Aged garlic extract and its constituent, S‑allyl‑L‑cysteine, induce the apoptosis of neuroblastoma cancer cells due to mitochondrial membrane depolarization

Kanamori,Yuta; Dalla Via,Lisa; Macone,Alberto; Canettieri,Gianluca; Greco,Antonio; Toninello,Antonio; Agostinelli,Enzo

doi:10.3892/etm.2019.8383

Aged garlic extract and its constituent, S‑allyl‑L‑cysteine, induce the apoptosis of neuroblastoma cancer cells due to mitochondrial membrane depolarization

Authors:
- Yuta Kanamori
- Lisa Dalla Via
- Alberto Macone
- Gianluca Canettieri
- Antonio Greco
- Antonio Toninello
- Enzo Agostinelli
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Affiliations: Department of Biochemical Sciences ‘A. Rossi Fanelli’, Sapienza University of Rome, I‑00185 Rome, Italy, Department of Pharmaceutical and Pharmacological Sciences, University of Padua, I‑35131 Padua, Italy, Pasteur Laboratory, Department of Molecular Medicine, Sapienza University of Rome, I‑00161 Rome, Italy, Department of Sensory Organs, Sapienza University of Rome, I‑00161 Rome, Italy, International Polyamines Foundation‑ONLUS, I‑00159 Rome, Italy
Published online on: December 27, 2019 https://doi.org/10.3892/etm.2019.8383
Pages: 1511-1521
Copyright: © Kanamori et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Aged garlic extract (AGE) has been demonstrated to have therapeutic properties in tumors; however its mechanisms of action have not yet been fully elucidated. A previous study revealed that AGE exerts an anti‑proliferative effect on a panel of both sensitive [wild‑type (WT)] and multidrug‑resistant (MDR) human cancer cells. Following treatment of the cells with AGE, cytofluorimetric analysis revealed the occurrence of dose‑dependent mitochondrial membrane depolarization (MMD). In this study, in order to further clarify the mechanisms of action of AGE, the effects of AGE on mitochondria isolated from rat liver mitochondria (RLM) were also examined. AGE induced an effect on the components of the electrochemical gradient (ΔµH+), mitochondrial membrane potential (ΔΨm) and mitochondrial electrochemical gradient (ΔpHm). The mitochondrial membrane dysfunctions of RLM induced by AGE, namely the decrease in both membrane potential and chemical gradient were associated with a higher oxidation of both the endogenous glutathione and pyridine nucleotide content. To confirm the anti‑proliferative effects of AGE, experiments were performed on the human neuroblastoma (NB) cancer cells, SJ‑N‑KP and the MYCN‑amplified IMR5 cells, using its derivative S‑allyl‑L‑cysteine (SAC), with the aim of providing evidence of the anticancer activity of this compound and its possible molecular mechanism as regards the induction of cytotoxicity. Following treatment of the cells with SAC at 20 mM, cell viability was determined by MTT assay and apoptosis was detected by flow cytometry, using Annexin V‑FITC labeling. The percentages of cells undergoing apoptosis was found to be 48.0% in the SJ‑N‑KP and 50.1% in the IMR5 cells. By cytofluorimetric analysis, it was suggested that the target of SAC are the mitochondria. Mitochondrial activity was examined by labeling the cells with the probe, 5,5',6,6'‑tetrachloro‑1,1',3,3'‑tetraethylimidacarbocyanine iodide (JC‑1). Following treatment with SAC at 50 mM, both NB cell lines exhibited a marked increase in MMD. On the whole, the findings of this study indicate that both natural products, AGE and SAC, cause cytotoxicity to tumor cells via the induction of mitochondrial permeability transition (MPT).

