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ALKALOIDS EXTRACTED FROM Pterogyne nitens MIGHT INDUCE APTOSIS IN BREAST ADENOCARCINOMA CELL LINE

Roberta A. Duarte1, Elaine R. Mello1, Tarsia G. A. Silva1, Camila Araki1, Vanderlan S. Bolzani2, Dulce H. S. Silva2, Luis O. Regasini2, Valdecir F. Ximenes3, Christiane P. Soares* 1.

1 Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista,14801-902 Araraquara-SP, Brasil.

2 NuBBE - Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Instituto de Química,Universidade Estadual Paulista, 14800-900 Araraquara-SP, Brasil.

3 Faculdade de Ciências de Bauru, UNESP, Universidade Estadual Paulista, 17033-360 Bauru-SP, Brasil.

* E-mail: soarescp@fcfar.unesp.br. Tel: +55 16 3301 6554, Fax +55 16 3301 6559.

RESUMO: O câncer de mama é considerado a maior causa de mortalidade entre as mulheres no mundo. Sua progressão está associada ao rompimento do equilíbrio entre o crescimento e morte celular. O desenvolvimento de novas estratégias para o tratamento do câncer, é necessário, assim como, a elucidação da integração dos múltiplos caminhos de morte celular. Produtos naturais têm contribuído intensamente para o desenvolvimento da terapêutica moderna, principalmente extratos ou substâncias isoladas de plantas têm sido freqüentemente avaliados quanto ao seu potencial anticarcinogênico. Dois alcalóides (pteroginina e pteroginidina) isolados da planta Pterogyne nitens foram os alvos do estudo citotóxico e apoptótico sobre linhagem de câncer de mama MCF-7. Os resultados obtidos após tratamento de 24 horas e tratamento seguido de recuperação de 24 horas (MTT) com ambos os alcalóides mostraram efeito dose resposta. A substância pteroginina mostrou maior porcentagem de apoptose nos ensaios de Anexina V e Hoesch iodeto, na qual foi diferenciada em apoptose precoce, enquanto que a pteroginidina apresentou maior porcentagem de apoptose tardia e necrose. Para melhor investigação das vias apoptóticas o ensaio de caspase revelou não induziu ativação enzimática para via 3/7 da caspase. Dessa maneira os resultados sugerem o potencial efeito apoptótico de alcalóides isolados de P. nitens sobre linhagem de câncer de mama, fortalecendo a perspectiva para novos estudos in vivo.

Unitermos: Pterogyne nitens, apoptose, câncer de mama, alcalóides, citotoxicidade, necrose.

ABSTRACT: “Alkaloids extracted from Pterogyne nitens induce apoptosis in breast adenocarcinoma cell line”. Breast cancer is considered the major cause of mortality among women worldwide. Its progression is associated with disruption of the balance between growth and cell death. The development of new strategies for cancer treatment, it is necessary, as well as the elucidation of the integration of multiple pathways of cell death. Natural products have contributed immensely to the development of modern therapy, mainly extracts or compounds isolated from plants have often been evaluated for their anticancer potential. Two alkaloids (pterogynine and pterogynidine) isolated from the plant Pterogyne nitens were the targets of the cytotoxic and apoptotic study on breast cancer line MCF-7. The results after 24 hours of treatment and treatment followed by recovery of 24 hours (MTT) with both alkaloids showed a dose response. The substance pteroginine showed a higher percentage of apoptosis in the Annexin V tests and Hoesch iodide, which was different in early apoptosis, whereas pterogynidine had a higher percentage of late apoptosis and necrosis. To better investigate the apoptotic pathways showed the test did not induce caspase activation via enzymatic 3/7 caspase. Thus the results suggest the potential apoptotic effect of alkaloids isolated from P. nitens strain on breast cancer, increasing the prospect for further studies in vivo.

Keywords: Pterogyne nitens, apoptose, breast cancer, alkaloids, cytotoxicity, necrose.

INTRODUCTION

Breast cancer is considered the most common neoplasm among women regarding incidence, prevalence and mortality. (Bernstein et al., 1999; Brasil, 2009; Bray et al., 2004; Ferrini et al., 2001; Malorni et al., 2006; Koifman et al., 1998; Silva et al., 2002).

