Abstract
The aim of this study was to investigate the anticancer potential of blueberry extract (Vaccinium virgatum) against a C6 rat glioma lineage. Cultures of the C6 cells were exposed to blueberry extract at concentrations of 50 to 600 µg/mL for 12, 24, 48, or 72 h and then evaluated for cell viability, proliferation, migration, colony formation and oxidative stress. We also evaluated the effects of blueberry extract on primary rat cortical astrocytes. Our results show that treatment with blueberry extract did not alter the viability or proliferation of normal primary astrocytes but it did significantly reduce the viability in 21.54 % after 48 h and proliferation in 8.59 % after 24 h of C6 cells at 200 µg/mL. We also observed a reduction in the size of the colonies of 29.99 % at 100 µg/mL when compared to the control cells and cell migration was also reduced at 50 µg/mL. After 72 h, there was a reduction in the reactive oxygen species levels ranging from 46.26 to 34.73 %, in addition to a 380.2 % increase in total thiol content. Superoxide dismutase, catalase, and glutathione S-transferase activities were also enhanced when compared to the control. Taken together this data suggests that blueberry extract exerts some selective anticancer activity in C6 glioma cells.
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References
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126. https://doi.org/10.1016/s0076-6879(84)05016-3
Aksenov MY, Markesbery WR (2001) Changes in thiol content and expression of glutathione redox system genes in the hippocampus and cerebellum in Alzheimer’s disease. Neurosci Lett 302:141–145. https://doi.org/10.1016/s0304-3940(01)01636-6
Alexander BM, Cloughesy TF (2017) Adult Glioblastoma. J Clin Oncol 35:2402–2409. https://doi.org/10.1200/JCO.2017.73.0119
Ali SF, Lebel CP, Bondy SC (1992) Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicology 13:637–648
Aroui S, Fetoui H, Kenani A (2020) Natural dietary compound naringin inhibits glioblastoma cancer neoangiogenesis. BMC Pharmacol Toxicol 21(1):46. https://doi.org/10.1186/s40360-020-00426-1
Barreiros ALBS, David JM, David JP (2006) Estresse oxidativo: relação entre geração de espécies reativas e defesa do organismo. Quim Nova 29:113–123. https://doi.org/10.1590/S0100-40422006000100021
Bona NP, Pedra NS, Azambuja JH, Soares MSP, Spohr L, Gelsleichter N, Meine BM, Sekine FG, Mendonça LT, Oliveira FH, Braganhol E, Spanevello RM, Silveira EF (2019) Tannic acid elicits selective antitumoral activity in vitro and inhibits cancer cell growth in a preclinical model of glioblastoma multiforme. Metab Brain Dis 35:283–293. https://doi.org/10.1007/s11011-019-00519-9
Bordignon CL, Francescatto V, Nienow AA, Calvete E, Reginatto FH (2009) Influência do pH da solução extrativa no teor de antocianinas em frutos de morango. Cienc Tecnol Aliment 29:183–188. https://doi.org/10.1590/S0101-20612009000100028
Campian JL, Ye X, Gladstone DE, Ambady P, Nirschl TR, Borrello I, Golightly M, King KE, Holdhoff M, Karp J, Drake CG, Grossman SA (2015) Pre-radiation lymphocyte harvesting and post-radiation reinfusion in patients with newly diagnosed high grade gliomas. J Neuro-Oncol 124:307–316. https://doi.org/10.1007/s11060-015-1841-y
Chaichana KL, Parker SL, Olivi A, Quiñones-Hinojosa A (2009) Long-term seizure outcomes in adult patients undergoing primary resection of malignant brain astrocytomas: Clinical article. J Neurosurg 111:282–292. https://doi.org/10.3171/2009.2.JNS081132
Ciccarese F, Ciminale V (2017) Escaping death: Mitochondrial redox homeostasis in cancer cells. Front Oncol 7:117. https://doi.org/10.3389/fonc.2017.00117