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1	Ohkubo S, Dalla Via L, Grancara S, Kanamori Y, García-Argáez AN, Canettieri G, Arcari P, Toninello A and Agostinelli E: The antioxidant, aged garlic extract, exerts cytotoxic effects on wild-type and multidrug-resistant human cancer cells by altering mitochondrial permeability. Int J Oncol. 53:1257–1268. 2018.PubMed/NCBI
2	Fleischauer AT, Poole C and Arab L: Garlic consumption and cancer prevention: Meta-analyses of colorectal and stomach cancers. Am J Clin Nutr. 72:1047–1052. 2000. View Article : Google Scholar : PubMed/NCBI
3	Rahman K and Lowe GM: Garlic and cardiovascular disease: A critical review. J Nutr. 136 (Suppl 3):736S–740S. 2006. View Article : Google Scholar : PubMed/NCBI
4	Banerjee SK and Maulik SK: Effect of garlic on cardiovascular disorders: A review. Nutr J. 1:42002. View Article : Google Scholar : PubMed/NCBI
5	Sundaresan S and Subramanian P: Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine. Mol Cell Biochem. 310:209–214. 2008. View Article : Google Scholar : PubMed/NCBI
6	Thomson M and Ali M: Garlic [Allium sativum]: A review of its potential use as an anti-cancer agent. Curr Cancer Drug Targets. 3:67–81. 2003. View Article : Google Scholar : PubMed/NCBI
7	Ray B, Chauhan NB and Lahiri DK: Oxidative insults to neurons and synapse are prevented by aged garlic extract and S-allyl-L-cysteine treatment in the neuronal culture and APP-Tg mouse model. J Neurochem. 117:388–402. 2011. View Article : Google Scholar : PubMed/NCBI
8	Amagase H, Petesch BL, Matsuura H, Kasuga S and Itakura Y: Intake of garlic and its bioactive components. J Nutr. 131:955S–962S. 2001. View Article : Google Scholar : PubMed/NCBI
9	Cemil B, Gokce EC, Kahveci R, Gokce A, Aksoy N, Sargon MF, Erdogan B and Kosem B: Aged garlic extract attenuates neuronal injury in a rat model of spinal cord ischemia/reperfusion injury. J Med Food. 19:601–606. 2016. View Article : Google Scholar : PubMed/NCBI
10	Hu X, Cao BN, Hu G, He J, Yang DQ and Wan YS: Attenuation of cell migration and induction of cell death by aged garlic extract in rat sarcoma cells. Int J Mol Med. 9:641–643. 2002.PubMed/NCBI
11	Fallah-Rostami F, Tabari MA, Esfandiari B, Aghajanzadeh H and Behzadi MY: Immunomodulatory activity of aged garlic extract against implanted fibrosarcoma tumor in mice. N Am J Med Sci. 5:207–212. 2013. View Article : Google Scholar : PubMed/NCBI
12	Li G, Qiao C, Lin R, Pinto J, Osborne M and Tiwari R: Antiproliferative effects of garlic constituents in cultured human breast-cancer cells. Oncol Rep. 2:787–791. 1995.PubMed/NCBI
13	Shirin H, Pinto JT, Kawabata Y, Soh JW, Delohery T, Moss SF, Murty V, Rivlin RS, Holt PR and Weinstein IB: Antiproliferative effects of S-allylmercaptocysteine on colon cancer cells when tested alone or in combination with sulindac sulfide. Cancer Res. 61:725–731. 2001.PubMed/NCBI
14	Hosono T, Fukao T, Ogihara J, Ito Y, Shiba H, Seki T and Ariga T: Diallyl trisulfide suppresses the proliferation and induces apoptosis of human colon cancer cells through oxidative modification of beta-tubulin. J Biol Chem. 280:41487–41493. 2005. View Article : Google Scholar : PubMed/NCBI
15	Howard EW, Ling MT, Chua CW, Cheung HW, Wang X and Wong YC: Garlic-derived S-allylmercaptocysteine is a novel in vivo antimetastatic agent for androgen-independent prostate cancer. Clin Cancer Res. 13:1847–1856. 2007. View Article : Google Scholar : PubMed/NCBI
16	Sriram N, Kalayarasan S, Ashokkumar P, Sureshkumar A and Sudhandiran G: Diallyl sulfide induces apoptosis in Colo 320 DM human colon cancer cells: Involvement of caspase-3, NF-kappaB, and ERK-2. Mol Cell Biochem. 311:157–165. 2008. View Article : Google Scholar : PubMed/NCBI
17	Lai KC, Kuo CL, Ho HC, Yang JS, Ma CY, Lu HF, Huang HY, Chueh FS, Yu CC and Chung JG: Diallyl sulfide, diallyl disulfide and diallyl trisulfide affect drug resistant gene expression in colo 205 human colon cancer cells in vitro and in vivo. Phytomedicine. 19:625–630. 2012. View Article : Google Scholar : PubMed/NCBI
18	Yan JY, Tian FM, Hu WN, Zhang JH, Cai HF and Li N: Apoptosis of human gastric cancer cells line SGC 7901 induced by garlicderived compound S-allylmercaptocysteine (SAMC). Eur Rev Med Pharmacol Sci. 17:745–751. 2013.PubMed/NCBI
