A carregar...
Projeto de investigação
Sem título
Financiador
Autores
Publicações
Development of new alkaloid derivatives as multidrug resistance reversers in cancer cells
Publication . Vieira, Rita Mestre; Ferreira, Maria José Umbelino; Gonçalves, Bruno Miguel Ferreira
A multirresistência a fármacos (MDR) tem sido apontada como um dos principais motivos para a ineficácia da quimioterapia no tratamento do cancro. Um dos fatores responsáveis pelo desenvolvimento de MDR é a sobre-expressão da glicoproteína-P (P-gp/ABCB1) nas células tumorais, levando ao aumento do efluxo do fármaco antitumoral do interior para o exterior da célula.
Neste trabalho, o principal objetivo foi explorar a capacidade dos alcalóides quinolizidínicos de reverterem a MDR mediada pela P-gp. Neste sentido, foram preparados catorze derivados da matrine (2.1 – 2.14), um alcalóide quinolizidínico encontrado em espécies do género Sophora (Fabaceae). A primeira transformação química consistiu na abertura do anel lactama, seguida da esterificação do ácido carboxílico resultante. Posteriormente, a reação com diferentes isocianatos aromáticos permitiu a introdução do grupo ureia no esqueleto quinolizidínico. Os compostos foram caracterizados por ressonância magnética nuclear unidimensional (1H RMN e 13C RMN) e bidimensional (HSQC e HMBC), e espectrometria de massa (MS).
A capacidade dos compostos para inibir a P-gp foi avaliada em células resistentes de linfoma de rato (T L5178Y), transfetadas com o gene ABCB1 humano, e nas células sensíveis correspondentes, através do ensaio de acumulação da rodamina-123, por citometria de fluxo. Os compostos 2.13 e 2.14 mostraram forte atividade moduladora da P-gp tento sido mais ativos do que o controlo positivo. Foram também avaliadas a atividade citotóxica e antiproliferativa de todos os compostos, incluindo o produto natural matrine, não tendo sido observados valores de IC50 significativos nestas linhas celulares.
Foram ainda realizados ensaios de combinação para avaliar o tipo de interação de alguns compostos com a doxorrubicina, um substrato da P-gp. Todos os compostos interagiram sinergicamente com este fármaco antitumoral. No entanto, os melhores resultados foram obtidos com o composto 2.14, que mostrou um sinergismo forte (índice de combinação < 0,3), fornecendo evidências do seu potencial como reversor da MDR.
Plant-derived compounds for targeting multidrug resistance in cancer
Publication . Cardoso, David; Ferreira, Maria José Umbelino; Santos, Daniel José Viegas Antunes dos
Bioactive terpenoids from Euphorbia pubescens: isolation and derivatization
Publication . Carréu, João Paulo Magalhães; Duarte, Noélia Maria da Silva Dias; Ferreira, Maria José Umbelino
As espécies do género Euphorbia têm sido utilizadas na medicina tradicional, desde a Antiguidade, para o tratamento de verrugas, tumores, cancros e infeções intestinais, entre outras patologias.
Nas últimas décadas, o estudo fitoquímico destas espécies tem merecido uma especial atenção, devido ao isolamento de um enorme número de compostos biologicamente ativos, dos quais se destacam os diterpenos policíclicos e macrocíclicos, com esqueletos do tipo jatrofano e latirano, triterpenos tetracíclicos e pentacíclicos e esteroides. Das diversas atividades biológicas demonstradas por estes compostos, são de salientar os resultados obtidos com os diterpenos macrocíclicos, que revelaram uma importante atividade moduladora da glicoproteína-P, uma das principais proteínas transmembranares envolvidas na multirresistência das células tumorais aos fármacos anti-neoplásicos.
Esta dissertação teve como principal objetivo a pesquisa de novos compostos bioativos a partir de Euphorbia pubescens. Para tal, foram utilizadas duas abordagens: i) o isolamento de compostos a partir três frações do extrato metanólico; ii) derivatização molecular de compostos isolados em maior quantidade. O estudo fitoquímico foi realizado recorrendo a técnicas cromatográficas, nomeadamente cromatografia em coluna ou cromatografia preparativa em camada fina.