Treating it is one of today’s biggest medicine challenges. The clinically modalities currently employed; as surgical removal, radiotherapy, and specially chemotherapy or hormone therapy; are systemic antiproliferative agents which disrupt cell division. (Bracke, et al., 2008; Komen et. al., 2008; Marsh e Liu, 2009; Xu & Mcleod 2001). These drugs, however, are not selective of cancerous cells and their therapeutic efficiency is limited due to the damage they can cause to healthy cells and tissues. .

Conventional chemotherapy cause intense side effects besides promoting more resistant tumors, especially in the cases of metastasis. Some strategies have been used to overcome the limitations of these agents so they can be selectively activated in the tumoral tissue. (Xu & Mcleod 2001). Thus, studies with natural substances can lead to a better understanding of these substances ability to allow the modulation of apoptosis signals. (Nobili et al., 2009).

The world nowadays is geared toward the use of natural substances present in fruits, vegetables and essential herb oils (Farr, 1997; Wang et al., 1997; Kitts et al., 2000; Son et. al., 2003; Arkin, 2005; Dumont et al., 2007).

There is a fundamental relevance in the study of the anti cancerous activity of alkaloids extracted from plants from the Brazilian Cerrado and Atlantic Forest, once these compounds seem to demonstrate a powerful antiproliferative and cytotoxic activity. (Liu et al., 2004; Kitajima et al., 2006). Purified substances extracted from plants found in the Brazilian Cerrado have demonstrated a varied amount of alkaloids, terpenes and flavonoids. (Urrea-Bulla, et al., 2004, Neheme et al, 2002). Previous studies made with plants from this Brazilian region point that alkaloids and terpenes possess an anti fungus and antiproliferative biological activity besides inducing genotoxicity and anti mutagenicity. (Maistro, et al., 2004; Carvalho et al, 1998; Ferreira et al., 2009).

Among these Brazilian plants, one in specific, the Pterogyne nitens, which belongs to the Fabaceae family and Caesalpinioideae subfamily, is the only species of its kind and is source of numerous cytotoxic guanidinic alkaloids. (Bolzani & Kingston, 1995). It is a plant which has been very little studied and which possesses a variable amount of flavonoids and alkaloids. In Brazil it is poppularly called: “amendoim” (peanut), “amendoim-bravo”, “viraró”, “madeira nova”, “pau-amendoim”, “óleo-branco” or “pau-de-fava” (Fernandes et al., 2008, Regasini et al., 2008). It is a perennial straight tree with glabrous leaves with a height of 10 to 15 meters, originally from Brazil. This plant can be found in Latin America, more specifically in Argentina, Bolivia, Brazil and Paraguay. In Brazil it can be found in the region of the Cerrado and the Atlantic Forest in an area that covers from the state of Ceará until the state of Paraná.

The connection between apoptosis and cancer has been recently emphasized due to the increasing evidences suggesting the processes related to neoplasm transformations, progressions and metastasis is linked to alterations of the apoptotic pathways. (Hanahan & Weinberg, 2000; Morrison et al., 2008)

Apoptosis is a highly regulated physiological process of programmed cell death. It is characterized by several morphological and biochemical cell alterations, (Bergantini et al., 2005; Bras et al., 2005), alterations in the cytoskeleton inducing cell contraction, DNA fragmentation, chromatin condensation leading to the appearance of pycnotic nuclei, formation of vesicle with no membrane loss and no inflammatory response. (Lavin, 1993; Liao et. al., 2005; Yasuhara et al., 2003).

The apoptosis events can be evaluated by the externalization of phosfatidilserine (PS) in the plasmatic membrane (Chinkwo, 2005). The cells in which apoptosis have occurred expose phosfatidilserine in their surfaces (PS), while viable cells keep this in the internal surface of the plasmatic membrane (Boersma et al., 2005, Fadok et al., 2001a, Tibrewal & Raymond, 2006). The present study has as its aim to evaluate the potential apoptotic effect of alkaloids isolated from P. nitens strain on breast cancer (MCF-7).

MATERIALS E METHODS

Extraction and preparations of pure substances (Bolzani, Guanatilaka & Kingston, 1995).