19	Zhang H, Wang K, Lin G and Zhao Z: Antitumor mechanisms of S-allyl mercaptocysteine for breast cancer therapy. BMC Complement Altern Med. 14:2702014. View Article : Google Scholar : PubMed/NCBI
20	Ng KT, Guo DY, Cheng Q, Geng W, Ling CC, Li CX, Liu XB, Ma YY, Lo CM, Poon RT, et al: A garlic derivative, S-allylcysteine (SAC), suppresses proliferation and metastasis of hepatocellular carcinoma. PLoS One. 7:e316552012. View Article : Google Scholar : PubMed/NCBI
21	Liu Z, Li M, Chen K, Yang J, Chen R, Wang T, Liu J, Yang W and Ye Z: S-allylcysteine induces cell cycle arrest and apoptosis in androgen-independent human prostate cancer cells. Mol Med Rep. 5:439–443. 2012.PubMed/NCBI
22	Chung LY: The antioxidant properties of garlic compounds: Allyl cysteine, alliin, allicin, and allyl disulfide. J Med Food. 9:205–213. 2006. View Article : Google Scholar : PubMed/NCBI
23	García E, Santana-Martínez R, Silva-Islas CA, Colín-González AL, Galván-Arzate S, Heras Y, Maldonado PD, Sotelo J and Santamaría A: S-allyl cysteine protects against MPTP-induced striatal and nigral oxidative neurotoxicity in mice: Participation of Nrf2. Free Radic Res. 48:159–167. 2014. View Article : Google Scholar : PubMed/NCBI
24	Xu YS, Feng JG, Zhang D, Zhang B, Luo M, Su D and Lin NM: S-allylcysteine, a garlic derivative, suppresses proliferation and induces apoptosis in human ovarian cancer cells in vitro. Acta Pharmacol Sin. 35:267–274. 2014. View Article : Google Scholar : PubMed/NCBI
25	Compston A and Coles A: Multiple sclerosis. Lancet. 372:1502–1517. 2008. View Article : Google Scholar : PubMed/NCBI
26	Lassmann H: Pathophysiology of inflammation and tissue injury in multiple sclerosis: What are the targets for therapy. J Neurol Sci. 306:167–169. 2011. View Article : Google Scholar : PubMed/NCBI
27	Zeinali H, Baluchnejadmojarad T, Fallah S, Sedighi M, Moradi N and Roghani M: S-allyl cysteine improves clinical and neuropathological features of experimental autoimmune encephalomyelitis in C57BL/6 mice. Biomed Pharmacother. 97:557–563. 2018. View Article : Google Scholar : PubMed/NCBI
28	Kyo E, Uda N, Kasuga S and Itakura Y: Immunomodulatory effects of aged garlic extract. J Nutr. 131:1075S–1079S. 2001. View Article : Google Scholar : PubMed/NCBI
29	Morihara N, Sumioka I, Moriguchi T, Uda N and Kyo E: Aged garlic extract enhances production of nitric oxide. Life Sci. 71:509–517. 2002. View Article : Google Scholar : PubMed/NCBI
30	Imai J, Ide N, Nagae S, Moriguchi T, Matsuura H and Itakura Y: Antioxidant and radical scavenging effects of aged garlic extract and its constituents. Planta Med. 60:417–420. 1994. View Article : Google Scholar : PubMed/NCBI
31	Timeus F, Crescenzio N, Doria A, Foglia L, Pagliano S, Ricotti E, Fagioli F, Tovo PA and Cordero di Montezemolo L: In vitro anti-neuroblastoma activity of saquinavir and its association with imatinib. Oncol Rep. 27:734–740. 2012.PubMed/NCBI
32	van Engeland M, Nieland LJ, Ramaekers FC, Schutte B and Reutelingsperger CP: Annexin V-affinity assay: A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 31:1–9. 1998. View Article : Google Scholar : PubMed/NCBI
33	Nicoletti I, Migliorati G, Pagliacci MC, Grignani F and Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 139:271–279. 1991. View Article : Google Scholar : PubMed/NCBI
34	Jiménez-Martín E, Ruiz J, Pérez-Palacios T, Silva A and Antequera T: Gas chromatography-mass spectrometry method for the determination of free amino acids as their dimethyl-tert-butylsilyl (TBDMS) derivatives in animal source food. J Agric Food Chem. 60:2456–2463. 2012. View Article : Google Scholar : PubMed/NCBI
35	Frezza C, Cipolat S and Scorrano L: Organelle isolation: Functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat Protoc. 2:287–295. 2007. View Article : Google Scholar : PubMed/NCBI
36	Gornall AG, Bardawill CJ and David MM: Determination of serum proteins by means of the biuret reaction. J Biol Chem. 177:751–766. 1949.PubMed/NCBI
37	Kamo N, Muratsugu M, Hongoh R and Kobatake Y: Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J Membr Biol. 49:105–121. 1979. View Article : Google Scholar : PubMed/NCBI