Debom G, Gazal M, Soares MSP, Couto CAT, Mattos B, Lencina C, Kaster MP, Ghisleni GC, Tavares R, Braganhol E, Chaves VC, Reginatto FH, Stefanello F, Spanevello RM (2016) Preventive effects of blueberry extract on behavioral and biochemical dysfunctions in rats submitted to a model of manic behavior induced by ketamine. Brain Res Bull 127:260–269. https://doi.org/10.1016/j.brainresbull.2016.10.008
Dias KE (2016) Avaliação da atividade antimicrobiana, imunomoduladora e anticancerígena de extratos de mirtilo e de framboesa. Dissertation, Universidade de Lisboa
Diehl A, Yarchoan M, Hopkins A, Jaffee E, Grossman SA (2017) Relationships between lymphocyte counts and treatmentrelated toxicities and clinical responses in patients with solid tumors treated with PD-1 checkpoint inhibitors. Oncotarget 8:114268–114280. https://doi.org/10.18632/oncotarget.23217
Eberhardt MV, Lee CY, Liu RH (2000) Antioxidant activity of fresh apples. Nature 405:903–904. https://doi.org/10.1038/35016151
Fang X, Wang C, Balgley BM, Zhao K, Wang W, He F, Weil RJ, Lee CS (2012) Targeted tissue proteomic analysis of human astrocytomas. J Proteome Res 11:3937–3946. https://doi.org/10.1021/pr300303t
Franken NAP, Rodermond HM, Stap J, Haveman J, Bree CV (2006) Clonogenic assay of cells in vitro. Nature 1:2315–2319. https://doi.org/10.1038/nprot.2006.339
Gosslau A, Jao DL, Huang MT, Ho CT, Evans D, Rawson NE, Chen KY (2011) Effects of the black tea polyphenol theaflavin-2 on apoptotic and inflammatory pathways in vitro and in vivo. Mol Nutr Food Res 55:198–208. https://doi.org/10.1002/mnfr.201000165
Gottfried C, Valentim L, Salbego C, Karl J, Wofchuk ST, Rodnight R (1999) Regulation of protein phosphorylation in astrocyte cultures by external calcium ions: Specific effects on the phosphorylation of glial fibrillary acidic protein (GFAP), vimentin and heat shock protein 27 (HSP27). Brain Res 833:142–149. https://doi.org/10.1016/s0006-8993(99)01503-6
Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139
Halliwell B (2006) Oxidative stress and neurodegeneration: Where are we now? J Neurochem 97:1634–1658. https://doi.org/10.1111/j.1471-4159.2006.03907.x
Hanif F, Muzaffar K, Perveen K, Malhi SM, Simjee SU (2017) Glioblastoma multiforme: A review of its epidemiology and pathogenesis through clinical presentation and treatment. Asian Pac J Cancer Prev 18:3–9. https://doi.org/10.22034/APJCP.2017.18.1.3
Heinonen IM, Meyer AS, Frankel EN (1998) Antioxidant Activity of Berry Phenolics on Human Low-Density Lipoprotein and Liposome Oxidation. J Agric Food Chem 46:4107–4112. https://doi.org/10.1021/jf980181c
Huang WC, Lin YS, Wang CY, Tsai CC, Tseng HC, Chen CL, Lu PJ, Chen PS, Qian L, Hong JS, Lin CF (2008) Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immunology 128. https://doi.org/10.1111/j.1365-2567.2008.02959.x
Janiszewska M, Primi MC, Izard T (2020) Cell adhesion in cancer: Beyond the migration of single cells. J Biol Chem 295:2495–2505. https://doi.org/10.1074/jbc.REV119.007759
Johnson DR, Giannini C, Kaufmann TJ (2020) Review of WHO 2016 changes to classification of gliomas; Incorporation of molecular markers. Glioma Imaging 127–138. https://doi.org/10.1007/978-3-030-27359-0_8
Khoo HE, Azlan A, Tang ST, Lim SM (2017) Anthocyanidins and anthocyanins: Colored pigments as food, pharmaceutical ingredients, and the potential health benefits. Food Nutr Res 61. https://doi.org/10.1080/16546628.2017.1361779
Kong JM, Chia LS, Goh NK, Chia TF, Brouillard R (2003) Analysis and biological activities of anthocyanins. Phytochemistry 64:923–933. https://doi.org/10.1016/s0031-9422(03)00438-2
Kou X, Han L, Li X, Xue Z, Zhou F (2016) Antioxidant and antitumor effects and immunomodulatory activities of crude and purified polyphenol extract from blueberries. Front Chem Sci Eng 10:108–119. https://doi.org/10.1007/s11705-016-1553-7
Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschläger M, Dolznig H (2013) In vitro cell migration and invasion assays. Mutat Res 752:10–24. https://doi.org/10.1016/j.mrrev.2012.08.001
Lamy S, Lafleur R, Bédard V, Moghrabi A, Barrette S, Gingras D, Béliveau R (2007) Anthocyanidins inhibit migration of glioblastoma cells: structure-activity relationship and involvement of the plasminolytic system. J Cell Biochem 100:100–111. https://doi.org/10.1002/jcb.21023
Lazze MC, Savio M, Pizzala R, Cazzalini O, Perucca P, Scovassi AI, Stivala LA, Bianchi L (2004) Anthocyanins induce cell cycle perturbations and apoptosis in different human cell lines. Carcinogenesis 25:1427–1433. https://doi.org/10.1093/carcin/bgh138
Lee SY (2016) Temozolomide resistance in glioblastoma multiforme. Genes Dis 3:198–210. https://doi.org/10.1016/j.gendis.2016.04.007
Levites Y, Weinreb O, Maor G, Youdim MBH, Mandel S (2001) Green tea polyphenol (-)-epigallocatechin-3-gallate prevents N-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration. J Neurochem 78:1073–1082. https://doi.org/10.1046/j.1471-4159.2001.00490.x
Liu W, Long H, Zhang M, Wang Y, Lu Q, Yuan H, Qu Q, Qu J (2019) Glutathione S-transferase genes variants and glioma risk: A case-control and meta-analysis study. J Cancer 10:4679–4688. https://doi.org/10.7150/jca.29398
Lowry OH, RosebroughNJ Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Magalhães M, Manadas B, Efferth T, Cabral C (2021) Chemoprevention and therapeutic role of essential oils and phenolic compounds: Modeling tumor microenvironment in glioblastoma. Pharmacol Res 169:105638. https://doi.org/10.1016/j.phrs.2021.105638
Manganaris GA, Goulas V, Vicente AR, Terry LA (2014) Berry antioxidants: Small fruits providing large benefits. J Sci Food Agric 94:825–833. https://doi.org/10.1002/jsfa.6432
Michaelson JS, Silverstein M, Wyatt J, Weber G, Moore R, Halpern E, Kopans DB, Hughes K (2002) Predicting the survival of patients with breast carcinoma using tumor size. Cancer 95:713–723. https://doi.org/10.1002/cncr.10742
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 247:3170–3175
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63. https://doi.org/10.1016/0022-1759(83)90303-4
Niewald M, Berdel C, Fleckenstein J, Licht N, Ketter R, Rübe C (2011) Toxicity after radiochemotherapy for glioblastoma using temozolomide - a retrospective evaluation. Radiat Oncol 6:141. https://doi.org/10.1186/1748-717X-6-141
Nowak R, Gawlik-Dziki U (2007) Polyphenols of Rosa L. leaves extracts and their radical scavenging activity. Z Naturforschung C J Biosci 62:32–38. https://doi.org/10.1515/znc-2007-1-206
Oliveira PS, Gazal M, Flores NP, Zimmer AR, Chaves VC, Reginatto FH, Kaster MP, Tavares RG, Spanevello RM, Lencina CL, Stefanello FM (2017) Vaccinium virgatum fruit extract as an important adjuvant in biochemical and behavioral alterations observed in animal model of metabolic syndrome. Biomed Pharmacother 88:939–947. https://doi.org/10.1016/j.biopha.2017.01.121
Pauwels B, Korst AE, de Pooter CM, Pattyn GG, Lambrechts HA, Baay MF, Lardon F, Vermorken JB (2003) Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies. Cancer Chemother Pharmacol 51:221–6. https://doi.org/10.1007/s00280-002-0557-9
Pedra NS, Galdino KCA, Silva DS, Ramos PT, Bona NP, Soares MSP, Azambuja JH, Canuto KM, Brito ES, Ribeiro PRV, Souza ASQ, Cunico W, Stefanello FM, Spanevello RM, Braganhol E (2018) Endophytic fungus isolated from Achyrocline satureioides exhibits selective Antiglioma activity - The role of Sch-642305. Front Oncol 8:476. https://doi.org/10.3389/fonc.2018.00476
Reque PM (2012) Frutos de mirtilo (Vaccinium spp.) e produtos derivados: caracterização e estabilidade de suas propriedades bioativas. Dissertantion, Universidade Federal do Rio Grande do Sul
