Euphol, a tetracyclic triterpene, from Euphorbia tirucalli induces autophagy and sensitizes temozolomide cytotoxicity on glioblastoma cells
- 253 Downloads
Glioblastoma (GBM) is the most frequent and aggressive type of brain tumor. There are limited therapeutic options for GBM so that new and effective agents are urgently needed. Euphol is a tetracyclic triterpene alcohol, and it is the main constituent of the sap of the medicinal plant Euphorbia tirucalli. We previously identified anti-cancer activity in euphol based on the cytotoxicity screening of 73 human cancer cells. We now expand the toxicological screening of the inhibitory effect and bioactivity of euphol using two additional glioma primary cultures. Euphol exposure showed similar cytotoxicity against primary glioma cultures compared to commercial glioma cells. Euphol has concentration-dependent cytotoxic effects on cancer cell lines, with more than a five-fold difference in the IC50 values in some cell lines. Euphol treatment had a higher selective cytotoxicity index (0.64–3.36) than temozolomide (0.11–1.13) and reduced both proliferation and cell motility. However, no effect was found on cell cycle distribution, invasion and colony formation. Importantly, the expression of the autophagy-associated protein LC3-II and acidic vesicular organelle formation were markedly increased, with Bafilomycin A1 potentiating cytotoxicity. Finally, euphol also exhibited antitumoral and antiangiogenic activity in vivo, using the chicken chorioallantoic membrane assay, with synergistic temozolomide interactions in most cell lines. In conclusion, euphol exerted in vitro and in vivo cytotoxicity against glioma cells, through several cancer pathways, including the activation of autophagy-associated cell death. These findings provide experimental support for further development of euphol as a novel therapeutic agent for GBM, either alone or in combination chemotherapy.
KeywordsGlioblastoma Anticancer Cytotoxic activity and euphol
The authors would like to thank Dr. Jeremy Squire for carefully proof-reading the English and for providing constructive criticism of the manuscript.
The work was supported by the Amazônia Fitomedicamentos (FITO05/2012) Ltda. and Barretos Cancer Hospital, all from Brazil.
Compliance with ethical standards
Conflict of interest
Viviane A O Silva declares that she has conflict of interest. Marcela N Rosa declares that she has conflict of interest. Vera Miranda-Gonçalves declares that she no has conflict of interest. Angela M Costa declares that she no has conflict of interest. Aline Tansini declares that she no has conflict of interest. Adriane F. Evangelista declares that she no has conflict of interest. Olga Martinho declares that she no has conflict of interest. Adriana C. Carloni declares that she no has conflict of interest. Chris Jones declares that he no has conflict of interest. João Paulo Lima declares that he no has conflict of interest. Luiz F Pianowski declares that he has conflict of interest. Rui M Reis declares that he has conflict of interest.
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.
Informed consent was obtained from all individual participants included in the study.
- 2.Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW (2016) The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131(6):803–820. https://doi.org/10.1007/s00401-016-1545-1 CrossRefGoogle Scholar
- 5.Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, Maira G, Parati EA, Stassi G, Larocca LM, De Maria R (2010) Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 468(7325):824–828. https://doi.org/10.1038/nature09557 CrossRefGoogle Scholar
- 7.Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63(18):5821–5828Google Scholar
- 8.Weller M, van den Bent M, Tonn JC, Stupp R, Preusser M, Cohen-Jonathan-Moyal E, Henriksson R, Le Rhun E, Balana C, Chinot O, Bendszus M, Reijneveld JC, Dhermain F, French P, Marosi C, Watts C, Oberg I, Pilkington G, Baumert BG, Taphoorn MJB, Hegi M, Westphal M, Reifenberger G, Soffietti R, Wick W (2017) European Association for Neuro-Oncology (EANO) guideline on the diagnosis and treatment of adult astrocytic and oligodendroglial gliomas. The Lancet Oncology 18 (6):e315-e329. doi: https://doi.org/10.1016/s1470-2045(17)30194-8
- 11.Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10):987–996. https://doi.org/10.1056/NEJMoa043330 CrossRefGoogle Scholar
- 13.Pozo-Guisado E, Merino JM, Mulero-Navarro S, Lorenzo-Benayas MJ, Centeno F, Alvarez-Barrientos A, Fernandez-Salguero PM (2005) Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-kappaB. Int J Cancer 115(1):74–84. https://doi.org/10.1002/ijc.20856 CrossRefGoogle Scholar
- 15.Silva VAO, Rosa MN, Tansini A, Oliveira RJ, Martinho O, Lima J, Pianowski LF, Reis RM (2018) In vitro screening of cytotoxic activity of euphol from Euphorbia tirucalli on a large panel of human cancer-derived cell lines. Exp Ther Med. https://doi.org/10.3892/etm.2018.6244
