Potencial efeito epigenético do extrato de própolis brasileira e de seus componentes sobre linhagens celulares derivadas de carcinomas mamários
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Data
2017-09-29
Autores
Assumpção, João Henrique Maia
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Universidade Estadual Paulista (Unesp)
Resumo
A própolis é uma substância de origem natural, de aspecto resinoso, produzida pelas abelhas a partir de diferentes plantas, que determina a sua composição química. Centenas de componentes foram identificados em diferentes amostras de própolis, cujo extrato exerce uma diversidade de efeitos biológicos e farmacológicos. Alguns estudos demonstraram que este produto pode interferir em vias de sinalização de oncogenes, inibir o crescimento, reduzir a proliferação celular, induzir apoptose e apresentar efeito antiangiogênico. O câncer de mama é uma doença complexa, caracterizada por heterogeneidade em nível molecular, histológico e clínico. O câncer de mama triplo-negativo é caracterizado pela ausência de expressão dos receptores hormonais ER (receptor de estrogeno) e PgR (receptor de progesterona), bem como pela baixa expressão da proteína Her-2; esse subtipo de câncer de mama é um problema do ponto de vista clínico devido ao seu pior prognóstico quando comparado aos outros subtipos, alta agressividade e ausência de terapias que possuam alvos específicos. Estudos prévios demonstraram que os compostos de origem natural podem inibir a atividade de enzimas da maquinaria epigenética, como as DNA metiltransferases e, consequentemente, aumentar a expressão de genes supressores de tumor silenciados por hipermetilação no câncer. Portanto, o presente estudo investigou a hipótese de que os efeitos antitumorais da própolis são, em parte, mediados por mecanismos epigenéticos. Primeiramente, utilizou-se a abordagem in silico de docking molecular a fim de verificar se moléculas derivadas da própolis podem interagir com o domínio metiltransferase (MTase) da DNMT1 (DNA metiltransferase 1) humana. O ligantes foram selecionados a partir da composição química definida de uma amostra de própolis brasileira contendo: benzil benzoato, ácido cafeico, CAPE (éster de ácido cafeico), ácido dihidrocinâmico, (2E,6Z)-farnesol, ácido ferúlico, kaempferide, ácido p-cumárico, ácido hidrocinâmico e espatulenol. Foram selecionados 3 programas para as predições de docking molecular: AutoDock 4.2, Dock 6.7 e SwissDock . As estruturas químicas dos ligantes foram obtidas no banco de dados PubChem e o modelo cristalográfico da DNMT1 humana em complexo com S-adenosil-homocisteína (SAH) (PDBID:4WXX) foi utilizado como receptor, onde foi definido uma área de interesse no sítio de interação de SAH ao domínio MTase (resíduos de aminoácidos 1135-1605). As conformações calculadas foram agrupadas em clusters usando Gromacs. O programa LigPlot + para verificar os contatos com os resíduos de aminoácidos de DNMT1. Adicionalmente, analisamos a inibição da atividade da metilase M.SssI na presença de EEP (extrato etanólico de própolis), EGCG (epigalocatequina-3-galato) e ácido p-cumárico, bem como o efeito do tratamento in vitro com EEP , EGCG e três ácidos aromáticos (cafeico, hidrocinâmico e p-cumárico). A viabilidade celular foi determinada pelo ensaio de MTT [(3- (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazólio]; a análise de MS-PCR (Methylation-Specific Polymerase Chain Reaction) foi realizada para verificar a possível efeito hipometilante do ácido p-cumárico, EGCG e EEP em 3 linhagens celulares de câncer de mama. Os nossos resultados de docking indicaram que CAPE, (2E,6Z)-farnesol e o ácido p-cumárico foram os ligantes que apresentaram energias de ligação mais favoráveis, melhor ocupação do sítio de ligação e interações químicas comuns com o complexo SAH-DNMT1. O EEP reduziu a viabilidade celular das linhagens BT-20 e BT-549, enquanto o ácido p-cumárico e EGCG reduziram a viabilidade de todas as linhas celulares estudadas, de um modo dose e tempo dependentes. Tanto o EGCG como o ácido p-cumárico foram incapazes de inibir a metilase M.SssI; porém, os resultados da MS-PCR demonstraram que o EEP é capaz de induzir hipometilação locus-específica nas linhagens BT-549 e MDA-MB-436. EGCG também foi capaz de hipometilar as linhagens BT-549 e MDA-MB231. Em conclusão, a análise de docking molecular mostrou evidências de que os compostos derivados de própolis brasileira podem interagir com o sítio de ligação de SAH no domínio metiltransferase da DNMT1 humana e devem ser considerados em estudos futuros dedicados à identificação de novas drogas para a terapia epigenética.