Foram isolados e identificados onze compostos, incluindo cinco triterpenos pentacíclicos (taraxerona, taraxerol, acetato de taraxerol, isomultiflorenol e acetato de isomultiflorenol), um diterpeno macrocíclico do tipo jatrofano (euphopubescenol), três lactonas diterpénicas do tipo ent-abietano (helioscopinolidos A, B e E), um esteroide (β-sitosterol) e um composto fenólico (coniferaldeído).
A taraxerona foi isolada em maiores quantidades, pelo que se procedeu à derivatização da função cetona em C-3 por condensação com a hidrazina, fenil-hidrazina e a 2,4-dinitrofenilhidrazina, obtendo-se três novos derivados.
As estruturas químicas dos compostos foram deduzidas com base nas suas características físicas e espectroscópicas (IV, espectrometria de massa, ressonância magnética nuclear unidimensional - 1H-RMN e 13C-RMN - e bidimensional - 1H-1H-COSY, HMQC, HMBC) e por comparação com dados descritos na literatura.
Futuramente, serão realizados estudos adicionais que permitirão avaliar a capacidade dos compostos obtidos como moduladores da glicoproteína-P em células tumorais resistentes.
Amaryllidaceae-type alkaloids as anticancer hit/leads, targeting multidrug resistant cancer cells
Publication . Sancha, Shirley; Ferreira, Maria José Umbelino
Multidrug resistance (MDR) is the major challenge in cancer chemotherapy. The main objective of this study was to find new effective anticancer compounds, from two species of Amaryllidaceae family to target MDR cancer cells.
The phytochemical study of the methanol extract of the bulbs of Narcissus bulbocodium L. subsp. obesus gave rise to the Amaryllidaceae-type alkaloid tazettine (1) and β-sitosterol (3). In addition, from the methanol extract of the flowers, the alkamide N-trans-feroulyl-tyramine (2) and the steroids β-sitosterol (3), β-sitosterol-O-β-D-glucoside (4), β-sitostenone (5), and ergosterol peroxide (6) were also isolated. In turn, the study of the methanol extract of the bulbs and flowers of Pancratium maritimum L. yielded 8-O-demethyl-2α-hydroxyhomolycorine (8), a new Amaryllidaceae alkaloid, along with the known alkaloids lycorine (7), 2α-10bα-dihydroxy-9-O-demethylhomolycorine (9), 8-O-demethylhomolycorine (10), hippeastrine (11), haemanthamine (12), haemanthidine (13), epigalanthamine (14) and 11β-hydroxygalanthamine (15) and the phenolic compound 4,6-dimethoxy-2- hydroxy acetophenone (16).
Aiming at generating a small library of Amaryllidaceae-type alkaloids, the chemical derivatization of lycorine (7), 2α-10bα-dihydroxy-9-O-demethylhomolycorine (9), and haemanthidine (13), isolated in large amount from P. maritimum, allowed the preparation of seventy-five derivatives. In this way, the chemical reaction of lycorine (7) with carbonyldiimidazole and different aromatic and aliphatic amines, afforded thirty-one new mono- and di-carbamates (7.1–7.31). Furthermore, cleavage of ring D with ethyl chloroformate of the diacetylated lycorine derivative (7.32) gave rise to compounds 7.34–7.44, bearing carbamate and amine functions. In addition, lycorine was treated with strong base resulting in Hofman elimination with the opening of ring D and aromatization of ring C to afford the derivative 7.45. Acylation of compound 9 provided the diester 9.1 and the monoesters 9.2–9.5. Oxidation of haemanthidine (13) yielded compound 13.1, while the reaction with acetic anhydride gave rise to the diester 13.2. Moreover, reaction of haemanthidine with aliphatic and aromatic halides originated the conversion into tazettine (1) and tazettine-, and pretazettine-type N- and O-alkylated derivatives (1.1– 1.24). The chemical structures of the compounds were established from their physical and spectroscopic data, namely IR, 1D- ( 1H NMR, 13C NMR) and 2D-NMR (COSY, HMBC, HMQC, and NOESY) experiments and MS.
