).

Measurement of mitochondrial transmembrane potential
Mitochondrial transmembrane potential was determined by the retention of rhodamine 123 dye.Aliquots removed from wells were incubated with 200 μL of rhodamine 123 in the dark for 15 min and centrifuged at 2000 rpm/5 min.Subsequently, cells were harvested and incubated in PBS solution for 30 min.at 25 In order to predict activity of EEMUS towards an in vivo cancer model, ascite-bearing female mice between 7 and 9 days postinoculation were sacrificed by cervical dislocation and a suspension of Sarcoma 180 cells was harvested from the intraperitoneal cavity under aseptic conditions.The suspension was centrifuged at 500 X g for 5 min to obtain a cell pellet and washed three times with RPMI medium.Cell concentration was adjusted to 0. loids were performed according to Matos (2000).These tests are based on visual observation of color modification or precipitate formation after addition of specific reagents.

STATISTICAL ANALYSIS
For cytotoxicity assays, the IC 50 and EC 50 values and their 95% confidence intervals were obtained by nonlinear regression using the Graphpad program (Intuitive Software for Science, San Diego, CA).In order to determine differences, data (mean ± standard error mean) were compared by analysis of variance (ANOVA) followed by Newman-Keuls test (P<0.05).

RESULTS AND DISCUSSION
Drug discovery from medicinal plants has played an important role in the treatment of cancer and most new clinical applications of plant secondary metabolites and their derivatives have applied towards combating cancer (Butler 2004, Cragg andNewman 2005).In this work, of the 21 ethanolic extracts tested, the analyses by MTT assay showed that only the ethanolic extract obtained from M. urundeuva seeds showed cytotoxic potential against cancer cells (Table II), given that it was the sole extract that caused cell proliferation inhibition higher than 90% (Torres et al. 2005).Subsequently, we determined its IC 50 values on tumor lines by MTT assay in a similar way as described above with the bioactive extract concentration ranging from 0.09 to 50 μg/mL.According to the American National Cancer Institute, the IC 50 limit to consider a promising crude extract for further purification is a value lower than 30 μg/mL (Suffness and Pezzuto 1990).As seen in Table III, EEMUS was inactive against in vitro colon and melanoma tumors, while it demonstrated moderate activity on glioblastoma [SF-295, IC 50 of 25.1 (17.3.3-36.3)μg/mL] and especially on leukemia [HL-60, IC 50 of 12.5 (9.5-16.7)μg/mL] cells.On the other hand, the positive control doxorubicin presented high cytotoxicity against all cell lines (Table III).Myracrodruon urundeuva Fr.Allemao, 1881 (Anarcadiaceae), an endemic tree in Northeastern Caatinga, is a folk medicinal plant known as "aroeira do sertão" very used for treating bleeding gums and gynecological disorders (Viana et al. 2003, Monteiro et al. 2006).Different parts of M. urundeuva also possess hepatoprotective, anti-diarrheal, anti-ulcer, cicatrizing, colonic anastomotic wound healing properties and larvicidal activity (Viana et al. 2003, Goes et al. 2005, Souza et al. 2007, Sá et al. 2009).Recently, Sá et al. ( 2008) isolated a termiticidal lectin from M. urundeuva heartwood resistant to enzyme degradation to elucidate the resistance and durability of its wood to biodegradation by termites, a plague that has caused damages in several wooden parts in buildings and serious economical issues.Despite its diverse pharmacological applications, this is the first study showing a directed anticancer potentiality of this plant.
In an attempt to envisage an antitumor action upon in vivo assessments, it was determined the EEMUS activity on Sarcoma 180 cells using the Alamar Blue assay.Here, we also found antiproliferative action of EEMUS against these malignant cells, exhibiting an IC 50 of 38.1 (33.5-43.4)μg/mL, an additional findings that stimulates further investigation to understand its mechanism of action.
Cell type antiproliferative specificity is observed in some plant extracts and this is probably due to the presence of different classes of compounds (Cragg et al. 1994).Hence, the use of more than one cell line is considered necessary for detection of cytotoxic compounds.In the present work, EEMUS showed activity against leukemia line 2-fold higher than against melanoma and Sarcoma 180 cells (P<0.05).Then, we chose the HL-60 line to study underlying mechanisms involved in the in vitro cytotoxicity.The human HL-60 cell line, acute promyelocytic leukemia with prevailing of neuthophilic promyelocytes, is commonly used in the research for novel cytotoxic substances (Costa et al. 2008, Ferreira et al. 2010, Magalhães et al. 2010).
Extract-induced morphological alterations in HL-60 treated and untreated cells were examined following 72 h of treatment and staining by H&E.In comparison with the control cells and treated cells at the lowest concentration (6.25 μg/mL), both exhibiting a typical non-adherent morphology and dividing cells (Fig. 1A), EEMUS-treated cells at 12.5 μg/mL presented chromatin condensation and shrinking (Fig. 1D) while damage on plasmatic membrane was mainly seen at 25 μg/mL (Fig. 1E).Doxorubicin also induced reduction in cell volume, besides nuclear disintegration and chromatin condensation (Fig. 1B).*Data are presented as IC 50 values and 95% confidence intervals for leukemia (HL-60), melanoma (MDA/MB-435), glioblastoma (SF-295) and colon (HCT-8) cells.
An Acad Bras Cienc (2011) 83 (3) Growth-inhibitory effects of the EEMUS were also analyzed in HL-60-treated cells by flow cytometry.The propidium iodide intercalation test showed that EEMUS caused a decreasing in the cell number in a dose-dependent manner after 72 h of exposure (Fig. 2A), starting at the concentration of 12.5 μg/mL (IC 50 value).These results are consistent with the MTT findings and with reduction of cell viability (Fig. 2B) at 25 μg/mL (71.9 ± 2.4%) in comparison to the negative control (95.1 ± 1.1%) (P<0.01).Internucleosomal DNA evaluations showed that EEMUS led to a significant and dosedependent increase in the DNA fragmentation (P<0.01), with fragmentation percentages of 27.7 ± 2.4% and 45.5 ± 2.0%, for the concentrations of 12.5 and 25 μg/ mL, respectively (Fig. 3).On the other hand, statistically significant cellular mitochondrial depolarization (Fig. 2B) was detected only at the highest concentration 27.6 ± 4.5% (P<0.01),suggesting that cell death should be initially triggered by a mitochondrial independent pathway or it could be an apoptosis resulting from an early DNA fragmentation (Wang et al. 1999).
Morphological and flow cytometric studies revealed a concomitant deoxyribonucleic acid disintegration (increasing sub-G1 population cells), chromatin condensation with membrane integrity at 12.5 μg/mL and membrane disruption at higher concentrations.These results suggest dose-dependent apoptotic cell death activation by EEMUS, though other biochemical and morphological examinations are necessary to confirm it, such as phosphatidylserine externalization and caspase determinations (Strasser et al. 2000).
The phytochemical analyses detected the presence of steroids, alkaloids and phenols in EEMUS (data not shown).Some steroids, flavonoids and other phenolic substances are frequently associated with the aging process of the human body, production of free radicals due to metabolic processes, initiation and promotion of cancer and tissue injury by free radicals, which has induced the intake of antioxidant products as chemical factors that prevent the onset of diseases (Núñez-Sellés 2005).Previously, it was demonstrated that hydroalchoolic stem bark extracts from M. urundeuva exert anti-inflammatory effects attributed to chalcones (Viana et al. 2003, Souza et al. 2007), a compound belonging to the group of flavonoids naturally found in fruits, flowers, vegetables, teas and wines (Abdulla andGruber 2000, Ferreira et al. 2008).Besides the traditional antioxidant properties attributed to the flavonoids, some chalcones and their derivatives have reported to be potent cyclooxygenase inhibitors (Hsieh et al. 1998).This is an important approach to some kinds of cancers, since COX-2 blockage avoids the expression of NF-κB activation, a key nuclear transcription factor involved in controlling inflammation and tumorigenesis (Surh et al. 2001), since inflammation has been frequently found in premalignant lesions (Dranoff 2004).This study displays the antiproliferative action of the ethanolic extract of Myracrondruon urundeuva seeds on leukemia cells by death suggestive of apoptosis and also showed its potential against experimental in vivo tumors.Further studies to support these discoveries are in progress as well as phytochemical and molecular investigations to identify the bioactive compound(s) responsible for this cytotoxic activity.
An Acad Bras Cienc (2011) 83 (3) We wish to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), Financiadora de Estudos e Projetos (FINEP) and Banco do Nordeste do Brasil (BNB) for financial support in the form of grants and fellowship awards.We are grateful to Berenice Alves and Silvana França dos Santos for technical assistance.

