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
References
Parkin DM, Bray F, Ferlay J, Pisani P (2001) Estimating the world cancer burden: Globocan 2000. Int J Cancer 94(2):153–156
Cha CH, Saif MW, Yamane BH, Weber SM (2010) Hepatocellular carcinoma: current management. Curr Probl Surg 47(1):10–67
Stagos D, Amoutzias GD, Matakos A, Spyrou A, Tsatsakis AM, Kouretas D (2012) Chemoprevention of liver cancer by plant polyphenols. Food Chem Toxicol 50(6):2155–2170
El-Serag HB (2002) Hepatocellular carcinoma and hepatitis C in the United States. Hepatology 36(5):S74–S83
Neuschwander-Tetri BA, Caldwell SH (2003) Nonalcoholic steatohepatitis: summary of an AASLD single topic conference. Hepatology 37(5):1202–1219
Bosch FX, Ribes J, Diaz M, Cleries R (2004) Primary liver cancer: worldwide incidence and trends. Gastroenterology 127(5):S5–S16
Pang R, Tse E, Poon RTP (2006) Molecular pathways in hepatocellular carcinoma. Cancer Lett 240(2):157–169
Forner A, Llovet JM, Bruix J (2012) Hepatocellular carcinoma. Lancet 379:1245–1255
He C, Sun XP, Qiao H, Jiang X, Wang D, Jin X, Dong X, Wang J, Jiang H, Sun X (2012) Downregulating hypoxia-inducible factor-2α improves the efficacy of doxorubicin in the treatment of hepatocellular carcinoma. Cancer Sci 103(3):528–534
Puszyk WM, Le Thu T, Chapple SJ, Liu C (2013) Linking metabolism and epigenetic regulation in development of hepatocellular carcinoma. Lab Invest 93(9):983–990
Poon RT, Fan ST (2004) Hepatectomy for hepatocellular carcinoma: patient selection and postoperative outcome. Liver Transpl 10:S39–S45
Ikeda K, Kumada H, Saitoh S, Arase Y, Chayama K (1991) Effect of repeated transcatheter arterial embolization on the survival time in patients with hepatocellular carcinoma. An analysis by the Cox proportional hazard model. Cancer 68(10):2150–2154
Petraccia L, Onori P, Sferra R, Lucchetta MC, Liberati G, Grassi M, Gaudio E (2003) MDR (multidrug resistance) in hepatocarcinoma clinical-therapeutic implications. La Clinica Terapeutica 154(5):325–335
Fojo A, Akiyama S, Gottesman MM, Pastan I (1985) Reduced drug accumulation in multiply drug-resistant human-kb carcinoma cell-lines. Cancer Res 45(7):3002–3007
Marin JJG, Romero MR, Briz O (2010) Molecular bases of liver cancer refractoriness to pharmacological treatment. Curr Med Chem 17(8):709–740
Boumendjel A, Macalou S, Valdameri G, Pozza A, Gauthier C, Arnaud O, Nicolle R, Magnard S, Falson P, Terreux R, Carrupt PA, Payen L, Di Pietro A (2011) Targeting the multidrug ABCG2 transporter with flavonoidic inhibitors: in vitro optimization and in vivo validation. Curr Med Chem 18(22):3387–3401
Ren W, Qiao Z, Wang H, Zhu L, Zhang L (2003) Flavonoids: promising anticancer agents. Med Res Rev 23(4):519–534
Teillet F, Boumendjel A, Boutonnat J, Ronot X (2008) Flavonoids as RTK inhibitors and potential anticancer agents. Med Res Rev 28(5):715–745
Boumendjel A, Ronot X, Boutonnat J (2009) Chalcones derivatives acting as cell cycle blockers: potential anti cancer drugs? Curr Drug Targets 10(4):363–371
Kilani-Jaziri S, Frachet V, Bhouri W, Ghedira K, Chekir-Ghedira L, Ronot X (2012) Flavones inhibit the proliferation of human tumor cancer cell lines by inducing apoptosis. Drug Chem Toxicol 35(1):1–10
Aggarwal BB, Shishodia S (2006) Molecular targets of dietary agents for prevention and therapy of cancer. Biochem Pharmacol 71(10):1397–1421
