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On-line version ISSN 1678-2690
An. Acad. Bras. Ciênc. vol.80 no.3 Rio de Janeiro Sept. 2008
Magdalena N. RennóI; Gleyce M. BarbosaI; Patricia ZancanII; Venicio F. VeigaIII; Celuta S. AlvianoIV; Mauro Sola-PennaII; Fábio S. MenezesV; Carla HolandinoI
ILaboratório Multidisciplinar de Ciências Farmacêuticas, Departamento de Medicamentos, Faculdade de Farmácia, Centro de Ciências da Saúde, UFRJ, Rua Professor Rodolfo Rocco s/n, Bloco B, sub-solo, sala 11, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
IILaboratório de Enzimologia e Controle do Metabolismo (LabECoM), Departamento de Fármacos, Faculdade de Farmácia, Centro de Ciências da Saúde, UFRJ, Rua Professor Rodolfo Rocco s/n, Bloco B, sub-solo, sala 27, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
IIIInstituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saúde, UFRJ, Rua Professor Rodolfo Rocco s/n, Bloco I, sub-solo, sala 27, Ilha do Fundão, 21941-590 Rio de Janeiro, RJ, Brasil
IV Instituto de Microbiologia Professor Paulo de Góes, Centro de Ciências da Saúde, UFRJ, Rua Professor Rodolfo Rocco s/n, Bloco I, 1º andar, sala 50, Ilha do Fundão, 21941-590, Rio de Janeiro, RJ, Brasil
VSchool of Pharmacy and Pharmaceutical Sciences, Trinity College, Dublin, 23, Westland Row, Dublin 2, Ireland
Plant-derived substances have been considered as important sources of drugs, including antineoplasic agents. Babassu mesocarp is popularly used in Brazil as a food additive, and in popular medicine against several conditions, such as inflammations, menstrual pains and leukaemia. From babassu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] epicarp/mesocarp, an ethanol extract was prepared and named OSEME, which was tested on the viability,morphology and metabolism of several cell lines, such as the leukaemic cell lines, HL-60, K562 and the latter multidrug resistant counterpart K562-Lucena 1, the human breast cancer cell line MCF-7, the mouse fibroblast cell line 3T3-L1 and fresh human lymphocytes. OSEME promoted a dose-dependent decrease on the viability of all cells. This effect was much more pronounced on the tumoral cell lines than on non-tumoral cells, a phenomenon revealed by the dose of OSEME which promotes half of maximal effect (ID50). The decrease on viability was followed by shrinkage of cells, alteration on their morphology, and a markedly nuclear condensation. Curiously, stimulation of 6-phosphofructokinase activity (6.6-times) was observed on HL-60 cells, treated with OSEME, when compared to control treated with ethanol (vehicle). These results support evidences to suggest OSEME as a promising source of novel antineoplasic agents.
Key words: antitumoral agents, ethanol extract, Orbignya speciosa.
Substâncias derivadas de plantas têm sido usadas como importante fonte de agentes antineoplásicos. O mesocarpo do babaçu é popularmente usado no Brasil como suplemento alimentar e na medicina popular para o tratamento de várias afecções, tais como: inflamações, cólicas menstruais e leucemia. A partir do epicarpo/mesocarpo do babaçu Orbignya speciosa (Mart.) Barb. Rodr. [Arecaceae (Palmae)] foi preparado um extrato etanólico, denominado OSEME, o qual foi incubado com as seguintes linhagens humanas leucêmicas: HL-60, K562 e a sua derivada resistente a múltiplas drogas, K562-Lucena 1; além destas, foram testadas a linhagem humana de câncer de mama, MCF-7; a linhagem de fibroblastos de camundongo, 3T3-L1 e linfócitos humanos de sangue periférico. OSEME promoveu diminuição da viabilidade em todas as linhagens celulares testadas de maneira dose-dependente. Este efeito foi mais pronunciado sobre as linhagens celulares tumorais quando comparado às não tumorais, o que foi revelado pela dose de OSEME capaz de promover metade do efeito máximo (ID50). A diminuição da viabilidade foi acompanhada por danos sobre a morfologia celular com pronunciada condensação citoplasmática e nuclear. Curiosamente, quando a linhagem HL-60 foi tratada com OSEME, foi detectado um aumento de 6,6 vezes da atividade da enzima 6-fosfofrutoquinase, quando comparado ao grupo controle (células tratadas com o veículo etanol). Esses resultados sugerem que OSEME pode ser uma promissora fonte de novos agentes antineoplásicos.