Santos M, Corrêa TS, Faria LDBB, Siqueira GSM, Reis PED, Pinheiro RN (2019) Diretrizes oncológicas 2. Doctor Press Ed. Científica, São Paulo
Schreck KC, Grossman SA (2018) Role of temozolomide in the treatment of cancers involving the central nervous system. Oncology (Williston Park) 32:555–560
Seeram NP (2008) Berry fruits: Compositional elements, biochemical activities, and the impact of their intake on human health, performance, and disease. J Agric Food Chem 56:627–629. https://doi.org/10.1021/jf071988k
Shih PH, Yeh CT, Yen GC (2007) Anthocyanins induce the activation of phase II enzymes through the antioxidant response element pathway against oxidative stress-induced apoptosis. J Agric Food Chem 55:9427–9435. https://doi.org/10.1021/jf071933i
Sivaramakrishna R, Gordon R (1997) Detection of breast cancer at a smaller size can reduce the likelihood of metastatic spread: a quantitative analysis. Acad Radiol 4:8–12. https://doi.org/10.1016/S1076-6332(97)80154-7
Sopik V, Narod SA (2018) The relationship between tumour size, nodal status and distant metastases: on the origins of breast cancer. Breast Cancer Res Treat 170:647–656. https://doi.org/10.1007/s10549-018-4796-9
Spohr L, Soares MSP, Oliveira PS, Mattos BS, Bona NP, Pedra NS, Teixeira FC, Couto CAT, Chaves VC, Reginatto FH, Lisboa MT, Ribeiro AS, Lencina CL, Stefanello FM, Spanevello RM (2018) Combined actions of blueberry extract and lithium on neurochemical changes observed in an experimental model of mania: exploiting possible synergistic effects. Metab Brain Dis 34:605–619. https://doi.org/10.1007/s11011-018-0353-9
Spohr L, Luduvico KP, Soares MSP, Bona NP, Oliveira PS, Mello JE, Alvez FL, Teixeira FC, Felix AOC, Stefanello FM, Spanevello RM (2020) Blueberry extract as a potential pharmacological tool for preventing depressive-like behavior and neurochemical dysfunctions in mice exposed to lipopolysaccharide. Nutr Neurosci 1-14. https://doi.org/10.1080/1028415X.2020.1819104
Stupp R, Mason WP, Bent MJ, Weller M, Fisher B, Taphoorn MJB, 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 352:987–996. https://doi.org/10.1056/NEJMoa043330
Su YB, Sohn S, Krown SE, Livingston P, Wolchok JD, Quinn C, Williams L, Foster T, Sepkowitz KA, Chapman PB (2004) Selective CD4+ lymphopenia in melanoma patients treated with temozolomide: A toxicity with therapeutic implications. J Clin Oncol 22:610–616. https://doi.org/10.1200/JCO.2004.07.060
Sun J, Chu Y, Wu X, Liu RH (2002) Antioxidant and antiproliferative activities of common fruits. J Agric Food Chem 50:7449–7454. https://doi.org/10.1021/jf0207530
Szajdek A, Borowska EJ (2008) Bioactive compounds and health-promoting properties of Berry fruits: A review. Plant Foods Hum Nutr 63:147–156. https://doi.org/10.1007/s11130-008-0097-5
Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholtz-Sloan JS, Villano JL (2014) Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 23:1985–1996. https://doi.org/10.1158/1055-9965.EPI-14-0275
Trivedi M, Laurence J, Siahaan T (2009) The role of thiols and disulfides on protein stability. Curr Protein Pept Sci 10:614–625
Virgili F, Marino M (2008) Regulation of cellular signals from nutritional molecules: a specific role for phytochemicals, beyond antioxidant activity. Free Radic Biol Med 45:1205–1216. https://doi.org/10.1016/j.freeradbiomed.2008.08.001
Wang J, Mazza G (2002) Effects of anthocyanins and other phenolic compounds on the production of tumor necrosis factor α in LPS/IFN-γ-activated RAW 264.7 macrophages. J Agric Food Chem 50:4183–4189. https://doi.org/10.1021/jf011613d
