RESUMEN

Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antioxidantes/farmacología , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Flavonas/farmacología , Neoplasias Hepáticas/patología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , Catalasa/biosíntesis , Catalasa/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Glutatión/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Células L , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Mitoxantrona/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Plantas Medicinales/metabolismo , ARN Mensajero/biosíntesis , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

A bioassay-guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira-Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane-type saponins, 3-O-ß-D-xylopyranosyl-(1→3)-ß-D-glucopyranosyl-2ß,3ß,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (2), 3-O-ß-D-glucopyranosyl-2ß,3ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester, (3) and 3-O-ß-D-xylopyranosyl-(1→3)-ß-D-glucopyranosyl-2ß,3ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (4), named grindeliosides A-C, respectively. Their structures were determined by extensive 1D- and 2D-NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN-γ-induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF-CEM and MRC-5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN-γ-induced NO production (IC50 51.4 µM), while grindeliosides A-C were found to be cytotoxic, with grindelioside C being the most active against both CCRF-CEM (IC50 4.2±0.1 µM) and MRC-5 (IC50 4.5±0.1 µM) cell lines.

Asunto(s)

Grindelia/química , Óxido Nítrico/biosíntesis , Saponinas/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estructura Molecular , Saponinas/química , Saponinas/aislamiento & purificación , Relación Estructura-Actividad

RESUMEN

The aim of this study was to determine whether eupafolin and hispidulin, flavones extracted from Eupatorium littorale Cabrera, Asteraceae, have the ability to change properties of biological membranes and promote cytotoxic effects. Eupafolin (50-200 µM) decreased approximately 30 percent the rate and total amplitude of valinomycin induced swelling and 60-100 percent the energy-dependent mitochondrial swelling. Moreover, eupafolin (200 µM) reduced 35 percent the mitochondrial permeability transition, and hispidulin did not change this parameter in any of the doses tested. The evaluation of phase transition of DMPC liposomes with the probe DPH demonstrated that hispidulin and eupafolin affect gel and fluid phase. With mitochondrial membrane as model, hispidulin increased the polarization of fluorescence when used DPH-PA probe. Eupafolin and hispidulin (100 µM) promoted a reduction of 40 percent in cellular viability of HeLa cells in 24 h. Our results suggest that eupafolin and hispidulin have cytotoxic effects that can be explained, in part, by alterations promoted on biological membranes properties and mitochondrial bioenergetics.

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O objetivo deste estudo foi avaliar se eupafolina e hispidulina, flavonas extraídas do Eupatorium littorale Cabrera, Asteraceae, possuíam a capacidade de alterar propriedades das membranas biológicas e promover efeitos citotóxicos. Eupafolina (50-200 µM) reduziu em aproximadamente 30 por cento a velocidade e amplitude do inchamento mitocondrial induzido por valinomicina e 60-100 por cento o inchamento mitocondrial dependente de substrato. Além disso, eupafolina na dose de 200 µM reduziu a transição de permeabilidade mitocondrial em 35 por cento entretanto, a hispidulina não alterou este parâmetro em todas as doses testadas. A avaliação da transição de fase dos lipossomas de DMPC com a sonda DPH demonstrou que ambas as flavonas afetam a fase gel e fluida. Quando lipossomas de membranas mitocondriais e a sonda DPH-PA foram utilizados, houve aumento da polarização de fluorescência promovido pela hispidulina. Eupafolina e hispidulina, na dose de 100 µM, promoveram 40 por cento de redução da viabilidade de células HeLa em 24 h. Nossos resultados sugerem que eupafolina e hispidulina têm efeitos citotóxicos que podem ser explicados em parte pelas alterações promovidas por estas flavonas sobre propriedades de membranas biológicas e sobre a bioenergética mitocondrial.