Palavras-chave: agentes antitumorais, extrato etanólico, Orbignya speciosa.
Babassu (Orbignya martiana, O. oleifera or O. speciosa) is a native palm tree from the North, Northeast and Central regions of Brazil, occurring mainly in the Maranhão, Piaui, Tocantins and Goias States in extensive forests (Emmerich and Luengo 1996). Traditionally, the babassu mesocarp has been used for the treatment of several conditions including inflammations of the uterus and ovarium, treatment of constipation, colitis, obesity, and leukaemias, among other tumors (Silva and Parente 2001).
The vast diversity of the Brazilian flora stimulates the exploitation of plant extracts with medical and biological activities. In this regard, studies of plant compounds with antioxidant activity has attracted great interest in the last years, due to the fact that free radicals have been clearly associated to pathological process, including cancer. Some authors have associated the prevention of cancer with the ingestion of plant extracts enriched in natural antioxidants (for a review see Ferguson and Philpott 2007). We have recently described that babassu extracts present antioxidant activity acting as free radical scavengers, evaluated by means of reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and protecting yeast cells from lethal oxidative stress (Silva et al. 2005). Moreover, other therapeutic properties of babassu have been reported, such as anti-inflammatory (Silva and Parente 2001), cicatrizant (Martins et al.2006), gastric healing (Batista et al. 2006), antithyroid (Gaitan et al. 1994), anti-thrombotic (Azevedo et al. 2007) and anti-microbial (Caetano et al. 2002).
The aim of the present study is to evaluate the effects of babassu extract on the viability, morphology and metabolism of leukaemic cell lines in culture, as well as on other tumor cells, non-tumor immortalized cell lines and freshly isolated human lymphocytes. Here we show a cytotoxic effect of babassu extract, which is indicative of a possible usage of babassu as a source of antineoplasic compounds with low damage to normal cells.
MATERIALS AND METHODS
The babassu (Orbignya speciosa (Mart.) Barb. Rodr.) fruits were collected in December 2002, in the Piauí State, Brazil. A voucher specimen was deposited in the Graziella Barroso Herbarium (Federal University of Piauí), under the number TEPB 18985.
The epicarp/mesocarp (at least 1200g) from the fruits was dried, powdered and submitted to successive extraction by maceration with 96% ethanol, at room temperature (Silva et al. 2005). All the extract was filtered through filter paper and then the crude ethanol extract of the epicarp/mesocarp was evaporated to dryness in a rotatory evaporator, with average yield from 3% of the starting material dry weight. After drying, the extract was suspended in ethanol. The ethanol extract of epicarp/mesocarp (OSEME) was further sterilized by filtration (0.22µm), for biological assays.
The erythroleukaemic cell line K562 and its multidrug-resistant (MDR) counterpart, K562-Lucena 1 and lymphocytes cells were kindly donated by Dr. Vivian M. Rumjanek (Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil). The human promyelocitic leukaemic cell line HL-60, 3T3-L1 mouse fibroblasts and MCF-7 cells (mammary duct carcinoma cell line) were obtained from Cell Bank of Hospital Universitário Clementino Fraga Filho, UFRJ, Brazil. Cells were grown at 37ºC in 25cm2 culture flasks containing Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% of fetal bovine serum (FBS-Gibco, Invitrogen, USA), 1% PSA (10,000 UI/mL penicillin G sodium, 10,000 g/mL streptomycin sulfate and 25µg/mL amphotericin B, as Fungizone®). D-MEM medium was previously supplemented with 3 g/L N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid) (HEPES), 0.3 g/L L-glutamine and 0.2 g/L NaHCO3 (pH 7.4), as previously described (Freshney 1994). K562-Lucena 1 cells were maintained in the presence of 60 nM vincristine (Maia et al. 1996), while the medium used for cultivation of MCF-7 was also supplemented with 5µg/mL insulin.