The Pterogyne nitens leaves were dried at room temperature and ground in a knife grinder. The ethanolic macerated (or raw extract) was dissolved in a solution of water:ethanol (1:4) and submitted to a liquid-liquid partition through the employing of ethyl acetate and n-butanol. The n-butanol fraction went through successive chromatographical steps (gel permeation chromatography and reversed-phase chromatography), leading to the isolation of the pterogynine and pterogynidine alkaloids. The isolation of the substances present in the extract was performed by usual chromatographical techniques. The substances were then submitted to spectrometric analysis. (IV, UV, RMN of 1H and 13C, and EM).

Cell culture

Cell lines of ATCC (American Type Culture Collection, USA) for breast cancer (MCF-7) were used to the biological tests with the extracts. The lines were cultivated in a mix 1:1 of DMEM and Ham´s F10 (Sigma), HEPES was added, as well as penicillin, streptomycin, sodium bicarbonate and fetal bovine serum (SFB, Cultilab). The cells were cultivated in bottles and kept at 5% CO2 at a temperature of 37 ºC.

Line treatment

For the cytotoxicity test (MTT), apoptosis/necrosis for Anexin V/propidium iodide (flow cytometry analysis), early and late apoptosis/necrosis by the Hoescht test/propidium iodide and caspase tests a suspension of 2.5x105 cells/cell line well was used. For the pterogynine and pterogynidine alkaloids the concentrations tested were 1.25, 2.5, 5 and 10mM. The cells were treated for 24 hours and evaluated immediately after the treatment (zero time, t0) for the parameters of cytotoxicity and apoptosis. The cells were also treated for 24 hours and then submitted to another 24 hours of post treatment recovery (recovery time, t24h), when the alkaloids were removed and the cytotoxicity and apoptosis tests were performed.

Citotoxicity (MTT)

The MTT method was employed in the evaluation of cytotoxicity, with the principle of determining the ability of the live cells to reduce the salt in 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma), forming insoluble formazan crystals of a violet color. After the treatment the cells were incubated with 10μL of MTT at 37ºC, away from light, until the observation of the presence of the Violet formazan crystals. Afterwards, the solubilization of the formazan crystals was made using 100 (L of isopropyl alcohol in each well and a spectophotometric reading in a wavelength of 540nm. The cytotoxicity of each treatment was expressed by the percentage of cell death in relation to the negative control as proposed by Zhang et al. 2004, Crawford & Bowen 2002.

Apoptosis Tests

Flow cytometry analysis (Anexin V)

To perform the test of anexin V an Anexin V –FITC detection kit was used (Alexis, Lausen, Switzerland). After treatment with alkaloids the cells were resuspended in 500(L ligation mix, 5(l of anexin V - FITC, and 5(l of propidium iodide. Reaction was incubated for 5 minutes at room temperature and kept away form sunlight. The fluorescence intensity (FITC and propidium iodide) was evaluated using a FACSCanto equipment (Bencton Dickinson, USA).

Hoechst method and Iodide

Evaluation of apoptotic and necrotic cells was performed by the method of fluocromos exclusion, through a fluorescent solution made of fluorescein diacetate (DAF), propidium iodide (IP), and Hoescht 33342 (HO) (Sigma). After treatment with the tested substance, the culture medium was taken (dead cells in suspension), the adherent cells were trypsinized, and both were centrifuged. In the cell suspension of 100μl a fluocromos solution was added in which there was 25% aqueous solution of propidium iodide (1mg/mL), 50% of fluorescein diacetate solution in DMSO (1,5mg/mL), 10% of aqueous Hoescht solution (1 mg/mL) and 15% of PBS. The reaction was incubated for 5 minutes at 37ºC and cells were then observed with fluorescence microscopy in an absorption spectrum of 360nm and 538nm.

Cells were classified as normal (blue spherical nuclei colored by the Hoescht, green cytoplasm, in an absorption spectrum of 360nm), apoptotic (blue nuclei with apoptotic bodies colored by the Hoescht, green cytoplasm, in absorption spectrum of 360nm), and necrotic (red spherical nuclei colored by the Propidium iodide, red cytoplasm, in absorption spectrum of 538nm.). Apoptosis was classified as Early (blue nuclei and nuclear morphology with apoptotic bodies) and Late (red colored nuclei with the presence of apoptotic bodies) and necrotic (red intact nuclei) as proposed by Korostoff et. al., 1998 and Hashimoto et al., 2003.

