Cytotoxic Compounds from the Fruits of Uapaca togoensis towards Multifactorial Drug-Resistant Cancer Cells

Abstract

Cancer cells may rapidly acquire multidrug resistance, mainly due to the presence of adenosine triphosphate-binding cassette transporters, epidermal growth factor receptor, or mutations in the p53 tumor suppressor gene. This work was designed to assess the cytotoxicity of the methanol crude extracts and compounds from the fruits of Uapaca togoensis, namely, β-amyryl acetate (1), 11-oxo-α-amyryl acetate (2), lupeol (3), pomolic acid (4), futokadsurin B (5), arborinin (6), and 3-O-β-D-glucopyranosyl sitosterol (7) against nine drug sensitive and multidrug-resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the fruits of U. togoensis and compounds, whilst the caspase-Glo assay was used to detect the activation of caspase enzymes by the fruits of U. togoensis and compound 6. Cell cycle, mitochondrial membrane potential, and levels of reactive oxygen species were all analyzed via flow cytometry. The acridone alkoid 6 and the crude extract from the fruits of U. togoensis were active on all of the nine tested cancer lines with IC₅₀ values below 32 µM and 30 µg/mL, respectively. Compounds 2 and 5 showed selective activities and IC₅₀ values below 99 µM or 42 µM, respectively, which were obtained towards 3/9 and 6/9 tested cancer cell lines. Compound 6 displayed IC₅₀ values below 10 µM towards seven of the nine tested cancer cell lines. The IC₅₀ values ranged from 3.55 µM (against CEM/ADR5000 cells) to 31.77 µM (against CCRF-CEM cells) for alkaloid 6 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. The crude extract of the fruits of U. togoensis induced apoptosis in the CCRF-CEM leukemia cells, which was mediated by the disruption of the mitochondrial membrane potential. Compound 6 also strongly induced apoptosis in CCRF-CEM cells and cell cycle arrest in the G0/G1 and S phases. The crude extract from the fruits of this plant as well as aborinin are potential antiproliferative natural products that deserve further investigation to develop novel cytotoxic drugs to fight sensitive and otherwise drug-resistant phenotypes.

Key words

Uapaca togoensis - Euphorbiaceae - alkaloid - arborinine - cytotoxicity - apoptosis