Oliveira BH, Nakashima T, Filho JDS, Frehse FL (2001) HPLC analysis of flavonoids in Eupatorium litorale. J Braz Chem Soc 12:243–246
Lee SJ, Chung HY, Maier CGA, Wood AR, Dixon RA, Mabry TJ (1998) Estrogenic flavonoids from Artemisia vulgaris L. J Agr Food Chem 46:3325–3329
Nikolova M, Petrova M, Zayova E, Vitkova A, Evstatieva L (2013) Comparative study of in vitro, ex vitro and in vivo grown plants of Arnica montana—polyphenols and free radical scavenging activity. Acta Bot Croat 72(1):13–22
He L, Wu Y, Lin L, Wang J, Chen Y, Yi Z, Liu M, Pang X (2011) Hispidulin, a small flavonoid molecule, suppresses the angiogenesis and growth of human pancreatic cancer by targeting vascular endothelial growth factor receptor 2-mediated PI3 K/Akt/mTOR signaling pathway. Cancer Sci 102(1):219–225
Yang JM, Hung CM, Fu CN, Lee JC, Huang CH, Yang MH, Lin CL, Kao JY, Way TD (2010) Hispidulin sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by AMPK activation leading to Mcl-1 block in translation. J Agric Food Chem 58(18):10020–10026
Gao H, Wang H, Peng J (2014) Hispidulin induces apoptosis through mitochondrial dysfunction and inhibition of P13 k/Akt signalling pathway in HepG2 cancer cells. Cell Biochem Biophys 69(1):27–34
Dabaghi-Barbosa P, Rocha AM, Lima AFD, Oliveira BH, Oliveira MBM, Carnieri EGS, Cadena SMS, Rocha MEM (2005) Hispidulin: antioxidant properties and effect on mitochondrial energy metabolism. Free Radic Res 39(12):1305–1315
Herrerias T, Oliveira AA, Belem ML, Oliveira BH, Carnieri EGS, Cadena SMS, Noleto GR, Martinez GR, Oliveira MBM, Rocha MEM (2010) Effects of natural flavones on membrane properties and citotoxicity of HeLa cells. Revista Brasileira de Farmacognosia 20(3):403–408
Arnaud O, Boumendjel A, Geze A, Honorat M, Matera J, Guitton EL, Stein WD, Bates SE, Falson P, Dumontet C, Di Pietro A, Payen L (2011) The acridone derivative MBLI-87 sensitizes breast cancer resistance protein-expressing xenografts to irinotecan. Eur J Cancer 47(4):640–648
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival—application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63
Kueng W, Silber E, Eppenberger U (1989) Quantification of cells cultured on 96-well plates. Anal Biochem 182(1):16–19
Wan CP, Myung E, Lau BHS (1993) An automated micro-fluorometric assay for monitoring oxidative burst activity of phagocytes. J Immunol Methods 159(1–2):131–138
Zhao JL, Zhao J, Jiao HJ (2014) Synergistic growth-suppressive effects of quercetin and cisplatin on HepG2 human hepatocellular carcinoma cells. Appl Biochem Biotechnol 172(2):784–791
Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione-reductase and 2-vinylpyridine. Anal Biochem 106(1):207–212
Rahman I, Kode A, Biswas SK (2006) Assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method. Nat Protoc 1(6):3159–3165
Aebi H (1984) Catalase invitro. Methods Enzymol 105:121–126
Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247(10):3170–3175
Sies H, Koch OR, Martino E, Boveris A (1979) Increased biliary glutathione disulfide release in chronically ethanol-treated rats. FEBS Lett 103(2):287–290
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Valdameri G, Trombetta-Lima M, Worfel PR, Pires AR, Martinez GR, Noleto GR, Cadena SM, Sogayar MC, Winnischofer SM, Rocha ME (2011) Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells. Chem Biol Interact 193(2):180–189