FRESH HUMAN LYMPHOCYTES PREPARATION
Peripheral blood mononuclear (lymphocytes) cells were collected from fresh heparinized blood of health volunteers (18-30 years old) by venopuncture and separated by density gradient centrifugation (Ficoll®). In 50 mL conical tubes, 20 mL of blood was layered over 15 mL of Ficoll®, followed by centrifugation at 1800 rpm for 30 min at room temperature. Buffy coats and serum were collected in phosphate-buffered saline and centrifuged at 1200 rpm for 7 min at room temperature (Baruque et al. 2005). Supernatants were discarded; cells were washed two times in 50 mL of phosphate-buffered saline, and re-suspended in the same culture medium described above.
For cytotoxicity assays, approximately 5×105 leukaemia cells were suspended in D-MEM/FBS and plated into the wells of 24-well culture plates. They were treated with different concentrations (150, 300, 600, 1200, 1500 and 2000mg/mL) of the OSEME or 1.2% ethanol (used as OSEME vehicle). The cells were incubated at 37ºC and viability was evaluated by permeability to trypan blue (Patterson 1979), after 1, 4, 8 and 24 hours. The effects of the OSEME on inhibition of cell proliferation were calculated by comparing extract-treated cells with untreated cells. The number of viable cells in the different systems was determined as previously described (Holandino et al. 2001), and the suspensions containing similar cell numbers cultivated in 24 well plates for 24 hours as described above. Aliquots of 25µl of the cell culture were then mixed with 25µl of trypan blue and the total number of cells determined in a Neubauer chamber. Non-viable cells were divided in trypan blue-stained cells that retained their shape and lyzed leukaemic cells (Veiga et al. 2005). Effects of the OSEME on lymphocytes were evaluated after a 24 h treatment, followed by staining with trypan blue. The cells were plated in 24 well culture plates in D-MEM/FBS, and treated with 300, 600, 1200µg/mL of the OSEME or 1.20% ethanol (used as OSEME vehicle) at 37ºC.
The confluent cell lines MCF-7 and 3T3-L1 were plated into the wells of 24-well plates in the presence of OSEME (300, 600 and 1200µg/mL) or control, 1.20% ethanol, and incubated for 24 hours at 37ºC. The supernatant was then removed and the cells were washed twice with PBS, followed by trypsinization and suspension in 1 mL DMEM. Cell viability was evaluated as described above.
RADIOASSAY FOR 6-PHOSPHOFRUCTO-1-KINASE ACTIVITY
The HL-60 cells were cultivated in presence of 300µg/ mL OSEME, 1.20% ethanol (vehicle control) or in medium alone for 24 hours at 37ºC. 6-Phosphofructo-1-kinase (PFK) activity was assayed by radiometric methods (Sola-Penna et al. 2002). Briefly, the assays were performed at 37ºC in a reaction medium containing50 mM Tris-HCl (pH 7.4), 5 mM (NH4)2SO4, 5 mM MgCl2, 1 mM [γ-32P] ATP (4µCi·µmol-1), and 1 mM fructose-6-phosphate. After different intervals, 0.4 mL aliquots were withdrawn and added to 1 mL of activated charcoal suspension (250 g/L) containing 0.1 N HCl and 0.5 M mannitol. The material was centrifuged at 1.500 g for 10 min, and an aliquot of the supernatant was counted in a liquid scintillation counter to evaluate the amount of [1-32P] fructose-1,6-bisphosphate formed. Activity rate was calculated by the linear regression of the amount of [1-32P] fructose-1,6-bisphosphate formed during the linear phase of the reaction as a function of reaction time. Quadruplicates were performed for all samples and blanks were obtained in the absence of fructose-6-phosphate.
The different cell line and fresh lymphocytes (5×105 cells/ mL) were incubated with different concentrations of OSEME for 24 h, centrifuged, washed and finally suspended in PBS. The morphological features of treated cells were assessed by the May-Grunwald-Giemsa's method. After fixation, structural cellular alterations were evaluated by optical microscopy. Images were acquired digitally (Veiga et al. 2005, Meira et al. 2005).
