Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus glutinosa) and Their Interaction with Anticancer Drugs

Abstract

Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI–H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC₅₀ values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.

Key words

diarylheptanoid - Alnus glutinosa - Betulaceae - curcumin - doxorubicin - cisplatin - reactive oxygen species (ROS)

• References

• 1 Brand S, Hölscher D, Schierhorn A, Svatos A, Schröder J, Schneider B. A type III polyketide synthase from Wachendorfia thyrsiflora and its role in diarylheptanoid and phenylphenalenone biosynthesis. Planta 2006; 224: 413-428
• 2 Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol 2009; 41: 40-59
• 3 Tung NH, Kim SK, Ra JC, Zhao YZ, Sohn DH, Kim YH. Antioxidative and hepatoprotective diarylheptanoids from the bark of Alnus japonica . Planta Med 2010; 76: 626-629
• 4 Lai YC, Chen CK, Lin WW, Lee SS. A comprehensive investigation of anti-inflammatory diarylheptanoids from the leaves of Alnus formosana . Phytochemistry 2012; 73: 84-94
• 5 Choi SE, Kim KH, Kwon JH, Kim SB, Kim HW, Lee MW. Cytotoxic activities of diarylheptanoids from Alnus japonica . Arch Pharm Res 2008; 31: 1287-1289
• 6 Matsuda H, Ishikado A, Nishida N, Ninomiya K, Fujiwara H, Kobayashi Y, Yoshikawa M. Hepatoprotective, superoxide scavenging, and antioxidative activities of aromatic constituents from the bark of Betula platyphylla var. japonica . Bioorg Med Chem Lett 1998; 8: 2939-2944
• 7 Lee MA, Lee HK, Kim SH, Kim YC, Sung SH. Chemical constituents of Alnus firma and their inhibitory activity on lipopolysaccharide-induced nitric oxide production in BV2 microglia. Planta Med 2010; 76: 1007-1010
• 8 Lewandowska U, Gorlach S, Owczarek K, Hrabec E, Szewczyk K. Synergistic interactions between anticancer chemotherapeutics and phenolic compounds and anticancer synergy between polyphenols. Postepy Hig Med Dosw (Online) 2014; 68: 528-540
• 9 Zingg JM, Hasan ST, Meydani M. Molecular mechanisms of hypolipidemic effects of curcumin. Biofactors 2013; 39: 101-121
• 10 Bae MK, Kim SH, Jeong JW, Lee YM, Kim HS, Kim SR, Yun I, Bae SK, Kim KW. Curcumin inhibits hypoxia-induced angiogenesis via down-regulation of HIF-1. Oncol Rep 2006; 15: 1557-1562
• 11 Choi H, Chun YS, Kim SW, Kim MS, Park JW. Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition. Mol Pharmacol 2006; 70: 1664-1671
• 12 Suresh A, Guedez L, Moreb J, Zucali J. Overexpression of manganese superoxide dismutase promotes survival in cell lines after doxorubicin treatment. Br J Haematol 2013; 120: 457-463
• 13 Doublier S, Belisario DC, Polimeni M, Annaratone L, Riganti C, Allia E, Ghigo D, Bosia A, Sapino A. HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast. BMC Cancer 2012; 12: 4
• 14 Yeung BH, Wong KY, Lin MC, Wong CK, Mashima T, Tsuruo T, Wong AS. Chemosensitisation by manganese superoxide dismutase inhibition is caspase-9 dependent and involves extracellular signal-regulated kinase 1/2. Br J Cancer 2008; 99: 283-293
• 15 OʼRourke C, Byres M, Delazar A, Kumarasamy Y, Nahar L, Stewart F, Sarker SD. Hirsutanonol, oregonin and genkwanin from the seeds of Alnus glutinosa (Betulaceae). Biochem Syst Ecol 2005; 33: 749-752
• 16 Smite E, Lundgren LN, Andersson R. Arylbutanoid and diarylheptanoid glycosides from inner bark of Betula pendula . Phytochemistry 1993; 32: 365-369
• 17 Novakovic M, Pesic M, Trifunovic S, Vuckovic I, Todorovic N, Podolski-Renic A, Dinic J, Stojkovic S, Tesevic V, Vajs V, Milosavljevic S. Diarylheptanoids from the bark of black alder inhibit the growth of sensitive and multi-drug resistant non-small cell lung carcinoma cells. Phytochemistry 2014; 97: 46-54
• 18 Novakovic M, Stankovic M, Vuckovic I, Todorovic N, Trifunovic S, Tesevic V, Vajs V, Milosavljevic S. Diarylheptanoids from Alnus glutinosa bark and their chemoprotective effect on human lymphocytes DNA. Planta Med 2013; 79: 499-505