Valdameri G, Gauthier C, Terreux R, Kachadourian R, Day BJ, Winnischofer SM, Rocha ME, Frachet V, Ronot X, Di Pietro A, Boumendjel A (2012) Investigation of chalcones as selective inhibitors of the breast cancer resistance protein: critical role of methoxylation in both inhibition potency and cytotoxicity. J Med Chem 55(7):3193–3200
Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72(1–2):248–254
Hu Y, Wang S, Wu X, Zhang J, Chen R, Chen M, Wang Y (2013) Chinese herbal medicine-derived compounds for cancer therapy: a focus on hepatocellular carcinoma. J Ethnopharmacol 149(3):601–612
Shukla S, Gupta S (2010) Apigenin: a promising molecule for cancer prevention. Pharmaceut Res 27(6):962–978
Valdameri G, Kenski JCN, Moure VR, Trombetta-Lima M, Martinez GR, Sogayar MC, Winnischofer SMB, Rocha MEM (2014) Flavone induces cell death in human hepatoma HepG2 Cells. Nat Prod Commun 9(10):1457–1460
Gupta S, Afaq F, Mukhtar H (2001) Selective growth-inhibitory, cell-cycle deregulatory and apoptotic response of apigenin in normal versus human prostate carcinoma cells. Biochem Biophys Res Commun 287(4):914–920
Chiang LC, Ng LT, Lin IC, Kuo PL, Lin CC (2006) Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer Lett 237(2):207–214
Lin YC, Hung CM, Tsai JC, Lee JC, Chen YL, Wei CW, Kao JY, Way TD (2010) Hispidulin potently inhibits human glioblastoma multiforme cells through activation of AMP-activated protein kinase (AMPK). J Agric Food Chem 58(17):9511–9517
Shim HY, Park JH, Paik HD, Nah SY, Kim DS, Han YS (2007) Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation. Mol Cells 24(1):95–104
Banerjee S, Zhang YX, Ali S, Bhuiyan M, Wang ZW, Chiao PJ, Philip PA, Abbruzzese J, Sarkar FH (2005) Molecular evidence for increased antitumor activity of gemcitabine by genistein in vitro and in vivo using an orthotopic model of pancreatic cancer. Cancer Res 65(19):9064–9072
Wu B, Zhang Q, Shen W, Zhu J (2008) Anti-proliferative and chemosensitizing effects of luteolin on human gastric cancer AGS cell line. Mol Cel Biochem 313(1–2):125–132
Davenport A, Frezza M, Shen M, Ge Y, Huo C, Chan TH, Dou QP (2010) Celastrol and an EGCG pro-drug exhibit potent chemosensitizing activity in human leukemia cells. Int J Mol Med 25(3):465–470
Liang G, Tang A, Lin X, Li L, Zhang S, Huang Z, Tang H, Li QQ (2010) Green tea catechins augment the antitumor activity of doxorubicin in an in vivo mouse model for chemoresistant liver cancer. Int J Oncol 37(1):111–123
Borska S, Chmielewska M, Wysocka T, Drag-Zalesinska M, Zabel M, Dziegiel P (2012) In vitro effect of quercetin on human gastric carcinoma: targeting cancer cells death and MDR. Food Chem Toxicol 50(9):3375–3383
Zhang S, Yang X, Morris ME (2004) Flavonoids are inhibitors of breast cancer resistance protein (ABCG2)-mediated transport. Mol Pharmacol 65:1208–1216
Ahmed-Belkacem A, Pozza A, Munoz-Martinez F, Bates SE, Castanys S, Gamarro F, Di Pietro A, Perez-Victoria JM (2005) Flavonoid structure–activity studies identify 6-prenyichrysin and tectochrysin as potent and specific inhibitors of breast cancer resistance protein ABCG2. Cancer Res 65(11):4852–4860
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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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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DOI: https://doi.org/10.1007/s11010-015-2518-8