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Dual properties of hispidulin: antiproliferative effects on HepG2 cancer cells and selective inhibition of ABCG2 transport activity

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Abstract

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.

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Abbreviations

7-AAD:

(7-Amino-actinomycin D)

CAT:

Catalase

DCFH–DA:

2′,7′-Dichlorofluorescin diacetate

DMSO:

Dimethyl sulfoxide

FBS:

Fetal bovine serum

Gred:

Glutathione reductase

GSH:

Glutathione

GPx:

Glutathione peroxidase

GSSG:

Glutathione disulfide

HCC:

Hepatocellular carcinoma

H2O2 :

Hydrogen peroxide

HCl:

Hydrochloric acid

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NAC:

N-acetylcysteine

NADPH:

Nicotinamide adenine dinucleotide phosphate

PBS:

Phosphate buffered saline

PCR:

Polymerase chain reaction

PE:

Phycoerythrin

PI:

Propidium iodide

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

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Acknowledgments

This study was supported by the Brazilian research funding agencies CNPq (Conselho Nacional para o Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Fundação Araucária, FINEP (CT-Infra), and INCT Redoxoma (FAPESP/CNPq/CAPES 573530/2008-4), and the French National League against Cancer (Equipe Labellisée 2014), CNRS, and University of Lyon 1 (UMR5086). G.V. was supported by a sandwich Ph.D. fellowship from the Brazilian Agency (CAPES) (process numbers 1203-10-8).

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Correspondence to Maria E. M. Rocha.

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Carina T. Scoparo and Glaucio Valdameri have contributed equally to this work.

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Scoparo, C.T., Valdameri, G., Worfel, P.R. et al. Dual properties of hispidulin: antiproliferative effects on HepG2 cancer cells and selective inhibition of ABCG2 transport activity. Mol Cell Biochem 409, 123–133 (2015). https://doi.org/10.1007/s11010-015-2518-8

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