• 19 Matsushima H, Yonemura K, Ohishi K, Hishida A. The role of oxygen free radicals in cisplatin-induced acute renal failure in rats. J Lab Clin Med 1998; 131: 518-526
• 20 Pommier Y, Leo E, Zhang H, Marchand C. DNA topoisomerases and their poisoning by anticancer and antibacterial drugs. Chem Biol 2010; 17: 421-433
• 21 Rao GV, Kumar S, Islam M, Mansour SE. Folk medicines for anticancer therapy – a current status. Cancer Ther 2008; 6: 913-922
• 22 Bhanot A, Sharma R, Noolvi MN. Natural sources as potential anti-cancer agents. Int J Phytomedicine 2011; 3: 9-26
• 23 Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz DA. Interference by doxorubicin with DNA unwinding in MCF-7 breast tumor cells. Mol Pharmacol 1994; 45: 649-656
• 24 Pang B, Qiao X, Janssen L, Velds A, Groothuis T, Kerkhoven R, Nieuwland M, Ovaa H, Rottenberg S, van Tellingen O, Janssen J, Huijgens P, Zwart W, Neefjes J. Drug-induced histone eviction from open chromatin contributes to the chemotherapeutic effects of doxorubicin. Nat Commun 2013; 4: 1908
• 25 Andjelkovic T, Pesic M, Bankovic J, Tanic N, Markovic ID, Ruzdijic S. Synergistic effects of the purine analog sulfinosine and curcumin on the multidrug resistant human non-small cell lung carcinoma cell line (NCI-H460/R). Cancer Biol Ther 2008; 7: 1024-1032
• 26 Ketron AC, Gordon ON, Schneider C, Osheroff N. Oxidative metabolites of curcumin poison human type II topoisomerases. Biochemistry 2013; 52: 221-227
• 27 Lopez-Lazaro M, Willmore E, Jobson A, Gilroy KL, Curtis H, Padget K, Austin CA. Curcumin induces high levels of topoisomerase I- and II-DNA complexes in K562 leukemia cells. J Nat Prod 2007; 70: 1884-1888
• 28 Jamieson ER, Lippard SJ. Structure, recognition, and processing of cisplatin-DNA adducts. Chem Rev 1999; 99: 2467-2498
• 29 Xu GP, Dave KR, Moraes CT, Busto R, Sick TJ, Bradley WG, Perez-Pinzon MA. Dysfunctional mitochondrial respiration in the wobbler mouse brain. Neurosci Lett 2001; 300: 141-144
• 30 Sardão VA, Oliveira PJ, Holy J, Oliveira CR, Wallace KB. Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide–characterization of morphological features of cell death. BMC Cell Biol 2007; 8: 11
• 31 Paniagua-Perez R, Madrigal-Bujaidar E, Reyes-Cadena S, Alvarez-Gonzalez I, Sanchez-Chapul L, Perez-Gallaga J, Hernandez N, Flores-Mondragon G, Velasco O. Cell protection induced by beta-sitosterol: inhibition of genotoxic damage, stimulation of lymphocyte production, and determination of its antioxidant capacity. Arch Toxicol 2008; 82: 615-622
• 32 Troyano A, Fernandez C, Sancho P, de Blas E, Aller P. Effect of glutathione depletion on antitumor drug toxicity (apoptosis and necrosis) in U-937 human promonocytic cells. The role of intracellular oxidation. J Biol Chem 2001; 276: 47107-47115
• 33 Miller AF. Superoxide dismutases: active sites that save, but a protein that kills. Curr Opin Chem Biol 2004; 8: 162-168
• 34 Weidemann A, Johnson RS. Biology of HIF-1alpha. Cell Death Differ 2008; 15: 621-627
• 35 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27-55
• 36 Larrea E, Beloqui O, Munoz-Navas MA, Civeira MP, Prieto J. Superoxide dismutase in patients with chronic hepatitis C virus infection. Free Radic Biol Med 1998; 24: 1235-1241
• 37 Nardinocchi L, Puca R, Sacchi A, DʼOrazi G. Inhibition of HIF-1alpha activity by homeodomain-interacting protein kinase-2 correlates with sensitization of chemoresistant cells to undergo apoptosis. Mol Cancer 2009; 8: 1
• 38 NicAmhlaoibh R, Heenan M, Cleary I, Touhey S, OʼLoughlin C, Daly C, Nunez G, Scanlon KJ, Clynes M. Altered expression of mRNAs for apoptosis-modulating proteins in a low level multidrug resistant variant of a human lung carcinoma cell line that also expresses mdr1 mRNA. Int J Cancer 1999; 82: 368-376
• 39 Wong H, Anderson WD, Cheng T, Riabowol KT. Monitoring mRNA expression by polymerase chain reaction: the “primer-dropping” method. Anal Biochem 1994; 223: 251-258