The antiproliferative effect of compounds 1-3, 5-16 was evaluated by the sulforhodamine B assay against the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, breast cancer cells MCF-7, and the non-malignant fibroblast (HFF-1) and breast (MCF12A) cell lines. The alkaloids 7, 9, 12, and 13 showed significant growth inhibitory effects against all breast cancer cell lines IC50 (0.73- 16.3 µM). The homolycorine-type alkaloid 9 was selected for further investigation of the mechanism of action in MDA-MB-231 cells. In the annexin-V assay, compound 9 increased cell death by apoptosis, which was substantiated, in western blot analyses, by the increased expression of the pro-apoptotic protein Bax, and the decreased expression of the anti-apoptotic protein Bcl-xL. Consistently, it further stimulated mitochondrial reactive oxygen species (ROS) generation. The antiproliferative effect of compound 9 was also associated with G2/M cell cycle arrest, which was supported by an increase in the p21 protein expression levels. In MDA-MB-231 cells, compound 9 also exhibited synergistic effects with conventional chemotherapeutic drugs such as etoposide.
The anticancer potential of the alkaloids 1–3 and 5–16 was also assessed in HCT116 colon cancer cells. The cytotoxicity of the compounds was evaluated by the MTS metabolism. Compounds 7, 12, and 13 exhibited the most potent cytotoxic activity with IC50 values of 2.07, 4.98, and 5.90 µM, respectively. The induced inhibition of proliferation of HCT116 cells by compound 12 was associated with G1 phase arrest, while compounds 7 and 13 induced G2/M cell cycle arrest.
Lycorine (7) and its carbamate derivatives 7.1–7.31 were evaluated as MDR reversers, through functional and chemosensitivity assays, in resistant human colon adenocarcinoma cancer cells (COLO 320), overexpressing P-glycoprotein (P-gp). Significant inhibition of P-gp efflux activity was observed for the di-carbamates, mainly those containing aromatic substituents, at non-cytotoxic concentrations. Compound 7.4, bearing a benzyl substituent, and compounds 7.8 and 7.24, with phenethyl moieties, were among the most active, exhibiting strong inhibition at 2 µM, being more active than verapamil at a 10-fold higher concentration. In drug combination assays, most compounds were able to synergize doxorubicin. Moreover, some derivatives showed a selective antiproliferative effect toward resistant cells, having a collateral sensitivity effect. In the ATPase assay, some selected compounds (7.1, 7.4, 7.8, 7.18, 7.24, and 7.25) behaved as inhibitors.
The effects of alkaloids 7, 9, and 13 and their derivatives (7.32–7.45, 9.1–9.5, 13.1, 13.2, and 1, 1.1–1.24) on the reversal of drug resistance were evaluated in resistant human ovarian carcinoma (HOC/ADR) cells, overexpressing P-gp. The derivatives 7.32–7.45, and 9.1–9.5 were not cytotoxic or showed moderate/weak cytotoxicity, however, lycorine (7) exhibited strong cytotoxicity (IC50 values of 1.2- 2.5 µM). In combination assays, most of the compounds synergized with the anticancer drug doxorubicin. Compounds 7.34, 7.35, 7.38–7.43, bearing both carbamate and aromatic amine moieties, showed the highest sensitization rate, reducing the dose of doxorubicin 5–35 times, thus highlighting their potential to reverse drug resistance in combination chemotherapy. Selected compounds (7.33–7.36, 7.38–7.43, and 9.5) that re-sensitize resistant cancer cells were further evaluated as P-gp inhibitors. Compound 7.40, with a para-methoxy-N-methylbenzylamine moiety, was the strongest inhibitor. In the ATPase assay, compounds 7.38–7.40 and 7.42 behaved as verapamil, suggesting competitive inhibition of P-gp.
Chemosensitivity and functional assays were also employed to assess the MDR reversal of haemanthidine (13), tazettine (1), and its derivatives (1.1–1.24) in resistant human ovarian carcinoma cells. Compounds 1.4, 1.14, 1.18, and 1.23, bearing aromatic moieties with methoxy and bromide substituents, exhibited the highest sensitization rate (up to 30-fold). Compounds 1.4, 1.12, and 1.13, sharing phenethyl moieties, with methoxy and bromide substituents, exhibited the strongest P-gp inhibitory activity P-glycoprotein. In conclusion, several Amaryllidaceae-type alkaloids of both natural origin and obtained by derivatization are promising potential lead structures as MDR reversers.
Keywords: Multidrug resistance; Amaryllidaceae-type alkaloids; Apoptosis induction; Cell cycle arrest, P-glycoprotein.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/MED-QUI/30591/2017