Caspase 3/7 activity test

The cell lines (1,0×105) were adhered in a plaque with 96 wells for luminometry. The plaque was prepared according to the instructions present in the kit, with blank (no cells), negative control, positive control and the treatments with the substances at the times t0 and t24. Subsequently it was added 25 µl (1:1) of the Caspase-Glo™ 3/7 reagent (Promega USA) in each well of the tested samples in culture medium, submitting them to agitation via a plaque shaker during 30 seconds. Plaques were incubated during one hour at room temperature. Then a luminescence reading was performed in a plaque luminometry equipment.

This test has a luminescence substrate with a tetraptide sequence recognized by Caspase 3 and 7 and thermostable luciferase ( Z-DEVD-AMC). When the Caspase-Glo™ reagent (Promega USA) is added to the MCF-7 treated and non treated lines, it promotes a cellular lyse, followed by a cleaveage of the substrate by the caspase 3/7 in the cells which were induced to its activation and liberating the aminoluciferines which afterwards will be consumed by the luciferase. The luminescent signal produced is proportional to the caspase 3/7 activity present in the sample.

Controls

For each test (MTT, Anexina e Hoescht) a positive control (line treated with doxorrubicin diluted in a DMEM medium in a concentration of 15(g/mL) and a negative control (cellular line not treated) were employed.

Statistical Analysis

The statistical analysis of the results obtained in the cytotoxicity and apoptosis tests was made as follows: first a normality test was applied, after the observation that the data presented a normal distribution, a parametric one way ANOVA test was applied followed by a Tukey post test. The software employed in the statistical tests was the Biostat 4.0.

RESULTS

Cytotoxicity Test

The MTT test was initially performed with the pterogynine and pterogynidine alkaloids for the determination of the IC50 (a concentration which allows the death of 50% of the treated cells), establishing the starting point for the next experiments, this procedure had to be adopted because there are no previous studies with these substances. The initial concentrations tested with both alkaloids were 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6 and 10mM (Figure 2). The IC50 of the alkaloids in the breast cancer line (MCF-7), was observed between 2.5mM and 4mM, for 24 hours (t0) of treatment (Figure 3). From the IC50 were established four concentrations of each substance for the cytotoxicity test and the apoptosis tests which was made through a flow cytometry analysis (Figure 4). After the cytotoxicity was determined, four concentrations were established (1.25, 2.5, 5 and 10mM for the 24 hours treatment (t0) and 24 hours recovery (t24)). The cytotoxicity showed a dose response and the higher concentration (10mM) presented a 70% death of the cells treated with Pteroginine for 24 hours (Figure 1 A)

The cytotoxicity in 24 hours of treatment, followed by more 24 hours of recovery (t24) presented an increase in the dose response cytotoxicity percentually similar to the ones found in the higher concentration (75%) and found in the positive control (78%) (Figure 3).

In 24 hours of treatment without recovery time (t0) an increase in the cytotoxicity dose response was also observed with maximum cell death of 74% (Figure 3).

Cytotoxicity in 24 hours of treatment, followed by more 24 hours of recovery time (t24) was determined for the pterogynidine alkaloid in the same cell line. For the performance of the cytotoxicity test with recovery time (t24) the same concentrations below and above of the IC50 value calculated in the previous test were used (t0). An increase in the dose response cytotoxicity was observed and the maximum cell death (73%) occurred in the 5.0mM concentration (Figure 3).

In figure 9 are showed the photomicrographies of the MCF-7 strains submitted to the treatments with pterogynine e pterogynidine in a culture medium DMEM at a 10X augmentation. The morphological alterations of the cells in death phase can be observed. The higher concentrations show a compromised morphology with a smaller number of cells when compared to the lower concentrations and the negative control.

Anexin V Test

For an assessment of the potential apoptotic effect of the pure substances from Pterogyne nitens, the anexin V by flow cytometric analysis test was performed in the MCF-7 lines treated with the pterogynine and pterogynidine alkaloids in concentrations similar to the established IC50 (Figure 4). The experiments were conducted for the evaluation of the apoptosis in cells treated for 24 hours without recovery time (t0) and cells treated for 24 hours followed by another 24 hours of recovery time (t24).

In the MCF-7 treatment with pterogynine without recovery time, t0 (Figure 4A), a smaller percentage of cells with early apoptosis was observed (12 to 30%) and a higher percentage of cells in late apoptosis/necrosis (6 to 82%). An increase in the dose response regarding late apoptosis/necrosis could be observed, presenting also a relevant difference (*p ................
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