• References

• 1 Holmes K, Egan B, Swan N, OʼMorain C. Genetic mechanisms and aberrant gene expression during the development of gastric intestinal metaplasia and adenocarcinoma. Curr Genomics 2007; 8: 379-397
• 2 Shen B, Li D, Dong P, Gao S. Expression of ABC transporters is an unfavorable prognostic factor in laryngeal squamous cell carcinoma. Ann Otol Rhinol Laryngol 2011; 120: 820-827
• 3 Biedler JL, Spengler BA. Reverse transformation of multidrug-resistant cells. Cancer Metastasis Rev 1994; 13: 191-207
• 4 Efferth T, Sauerbrey A, Olbrich A, Gebhart E, Rauch P, Weber HO, Hengstler JG, Halatsch ME, Volm M, Tew KD, Ross DD, Funk JO. Molecular modes of action of artesunate in tumor cell lines. Mol Pharmacol 2003; 64: 382-394
• 5 Efferth T, Sauerbrey A, Halatsch ME, Ross DD, Gebhart E. Molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree Cephalotaxus hainanensis in human tumor cell lines. Naunyn Schmiedebergs Arch Pharmacol 2003; 367: 56-67
• 6 el-Deiry WS. Role of oncogenes in resistance and killing by cancer therapeutic agents. Curr Opin Oncol 1997; 9: 79-87
• 7 Eichhorn T, Efferth T. P-glycoprotein and its inhibition in tumors by phytochemicals derived from Chinese herbs. J Ethnopharmacol 2012; 141: 557-570
• 8 Choumessi AT, Loureiro R, Silva AM, Moreira AC, Pieme AC, Tazoacha A, Oliveira PJ, Penlap VB. Toxicity evaluation of some traditional African spices on breast cancer cells and isolated rat hepatic mitochondria. Food Chem Toxicol 2012; 50: 4199-4208
• 9 Choumessi AT, Danel M, Chassaing S, Truchet I, Penlap VB, Pieme AC, Asonganyi T, Ducommun B, Valette A. Characterization of the antiproliferative activity of Xylopia aethiopica . Cell Div 2012; 7: 8
• 10 Dzoyem JP, Nkuete AH, Kuete V, Tala MF, Wabo HK, Guru SK, Rajput VS, Sharma A, Tane P, Khan IA, Saxena AK, Laatsch H, Tan NH. Cytotoxicity and antimicrobial activity of the methanol extract and compounds from Polygonum limbatum . Planta Med 2012; 78: 787-792
• 11 Dzoyem JP, Guru SK, Pieme CA, Kuete V, Sharma A, Khan IA, Saxena AK, Vishwakarma RA. Cytotoxic and antimicrobial activity of selected Cameroonian edible plants. BMC Complement Altern Med 2013; 13: 78
• 12 Kuete V, Tchakam PD, Wiench B, Ngameni B, Wabo HK, Tala MF, Moungang ML, Ngadjui BT, Murayama T, Efferth T. Cytotoxicity and modes of action of four naturally occuring benzophenones: 2,2′,5,6′-tetrahydroxybenzophenone, guttiferone E, isogarcinol and isoxanthochymol. Phytomedicine 2013; 20: 528-536
• 13 Kuete V, Sandjo L, Nantchouang Ouete J, Fouotsa H, Wiench B, Efferth T. Cytotoxicity and modes of action of three naturally occuring xanthones (8-hydroxycudraxanthone G, morusignin I and cudraxanthone I) against sensitive and multidrug-resistant cancer cell lines. Phytomedicine 2013; 21: 315-322
• 14 Kuete V, Sandjo L, Wiench B, Efferth T. Cytotoxicity and modes of action of four Cameroonian dietary spices ethno-medically used to treat cancers: Echinops giganteus, Xylopia aethiopica, Imperata cylindrica and Piper capense . J Ethnopharmacol 2013; 149: 245-253
• 15 Kuete V, Krusche B, Youns M, Voukeng I, Fankam AG, Tankeo S, Lacmata S, Efferth T. Cytotoxicity of some Cameroonian spices and selected medicinal plant extracts. J Ethnopharmacol 2011; 134: 803-812
• 16 Kuete V, Tankeo SB, Saeed ME, Wiench B, Tane P, Efferth T. Cytotoxicity and modes of action of five Cameroonian medicinal plants against multi-factorial drug resistance of tumor cells. J Ethnopharmacol 2014; 153: 207-219
• 17 Kuete V, Sandjo LP, Kwamou GM, Wiench B, Nkengfack AE, Efferth T. Activity of three cytotoxic isoflavonoids from Erythrina excelsa and Erythrina senegalensis (neobavaisoflavone, sigmoidin H and isoneorautenol) toward multi-factorial drug resistant cancer cells. Phytomedicine 2014; 21: 682-688
• 18 Mengome LE, Akue JP, Souza A, Feuya Tchoua GR, Nsi Emvo E. In vitro activities of plant extracts on human Loa loa isolates and cytotoxicity for eukaryotic cells. Parasitol Res 2010; 107: 643-650
• 19 Kone WM, Atindehou KK, Kacou-Nʼdouba A, Dosso M. Evaluation of 17 medicinal plants from Northern Cote dʼIvoire for their in vitro activity against Streptococcus pneumoniae . Afr J Tradit Complement Altern Med 2006; 4: 17-22
• 20 Kone WM, Atindehou KK, Terreaux C, Hostettmann K, Traore D, Dosso M. Traditional medicine in north Cote-dʼIvoire: screening of 50 medicinal plants for antibacterial activity. J Ethnopharmacol 2004; 93: 43-49