KINETIK CALCULATIONS AND STATISTICS
Kinetics parameters were calculated by non-linear regression as described previously (Meira et al. 2005) using the Sigma Plot/SigmaStat integrated software packages (Systat, CA, USA). The OSEME dose that induces half of maximal decrease on cells viability (ID50), was calculated through the equation:
where VC is the number of viable cell at a given dose of OSEME ([OSEME]) and C0 is the number of viable cells in the absence of OSEME. The incubation time to achieve half of the maximal inhibition (t0.5) was calculated through the equation:
where k is the rate constant calculated through equation:
where VC is the number of viable cells at a given incubation time (t) and Ci is the number of viable cells before incubation.
Student's t-test or one-tailed ANOVA were used to determine significance between means, considering P < 0.05 as statistical significant.
The parameter used to evaluate cell viability was the permeability to trypan blue dye. Results are presented as the number of non-permeable cells immediately after OSEME treatment or after a 24 hours incubation of treated cells. As demonstrated in Figure 1 OSEME promoted a dose-dependent decrease in the viability in all cell types. However, the magnitude of the inhibitory effect varied according with cell type. (ID50) determinations (Table I) demonstrate that OSEME was more effective in inhibiting HL-60 cells. The other tumoral cell lines K562, K562-Lucena 1 and MCF-7 were moderately sensitive to OSEME, while non-tumoral cells (3T3-L1 and fresh human lymphocytes) were relatively resistant (ID50 higher than 100µg/mL). These effects were also dependent on the time of incubation in the presence of OSEME. As a representative experiment, the effects of OSEME on the viability of HL-60 cells are presented in Figure 2A where it can be seen that cell viability decreases as the dose of OSEME increases. On the other hand, HL-60 incubated in the absence of OSEME grown normally (Fig. 2A, inset). From these experiments, we calculated the time to reach half of the maximal effect (t0.5) in the presence of different OSEME doses. This parameter decreases as the dose of OSEME increases (Table II), revealing a classical dose- and time-dependent effect. Figure 2B shows the profile of time-dependence when a 300µg/mL dose of OSEME was used in viability assays using HL-60, K562 and K562-Lucena 1 cells.The calculated t0.5 for these experiments, (7.0 ±0.7, 6.9 ±0.6 and 7.0 ±0.8, for HL60, K562 and K562-Lucena 1, respectively) were not significantly different (P > 0.05, ANOVA).
OSEME promoted alterations on cell morphology, size and shape, as microscopically observed in Giemsa-stained preparations of cells treated with a 1200µg/mL dose of OSEME (Fig. 3). After treatment with the extract for 24 hours, cells become smaller and rounded, and presented a marked nuclear condensation. Treatment with ethanol did not promote significant alterations in animal or human cells.
In order to evaluate the effects of OSEME on the cellular metabolism, we measured the activity of PFK in HL-60 cells incubated with 300µg/mL OSEME for 24 hours (Fig. 4). These cells were chosen because of their higher sensibility to the inhibitory effects of OSEME (ID50 = 9.3 ±0.8µg/mL). Curiously, in comparison with ethanol-treated controls, OSEME-treated cells presented a stimulated PFK activity. However, PFK activity of OSEME-treated cells did not differ from that observed in cells that were not exposed to any treatment, suggesting that exposure to ethanol was probably the cause of enzyme inhibition.
In the current anti-cancer therapy, around 60% of the currently used drugs are derived in one way or another from natural sources, including plants, marine organisms and microorganisms (Newman et al. 2003, Cragg et al. 2005). In addition, plant-derived compounds and semi-synthetic analogs have been used in combination with other chemotherapeutics for the treatment of a variety of cancers, including leukaemias, lymphomas, advanced testicular cancer, breast and lung cancers, and Kaposi's sarcoma (Cragg and Newman 2005). The search for new drugs able to overcome resistance mechanisms is of great interest for cancer therapy (Fernandes et al. 2006). Although the chemotherapeutic treatment of leukaemia has been dramatically improved due to newly discovered drugs, the rate of complete remission in most leukaemia patients is still not satisfactory. Additionally, the phenotype of multidrug resistance (MDR) represents the major cause of chemotherapy failure (Kartner et al. 1985, Pastan and Gottesman 1987, Ferte 2000).