Propolis is a natural resinous substance produced by honeybees from several parts of plants, which are related to its chemical composition. Hundreds of chemical substances have been identified in different samples of propolis, which extract shows a plethora of biological and pharmacological activities. Generally, it has been demonstrated that this complex mixture can disrupt oncogene signaling pathways, inhibit cell growth and proliferation, induces apoptosis, and shows antiangiogenic effects. Breast cancer is a complex disease characterized by molecular, histological, and clinical heterogeneity. Triple-negative breast cancer, characterized by absence of expression of the receptors ER (estrogen receptor), PgR (progesterone receptor), and HER2 (erb-b2 receptor tyrosine kinase 2), presents a significant clinical problem due to its poor prognosis, aggressiveness and lack of targeted therapies. Previous studies reported that compounds of natural origin target epigenetic modifying enzymes, such as DNA methyltransferases (DNMTs), and reactivate methylation-silenced genes in cancer. Thus, we hypothesized that the effects of propolis in cancer cell lines are, in part, mediated by epigenetic mechanisms. Therefore, this study aims to demonstrate the possible hypomethylating effect of propolis or its compounds on triple negative breast cancer cell lines. Initially, we used an in silico approach based on molecular docking to find propolis-derived molecules able to interact with the MTAse domain of human DNMT1. The potential ligands were selected based on the chemical composition of a defined Brazilian propolis sample: benzyl benzoate, caffeic acid, CAPE (caffeic acid phenethyl ester), dihydrocinnamic acid, farnesol, ferulic acid, kampeferide, p-coumaric acid, hydrocinnamic acid, and spathulenol. For molecular docking predictions three software were selected: AutoDock 4.2 (autodock.scripps.edu/), Dock 6.7 (http://dock.compbio.ucsf.edu/), and SwissDock (www.swissdock.ch). The chemical structures of probable ligands were retrieved from PubChem database (pubchem.ncbi.nlm.nih.gov) and the crystallographic model of human DNMT1 in complex with S-adenosyl-homocisteine (SAH) (PDBID: 4WXX) was used to refine the site of interest in the receptor [methyltransferase (Mtase) domain, amino acids residues 1135-1605]. All structures were prepared for docking using Chimera 1.11.2 (cgl.ucsf.edu/chimera). The known interaction of methyl group donator S-adenosylmethionine (SAM) and the intrinsic inhibitor (SAH) with DNMT1 were used as reference. The calculated conformations were clustered using Gromacs (www.gromacs.org) with the Gromos algorithm, using a RMSD (Root-Mean-Square Deviation) of atomic positions cutoff of 1Å. The middle structure of the 3 clusters with more conformations of each ligand was analyzed with LigPlot+ (www.ebi.ac.uk/thornton-srv/software/LigPlus/) to verify the contacts with the amino acids residues of DNMT1. Further, we have analyzed the inhibition of M.SssI methylase activity in the presence of EEP (ethanolic extract of propolis), EGCG and p-coumaric acid as well as the effect after in vitro treatment with EPP (concentrations of 6.25, 12.5, 25, 50 and 100mg/mL), EGCG, and three aromatic acids (cafeic, hydrocymanic, and p-coumaric) (concentration of 6.25, 12.5, 25, 50 and 100µM), during 24, 48, 72 and 96h in the BT-20, BT-549, MDA-MB-231 and MDA-MB-468 breast cancer cell lines. Cell viability was determined by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] test and Methylation Specific – Polymerase Chain Reaction (MS-PCR) was used to verify the possible hypomethylation effect of p-coumaric acid, EGCG and EEP three breast cancer cell-lines (BT-549, MDA-MB-231 and MDA-MB-436). Our results indicates that CAPE, (2E, 6Z)-farnesol and p-coumaric acid were the ligands that showed more negative free binding energies, better occupation of the binding site and common chemical interactions with the complex SAH-DNMT1. The EEP reduced the cell viability of BT-20 and BT-549, while p-coumaric acid and EGCG reduced the viability of all cell lines studied, in a dose and time dependent manners. Both EGCG and p-coumaric acid were unable to inhibit M.SssI methylase, however MS-PCR results demonstrated that EEP is able to induce locus-specific hypomethylation in the BT-549 and MDA-MB-436 cell lines and EGCG was also able to hypomethylate BT-549 and MDA-MB231. In conclusion, the molecular docking demonstrates that compounds derived from Brazilian propolis can interact with SAH binding pocket of methyltransferase domain of human DNMT1 and could be included in future studies of new drugs for epigenetic therapy.