- 17.Dutra RC, Bicca MA, Segat GC, Silva KA, Motta EM, Pianowski LF, Costa R, Calixto JB (2015) The antinociceptive effects of the tetracyclic triterpene euphol in inflammatory and neuropathic pain models: the potential role of PKCepsilon. Neuroscience 303:126–137. https://doi.org/10.1016/j.neuroscience.2015.06.051 CrossRefGoogle Scholar
- 20.Lin MW, Lin AS, Wu DC, Wang SS, Chang FR, Wu YC, Huang YB (2012) Euphol from Euphorbia tirucalli selectively inhibits human gastric cancer cell growth through the induction of ERK1/2-mediated apoptosis. Food Chem Toxicol 50(12):4333–4339. https://doi.org/10.1016/j.fct.2012.05.029 CrossRefGoogle Scholar
- 21.Martinho O, Silva-Oliveira R, Miranda-Goncalves V, Clara C, Almeida JR, Carvalho AL, Barata JT, Reis RM (2013) In vitro and in vivo analysis of RTK inhibitor efficacy and identification of its novel targets in glioblastomas. Transl Oncol 6 (2):187–196Google Scholar
- 22.Cruvinel-Carloni A, SilvaOliveira R, Torrieri R, Bidinotto LT, Berardinelli GN, Oliveira-Silva VA, Clara CA, de Almeida GC, Martinho O, Squire JA, Reis RM (2017) Molecular characterization of short-term primary cultures and comparison with corresponding tumor tissue of Brazilian glioblastoma patients. Trans Can Res 6(2):332–345. https://doi.org/10.21037/tcr.2017.03.32
- 23.Silva-Oliveira RJ, Silva VA, Martinho O, Cruvinel-Carloni A, Melendez ME, Rosa MN, de Paula FE, de Souza Viana L, Carvalho AL, Reis RM (2016) Cytotoxicity of allitinib, an irreversible anti-EGFR agent, in a large panel of human cancer-derived cell lines: KRAS mutation status as a predictive biomarker. Cell Oncol (Dordrecht) 39(3):253–263. https://doi.org/10.1007/s13402-016-0270-z CrossRefGoogle Scholar
- 24.Dutra RC, Simao da Silva KA, Bento AF, Marcon R, Paszcuk AF, Meotti FC, Pianowski LF, Calixto JB (2012) Euphol, a tetracyclic triterpene produces antinociceptive effects in inflammatory and neuropathic pain: the involvement of cannabinoid system. Neuropharmacology 63(4):593–605. https://doi.org/10.1016/j.neuropharm.2012.05.008 CrossRefGoogle Scholar
- 25.Konecny GE, Glas R, Dering J, Manivong K, Qi J, Finn RS, Yang GR, Hong KL, Ginther C, Winterhoff B, Gao G, Brugge J, Slamon DJ (2009) Activity of the multikinase inhibitor dasatinib against ovarian cancer cells. Br J Cancer 101(10):1699–1708. https://doi.org/10.1038/sj.bjc.6605381 CrossRefGoogle Scholar
- 26.Suffness M and Pezzuto JM (1990) Assays related to cancer drug discovery. In: Hostettmann K (ed) Methods in plant biochemistry: assays for bioactivity, vol 6. Academic Press, London, pp 71–133Google Scholar
- 27.Teixeira TL, Oliveira Silva VA, da Cunha DB, Polettini FL, Thomaz CD, Pianca AA, Zambom FL, da Silva Leitao Mazzi DP, Reis RM, Mazzi MV (2016) Isolation, characterization and screening of the in vitro cytotoxic activity of a novel L-amino acid oxidase (LAAOcdt) from Crotalus durissus terrificus venom on human cancer cell lines. Toxicon 119:203–217. doi: https://doi.org/10.1016/j.toxicon.2016.06.009
- 29.Miranda-Goncalves V, Honavar M, Pinheiro C, Martinho O, Pires MM, Pinheiro C, Cordeiro M, Bebiano G, Costa P, Palmeirim I, Reis RM, Baltazar F (2013) Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets. Neuro-Oncology 15(2):172–188. https://doi.org/10.1093/neuonc/nos298 CrossRefGoogle Scholar
- 30.Bruzzese F, Di Gennaro E, Avallone A, Pepe S, Arra C, Caraglia M, Tagliaferri P, Budillon A (2006) Synergistic antitumor activity of epidermal growth factor receptor tyrosine kinase inhibitor gefitinib and IFN-alpha in head and neck cancer cells in vitro and in vivo. Clin Cancer Res 12(2):617–625. https://doi.org/10.1158/1078-0432.ccr-05-1671 CrossRefGoogle Scholar
- 32.Passos GF, Medeiros R, Marcon R, Nascimento AF, Calixto JB, Pianowski LF (2013) The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice. Eur J Pharmacol 698(1–3):413–420. https://doi.org/10.1016/j.ejphar.2012.10.019 CrossRefGoogle Scholar
- 34.Paugh BS, Qu C, Jones C, Liu Z, Adamowicz-Brice M, Zhang J, Bax DA, Coyle B, Barrow J, Hargrave D, Lowe J, Gajjar A, Zhao W, Broniscer A, Ellison DW, Grundy RG, Baker SJ (2010) Integrated molecular genetic profiling of pediatric high-grade gliomas reveals key differences with the adult disease. J Clin Oncol 28 (18):3061–3068. doi: https://doi.org/10.1200/jco.2009.26.7252
- 39.Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I (2003) Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res 63(9):2103–2108Google Scholar
- 40.Santos OJ, Sauaia Filho EN, Nascimento FR, Junior FC, Fialho EM, Santos RH, Santos RA, Serra IC (2016) Use of raw Euphorbia tirucalli extract for inhibition of ascitic Ehrlich tumor. Revista do Colegio Brasileiro de Cirurgioes 43(1):18–21. https://doi.org/10.1590/0100-69912016001005 CrossRefGoogle Scholar
- 43.Azoitei N, Kleger A, Schoo N, Thal DR, Brunner C, Pusapati GV, Filatova A, Genze F, Möller P, Acker T, Kuefer R, Van Lint J, Baust H, Adler G, Seufferlein T (2011) Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation. Neuro Oncol 13(7):710–724. https://doi.org/10.1093/neuonc/nor084