In this regard, it is worthwhile to mention that the MDR cell line used in this study, K562-Lucena 1, was affected in the same extent as its non-resistant counterpart K562. This is indicative that OSEME can overcome the MDR phenotype. Additionally, the cytotoxic effects of OSEME are less prominent in non-tumor cells, indicating a potential selectivity for tumoral cells. This becomes clear after comparison of the OSEME ID50 calculated from the dose-response curves presented in Figure 1. For example, (ID50) on leukaemic cell line HL-60 is approximately 15 times smaller than the value obtained with fresh human lymphocytes. This difference is less pronounced in the other tumoral cells ( ~ 3-times), although it is still highly significant. The inhibitory effect occurs rapidly, as 1 hour after incubation, a significant reduction in cell viability can be observed for all doses of OSEME tested (Fig. 2, P < 0.05, Student's t-test).
Cancer cells present alterations on energetic metabolism to supply the increased demand of ATP and biosynthetic precursors to sustain their augmented cell cycle (El-Bacha and Sola-Penna 2002). These cells are characterized by a high glycolytic rate, which is not inhibited by the presence of oxygen such as in normal mammalian cells (Gatenby and Gillies 2004). The dependence on the glycolytic flux as energy source is higher in aggressive metastatic tumors than in less proliferative neoplasic cells (Kubota 2001). It is frequently observed that tumor malignancy is accompanied by an increase in glucose consumption, lactate production and media acidification (Ashrafian 2006). Classical anticancer agents, such as paclitaxel and vinca alkaloids, inhibit glycolysis in cancer cells (Vertessy et al. 1997, Glass-Marmor and Beitner 1999). Recently, we have demonstrated that clotrimazole decreases human breast cancer cells viability by inhibiting the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK) (Meira et al. 2005). These effects occur in parallel to alterations on cell morphology that includes reduction on cell volume, formation of rounded cells and a marked nuclear condensation, which was associated to induction of apoptosis (Vermes et al. 2000, Meira et al. 2005). In the present work, OSEME promoted similar cellular alterations. This effect is also seen on the MDR cell line K562-Lucena 1, supporting the evidences that OSEME can overcome the MDR phenotype of tumoral cells. However, 300mg/mL of OSEME did not inhibit PFK activity of HL-60 cells after 24 hours incubation. Since calculated PFK activity was corrected by the number of non-permeable cells, we may suggest that this phenomenon is due to the fact that the remaining cells present an enhanced metabolic activity in order to overcome the toxic effects of OSEME. Similar results have been reported elsewhere (Penso and Beitner 2002, 2003, Meira et al. 2005, Leite et al. 2007). We also tried to perform MTT experiments; however OSEME reduced MTT reagent, disguising the results (data not shown).
The presence of bioactive constituents such as, triterpenes, tannins, sugars, saponins and steroid compounds (Bandeira et al. 1986, Garcia et al. 1995) could be correlated to the pharmacological properties of babassu. Silva and Parente isolated from the mesocarp of fruits of Orbignya phalerata a polysaccharide with anti-inflammatory and immunomodulatory properties (Silva and Parente 2001) and our research group described the antioxidant activity of OSEME (Silva et al. 2005). All these properties reinforce the OSEME cytotoxicity, which additionally may present a multi-target action that is desirable in a chemotherapeutical.
The purpose of the present work is to assess in vitro anti-tumor properties of crude ethanol extract against a panel of selected tumoral and non-tumoral cells. In conclusion, results show that ethanol extract from epicarp/mesocarp of babassu exhibit cytotoxic activities against preferentially tumoral cells, comparing with non-tumoral cells, and provide preliminary evidences for the presence of one or more ethanol-soluble constituents with anti-tumor properties. Other studies are necessary to identify the active component(s), to elucidate the mechanisms to support the use of babassu as a source of new anti-tumor drugs.
We thank Dr. Marcio Rodrigues for critical reading of the manuscript and Dr. Ken Boylan for language review. This work was supported by Financiadora de Estudos e Projetos (FINEP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa no Estado do Rio de Janeiro (FAPERJ), Fundação José Bonifácio (FUJB), Fundação Ary Franzino/Fundação Educacional Charles Darwin (FAF/FECD/ONCO), Projetos de Pesquisa para o Sistema Único de Saúde (PPSUS/FAPERJ) and Programa de Apoio a Núcleos de Excelência (PRONEX).
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Dra. Carla Holandino
Manuscript received on August 14, 2007; accepted for publication on March 27, 2008; presented by SERGIO VERJOVSKI-ALMEIDA