• 21 Ma YM, Shi QH, Kong Y. Chemical constituents of stem bark from Periploca sepium . Chem Natural Compd 2007; 43: 497-498
• 22 Ogawa S, Wakatsuki Y, Makino M, Fujimoto Y, Yasukawa K, Kikuchi T, Ukiya M, Akihisa T, Iida T. Oxyfunctionalization of unactivated C–H bonds in triterpenoids with tert-butylhydroperoxide catalyzed by meso-5,10,15,20-tetramesitylporphyrinate osmium(II) carbonyl complex. Chem Phys Lipids 2010; 163: 165-171
• 23 Tour N, Talele G. Phytochemical studies of Calotropis procera stem bark. Chem Natural Compd 2012; 48: 708-709
• 24 Cheng DL, Cao XP. Pomolic acid derivatives from the root of Sanguisorba officinalis . Phytochemistry 1992; 31: 1317-1320
• 25 Konishi T, Konoshima T, Daikonya A, Kitanaka S. Neolignans from Piper futokadsura and their inhibition of nitric oxide production. Chem Pharm Bull (Tokyo) 2005; 53: 121-124
• 26 Pal C, Kundu MK, Bandyopadhyay U, Adhikari S. Synthesis of novel heme-interacting acridone derivatives to prevent free heme-mediated protein oxidation and degradation. Bioorg Med Chem Lett 2011; 21: 3563-3567
• 27 Bilia AR, Mendez J, Morelli I. Phytochemical investigations of Licania genus. Flavonoids and triterpenoids from Licania carii . Pharm Acta Helv 1996; 71: 191-197
• 28 Suffness M, Pezzuto J. Assays related to cancer drug discovery. London: Academic Press; 1990
• 29 Boik J. Natural compounds in cancer therapy. Princeton, Minnesota: Oregon Medical Press; 2001
• 30 Brahemi G, Kona FR, Fiasella A, Buac D, Soukupova J, Brancale A, Burger AM, Westwell AD. Exploring the structural requirements for inhibition of the ubiquitin E3 ligase breast cancer associated protein 2 (BCA2) as a treatment for breast cancer. J Med Chem 2010; 53: 2757-2765
• 31 Efferth T. The human ATP-binding cassette transporter genes: from the bench to the bedside. Curr Mol Med 2001; 1: 45-65
• 32 Gottesman MM, Ling V. The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research. FEBS Lett 2006; 580: 998-1009
• 33 Gillet JP, Efferth T, Remacle J. Chemotherapy-induced resistance by ATP-binding cassette transporter genes. Biochim Biophys Acta 2007; 1775: 237-262
• 34 Rethy B, Zupko I, Minorics R, Hohmann J, Ocsovszki I, Falkay G. Investigation of cytotoxic activity on human cancer cell lines of arborinine and furanoacridones isolated from Ruta graveolens . Planta Med 2007; 73: 41-48
• 35 Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW, Yuan J. Human ICE/CED-3 protease nomenclature. Cell 1996; 87: 171
• 36 Roseghini R, Falcão GM, Oliveira Costa JF, Clarencio J, Nascimento I, Velozo E, Schaer R, Vale V, Costa SL, Costa Mde F, Tardy M, Meyer R, Menezes Freire S. The flavonoid rutin but not the alkaloid arborinine induces apoptosis in a B-cell hybridoma cell line. Planta Med 2009; 75: 488-493
• 37 Wang Y, Gao D, Chen Z, Li S, Gao C, Cao D, Liu F, Liu H, Jiang Y. Acridone derivative 8a induces oxidative stress-mediated apoptosis in CCRF-CEM leukemia cells: application of metabolomics in mechanistic studies of antitumor agents. PLoS One 2013; 8: e63572
• 38 OʼBrien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000; 267: 5421-5426
• 39 Kimmig A, Gekeler V, Neumann M, Frese G, Handgretinger R, Kardos G, Diddens H, Niethammer D. Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells. Cancer Res 1990; 50: 6793-6799
• 40 Gillet J, Efferth T, Steinbach D, Hamels J, de Longueville F, Bertholet V, Remacle J. Microarray-based detection of multidrug resistance in human tumor cells by expression profiling of ATP-binding cassette transporter genes. Cancer Res 2004; 64: 8987-8993
• 41 Doyle LA, Yang W, Abruzzo LV, Krogmann T, Gao Y, Rishi AK, Ross DD. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc Natl Acad Sci U S A 1998; 95: 15665-15670
• 42 Kuete V, Fankam AG, Wiench B, Efferth T. Cytotoxicity and modes of action of the methanol extracts of six Cameroonian medicinal plants against multidrug-resistant tumor cells. Evid Based Complement Alternat Med 2013; 2013: 285903
• 43 Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol 1983; 130: 1910-1917
• 44 Cossarizza A, Ferraresi R, Troiano L, Roat E, Gibellini L, Bertoncelli L, Nasi M, Pinti M. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry. Nat Protoc 2009; 4: 1790-1797