Propolis is a natural resinous substance produced by honeybees from several parts of plants, which are related to its chemical composition. Hundreds of chemical substances have been identified in different samples of propolis, which extract shows a plethora of biological and pharmacological activities. Generally, it has been demonstrated that this complex mixture can disrupt oncogene signaling pathways, inhibit cell growth and proliferation, induces apoptosis, and shows antiangiogenic effects. Breast cancer is a complex disease characterized by molecular, histological, and clinical heterogeneity. Triple-negative breast cancer, characterized by absence of expression of the receptors ER (estrogen receptor), PgR (progesterone receptor), and HER2 (erb-b2 receptor tyrosine kinase 2), presents a significant clinical problem due to its poor prognosis, aggressiveness and lack of targeted therapies. Previous studies reported that compounds of natural origin target epigenetic modifying enzymes, such as DNA methyltransferases (DNMTs), and reactivate methylation-silenced genes in cancer. Thus, we hypothesized that the effects of propolis in cancer cell lines are, in part, mediated by epigenetic mechanisms. Therefore, this study aims to demonstrate the possible hypomethylating effect of propolis or its compounds on triple negative breast cancer cell lines. Initially, we used an in silico approach based on molecular docking to find propolis-derived molecules able to interact with the MTAse domain of human DNMT1. The potential ligands were selected based on the chemical composition of a defined Brazilian propolis sample: benzyl benzoate, caffeic acid, CAPE (caffeic acid phenethyl ester), dihydrocinnamic acid, farnesol, ferulic acid, kampeferide, p-coumaric acid, hydrocinnamic acid, and spathulenol. For molecular docking predictions three software were selected: AutoDock 4.2 (autodock.scripps.edu/), Dock 6.7 (http://dock.compbio.ucsf.edu/), and SwissDock (www.swissdock.ch). The chemical structures of probable ligands were retrieved from PubChem database (pubchem.ncbi.nlm.nih.gov) and the crystallographic model of human DNMT1 in complex with S-adenosyl-homocisteine (SAH) (PDBID: 4WXX) was used to refine the site of interest in the receptor [methyltransferase (Mtase) domain, amino acids residues 1135-1605]. All structures were prepared for docking using Chimera 1.11.2 (cgl.ucsf.edu/chimera). The known interaction of methyl group donator S-adenosylmethionine (SAM) and the intrinsic inhibitor (SAH) with DNMT1 were used as reference. The calculated conformations were clustered using Gromacs (www.gromacs.org) with the Gromos algorithm, using a RMSD (Root-Mean-Square Deviation) of atomic positions cutoff of 1Å. The middle structure of the 3 clusters with more conformations of each ligand was analyzed with LigPlot+ (www.ebi.ac.uk/thornton-srv/software/LigPlus/) to verify the contacts with the amino acids residues of DNMT1. Further, we have analyzed the inhibition of M.SssI methylase activity in the presence of EEP (ethanolic extract of propolis), EGCG and p-coumaric acid as well as the effect after in vitro treatment with EPP (concentrations of 6.25, 12.5, 25, 50 and 100mg/mL), EGCG, and three aromatic acids (cafeic, hydrocymanic, and p-coumaric) (concentration of 6.25, 12.5, 25, 50 and 100µM), during 24, 48, 72 and 96h in the BT-20, BT-549, MDA-MB-231 and MDA-MB-468 breast cancer cell lines. Cell viability was determined by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] test and Methylation Specific – Polymerase Chain Reaction (MS-PCR) was used to verify the possible hypomethylation effect of p-coumaric acid, EGCG and EEP three breast cancer cell-lines (BT-549, MDA-MB-231 and MDA-MB-436). Our results indicates that CAPE, (2E, 6Z)-farnesol and p-coumaric acid were the ligands that showed more negative free binding energies, better occupation of the binding site and common chemical interactions with the complex SAH-DNMT1. The EEP reduced the cell viability of BT-20 and BT-549, while p-coumaric acid and EGCG reduced the viability of all cell lines studied, in a dose and time dependent manners. Both EGCG and p-coumaric acid were unable to inhibit M.SssI methylase, however MS-PCR results demonstrated that EEP is able to induce locus-specific hypomethylation in the BT-549 and MDA-MB-436 cell lines and EGCG was also able to hypomethylate BT-549 and MDA-MB231. In conclusion, the molecular docking demonstrates that compounds derived from Brazilian propolis can interact with SAH binding pocket of methyltransferase domain of human DNMT1 and could be included in future studies of new drugs for epigenetic therapy.
Descrição
Palavras-chave
Ácido p-cumárico, Câncer, Terapia epigenética, DNA-metiltrasnferase, Docking molecular