Fig. 2 -
Fig. 2 -Effects of the ethanolic extract of Myracrodruon urundeuva seeds (EEMUS) on HL-60 cells analyzed by flow cytometry after 72 h exposure.A -Total of cells; B -Cell membrane integrity.Analyses were determined by exclusion of propidium iodide.Negative control (C) was treated with the vehicle used to dilute the tested substance.Doxorubicin (0.3 μg/mL) was used as positive control (D).Results are expressed as mean ± standard error of measurement (S.E.M.) from three independent experiments.*P<0.01 compared to control by ANOVA followed by Student Newman-Keuls test.

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Fig. 3 -DNA fragmentation on HL-60 cells determined by flow cytometry after 72 h of incubation with ethanolic extract of Myracrodruon urundeuva seeds (EEMUS).All evaluations were performed by nuclear fluorescence using propidium iodide, triton X-100 and citrate.Negative control (C) was treated with the vehicle used for diluting the tested substance.Doxorubicin (0.3 μg/mL) was used as positive control (D).Results are expressed as mean ± standard error of measurement (S.E.M.) from three independent experiments.*P<0.01 compared to control by ANOVA followed by Student Newman-Keuls test.

1047 TABLE I Plant species employed in this study and some ethnobotanical uses and pharmacological activities.
An Acad Bras Cienc (2011)83(3) 1048 PAULO MICHEL P. FERREIRA et al.

TABLE II Tumor cell proliferation inhibition (%) of ethanolic seed extracts of twenty-one plant species belonging to different families from Northeastern Brazil determined by MTT assay after 72h of incubation at the concentration of 50 μg
/mL.