Abstract
The effect of the carcinogen safrole on intracellular Ca2+ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca2+ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at a concentration of 325 μM started to increase [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 40% by removing extracellular Ca2+, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca2+-free medium, after pretreatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) barely induced a [Ca2+]i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca2+]i rise. Neither inhibition of phospholipase C with 2 μM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 1 μM safrole did not alter cell proliferation, but incubation with 10–1000 μM safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca2+ with 10 μM of the Ca2+ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca2+]i rise by causing release of stored Ca2+ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca2+ influx via nifedipine-sensitive Ca2+ entry. Furthermore, safrole can enhance cell growth in a Ca2+-independent manner.
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Annunziato L, Amoroso S, Pannaccione A, Cataldi M, Pignataro G, D’Alessio A, Sirabella R, Secondo A, Sibaud L, Di Renzo GF (2003) Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions. Toxicol Lett 139:125–133
Benedetti MS, Malnoe A, Broillet AL (1977) Absorption, metabolism and excretion of safrole in the rat and man. Toxicology 7:69–83
Bootman MD, Berridge MJ, Roderick HL (2002) Calcium signalling: more messengers, more channels, more complexity. Curr Biol 12:563–565
Chang MJ, Ko CY, Lin RF, Hsieh LL (2002) Biological monitoring of environment exposure to safrole and the Taiwanese betel quid chewing. Arch Environ Contam Toxicol 43:432–437
Chen CL, Chi CW, Chang KW, Liu TY (1999) Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history. Carcinogenesis 20:2331–2334
Daimon H, Sawada S, Asakura S, Sagami F (1997–98) Inhibition of sulfotransferase affecting in vivo genotoxicity and DNA adducts induced by safrole in rat liver. Teratog Carcinog Mutagen 17:327–337
Gilster J, Bacon K, Marlink K, Sheppard B, Deveney C, Rutten M (2004) Bismuth subsalicylate increases intracellular Ca2+, MAP-kinase activity, and cell proliferation in normal human gastric mucous epithelial cells. Dig Dis Sci 49:370–380
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260(6):3440–3450
Gupta KP, van Golen KL, Putman KL, Randerath K (1993) Formation and persistence of safrole-DNA adducts over a 10,000-fold dose range in mouse liver. Carcinogenesis. 14:1517–1521
Hsieh LL, Wang PF, Chen IH, Liao CT, Wang HM, Chen MC, Chang JT, Cheng AJ (2001) Characteristics of mutations in the p53 gene in oral squamous cell carcinoma associated with betel quid chewing and cigarette smoking in Taiwanese. Carcinogenesis 22:1497–1503
Huang TH, Tsai CY, Chen SL, Kao CT (2002) An evaluation of the cytotoxic effects of orthodontic bonding adhesives upon a primary human oral gingival fibroblast culture and a permanent, human oral cancer-cell line. J Biomed Mater Res 63:814–821
Hung SL, Chen YL, Chen YT (2003) Effects of safrole on the defensive functions of human neutrophils. J Periodontal Res 38:130–134
Iwase M, Takemi T, Manabe M, Nagumo M (2003) Hypercalcemic complication in patients with oral squamous cell carcinoma. Int J Oral Maxillofac Surg 32:174–180
Jan CR, Ho CM, Wu SN, Tseng CJ (1998) Bradykinin-evoked Ca2+ mobilization in Madin Darby canine kidney cells. Eur J Pharmacol 355:219–233
Jeurissen SM, Bogaards JJ, Awad HM, Boersma MG, Brand W, Fiamegos YC, van Beek TA, Alink GM, Sudholter EJ, Cnubben NH, Rietjens IM (2004) Human cytochrome P450 enzyme specificity for bioactivation of safrole to the proximate carcinogen 1′-hydroxysafrole. Chem Res Toxicol 17:1245–1250
Kuhlmann CR, Mist AK, Li F, Munz BM, Schaefer CA, Walther S, Raedle-Hurst T, Waldecker B, Piper HM, Tillmanns H, Wiecha J (2005) Endothelin-induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx. Acta Physiol Scand 183:161–169
Liu TY, Chen CC, Chen CL, Chi CW (1999) Safrole-induced oxidative damage in the liver of Sprague–Dawley rats. Food Chem Toxicol 37:697–702
Liu TY, Chung YT, Wang PF, Chi CW, Hsieh LL (2004) Safrole-DNA adducts in human peripheral blood–an association with areca quid chewing and CYP2E1 polymorphisms. Mutat Res 559:59–66
Merritt JE, Jacob R, Hallam TJ (1989) Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem 264:1522–1527
Michel FJ, Fortin GD, Martel P, Yeomans J, Trudeau LE (2005) M3-like muscarinic receptors mediate Ca2+ influx in rat mesencephalic GABAergic neurones through a protein kinase C-dependent mechanism. Neuropharmacology 48(6):796–809
Mukhopadhyay S, Munshi HG, Kambhampati S, Sassano A, Platanias LC, Stack MS (2004) Calcium-induced matrix metalloproteinase 9 gene expression is differentially regulated by ERK1/2 and p38 MAPK in oral keratinocytes and oral squamous cell carcinoma. J Biol Chem 279:33139–33146
Munshi HG, Wu YI, Ariztia EV, Stack MS (2002) Calcium regulation of matrix metalloproteinase-mediated migration in oral squamous cell carcinoma cells. J Biol Chem 277:41480–41488
Rietjens IM, Martena MJ, Boersma MG, Spiegelenberg W, Alink GM (2005) Molecular mechanisms of toxicity of important food-borne phytotoxins. Mol Nutr Food Res 49:131–158
Schiestl RH, Chan WS, Gietz RD, Mehta RD, Hastings PJ (1989) Safrole, eugenol and methyleugenol induce intrachromosomal recombination in yeast. Mutat Res 224:427–436
Shieh DH, Chiang LC, Shieh TY (2003) Augmented mRNA expression of tissue inhibitor of metalloproteinase-1 in buccal mucosal fibroblasts by arecoline and safrole as a possible pathogenesis for oral submucous fibrosis. Oral Oncol 39:728–735
Shieh DH, Chiang LC, Lee CH, Yang YH, Shieh TY (2004) Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan. J Oral Pathol Med 33:581–587
Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP (1990) Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A 87:2466–2470
Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher SK (1991) The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem 266:23856–23862
Acknowledgements
This work was supported by grants from Veterans General Hospital, Kaohsiung: VGHKS94G-11 and VGHKS94-054 to C.R. Jan, and NSC93-2314-B-075B-009 and VGHKS94G-12 to J.K. Huang.
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Huang, JK., Huang, CJ., Chen, WC. et al. Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells. Naunyn Schmied Arch Pharmacol 372, 88–94 (2005). https://doi.org/10.1007/s00210-005-1086-y
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DOI: https://doi.org/10.1007/s00210-005-1086-y