Wang S, Meckling KA, Marcone MF, Kakuda Y, Tsao R (2011) Can phytochemical antioxidant rich foods act as anti-cancer agents? Food Res Int 44:2545–2554. https://doi.org/10.1016/j.foodres.2011.05.021
Wei QY, Zhou B, Cai YJ, Yang L, Liu ZL (2006) Synergistic effect of green tea polyphenols with trolox on free radical-induced oxidative DNA damage. Food Chem 96:90–95. https://doi.org/10.1016/j.foodchem.2005.01.053
Wen PY, Kesari S (2008) Malignant gliomas in adults. New Engl J Med 359:492–507. https://doi.org/10.1056/NEJMra0708126
Yoshino M, Murakami K (1998) Interaction of iron with polyphenolic compounds: Application to antioxidant characterization. Anal Biochem 257:40–44. https://doi.org/10.1006/abio.1997.2522
Zamora-Ros R, Serafini M, Estruch R, Lamuela-Raventós RM, Martínez-González MA, Salas-Salvadó J, Fiol M, Lapetra J, Arós F, Covas MI, Andres-Lacueva C (2013) Mediterranean diet and non enzymatic antioxidant capacity in the PREDIMED study: Evidence for a mechanism of antioxidant tuning. Nutr Metab Cardiovasc Dis 23:1167–1174. https://doi.org/10.1016/j.numecd.2012.12.008
Zhang G, Li D, Chen H, Zhang J, Jin X (2018) Vitexin induces G2/M-phase arrest and apoptosis via Akt/mTOR signaling pathway in human glioblastoma cells. Mol Med Rep 17(3):4599–4604. https://doi.org/10.3892/mmr.2018.8394
Zhang GL, Liang Y, Zhu JY, Jia Q, Gan WQ, Sun LM, Hou HM (2015) Oxidative stress-mediated antiproliferative effects of furan-containing sulfur flavors in human leukemia Jurkat cells. Food Chem 180:1–8. https://doi.org/10.1016/j.foodchem.2015.01.122
Zhao X, Feng P, He W, Du X, Chen C, Suo L, Liang M, Zhang N, Na A, Zhang Y (2019) The prevention and inhibition effect of anthocyanins on colorectal cancer. Curr Pharm Des 25:4919–4927. https://doi.org/10.2174/1381612825666191212105145
Funding
This study did not receive specific funding. The general funding was supported by the Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES – Finance Code 001).
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Larissa Menezes da Silveira: Cells treatment, biochemical analysis, statistical analysis, and manuscript preparation.
Nathalia Stark Pedra: Astrocytes treatment and biochemical analysis.
Natália Pontes Bona: C6 treatment and biochemical analysis.
Luiza Spohr: Blueberry extract preparation and biochemical analysis.
Francieli da Silva dos Santos: Oxidative stress analysis.
Juliane Torchelsen Saraiva: Oxidative stress analysis.
Fernando Lopez Alvez: C6 treatment and biochemical analysis.
Bernardo de Moraes Meine: Oxidative stress analysis.
Roselia Maria Spanevello: Experimental design, results interpretation, discussion, and manuscript preparation.
Francieli Moro Stefanello: Experimental design, results interpretation, discussion, and manuscript preparation.
Mayara Sandrielly Pereira Soares: Experimental design, results interpretation, discussion, and manuscript preparation.
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All procedures involving animal models were approved by the Animal Ethics and Experimentation Committee of the Federal University of Pelotas (UFPel) under protocol number CEEA (31292-2018). The protocols complied with the Brazilian Guidelines for the Care and Use of Animals in Scientific Research Activities (DBCA), which is in accordance with the expectations described by the National Council for the Control of Animal Experimentation (CONCEA).
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da Silveira, L.M., Pedra, N.S., Bona, N.P. et al. Selective in vitro anticancer effect of blueberry extract (Vaccinium virgatum) against C6 rat glioma: exploring their redox status. Metab Brain Dis 37, 439–449 (2022). https://doi.org/10.1007/s11011-021-00867-5
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DOI: https://doi.org/10.1007/s11011-021-00867-5