38	Rottenberg H: Non-equilibrium thermodynamics of energy conversion in bioenergetics. Biochim Biophys Acta. 549:225–253. 1979. View Article : Google Scholar : PubMed/NCBI
39	Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal Biochem. 27:502–522. 1969. View Article : Google Scholar : PubMed/NCBI
40	Kanda Y: Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 48:452–458. 2013. View Article : Google Scholar : PubMed/NCBI
41	Ho JN, Kang M, Lee S, Oh JJ, Hong SK, Lee SE and Byun SS: Anticancer effect of S-allyl-L-cysteine via induction of apoptosis in human bladder cancer cells. Oncol Lett. 15:623–629. 2018.PubMed/NCBI
42	Hoehner JC, Gestblom C, Hedborg F, Sandstedt B, Olsen L and Påhlman S: A developmental model of neuroblastoma: Differentiating stromapoor tumors' progress along an extra-adrenal chromaffin lineage. Lab Invest. 75:659–675. 1996.PubMed/NCBI
43	Huang M and Weiss WA: Neuroblastoma and MYCN. Cold Spring Harb Perspect Med. 3:a0144152013. View Article : Google Scholar : PubMed/NCBI
44	Fonseka P, Liem M, Ozcitti C, Adda CG, Ang CS and Mathivanan S: Exosomes from N-Myc amplified neuroblastoma cells induce migration and confer chemoresistance to non-N-Myc amplified cells: Implications of intratumour heterogeneity. J Extracell Vesicles. 8:15976142019. View Article : Google Scholar : PubMed/NCBI
45	Lawson LD: Garlic: A review of its medicinal effects and indicated active compounds. Phytomedicines of Europe: Chemistry and biological activity. Lawson LD and Bauer R; ACS symposium series, : 691. American Chemical Society; Washington, DC, USA: pp. 176–209. 1998, View Article : Google Scholar
46	Kodera Y, Suzuki A, Imada O, Kasuga S, Sumioka I, Kanezawa A, Taru N, Fujikawa M, Nagae S, Masamoto K, et al: Physical, chemical, and biological properties of S-allylcysteine, an amino acid derived from garlic. J Agric Food Chem. 50:622–632. 2002. View Article : Google Scholar : PubMed/NCBI
47	Colín-González AL, Santana RA, Silva-Islas CA, Chánez-Cárdenas ME, Santamaría A and Maldonado PD: The antioxidant mechanisms underlying the aged garlic extract- and S-allylcysteine-induced protection. Oxid Med Cell Longev. 2012:9071622012. View Article : Google Scholar : PubMed/NCBI
48	Maldonado PD, Alvarez-Idaboy JR, Aguilar-González A, Lira-Rocha A, Jung-Cook H, Medina-Campos OM, Pedraza-Chaverri J and Galano A: Role of allyl group in the hydroxyl and peroxylradical scavenging activity of S-allylcysteine. J Phys Chem B. 115:13408–13417. 2011. View Article : Google Scholar : PubMed/NCBI
49	Dairam A, Fogel R, Daya S and Limson JL: Antioxidant and iron-binding properties of curcumin, capsaicin, and S-allylcysteine reduce oxidative stress in rat brain homogenate. J Agric Food Chem. 56:3350–3356. 2008. View Article : Google Scholar : PubMed/NCBI
50	Pinto JT, Qiao C, Xing J, Rivlin RS, Protomastro ML, Weisler ML, Tao Y, Thaler H and Heston WD: Effects of garlic thioallyl derivatives on growth, glutathione concentration, and polyamine formation of human prostate carcinoma cells in culture. Am J Clin Nutr. 66:398–405. 1997. View Article : Google Scholar : PubMed/NCBI
51	Kim KM, Chun SB, Koo MS, Choi WJ, Kim TW, Kwon YG, Chung HT, Billiar TR and Kim YM: Differential regulation of NO availability from macrophages and endothelial cells by the garlic component S-allyl cysteine. Free Radic Biol Med. 30:747–756. 2001. View Article : Google Scholar : PubMed/NCBI
52	Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ, Moon KM, Park MH, Park CH, Chung KW, et al: Anti-wrinkle and anti-inflammatory effects of active garlic components and the inhibition of MMPs via NF-κB signaling. PLoS One. 8:e738772013. View Article : Google Scholar : PubMed/NCBI
53	Mong MC and Yin MC: Nuclear factor κB-dependent anti-inflammatory effects of S-allyl cysteine and S-propyl cysteine in kidney of diabetic mice. J Agric Food Chem. 60:3158–3165. 2012. View Article : Google Scholar : PubMed/NCBI
54	Grancara S, Zonta F, Ohkubo S, Brunati AM, Agostinelli E and Toninello A: Pathophysiological implications of mitochondrial oxidative stress mediated by mitochondriotropic agents and polyamines: The role of tyrosine phosphorylation. Amino Acids. 47:869–883. 2015. View Article : Google Scholar : PubMed/NCBI