Altered gap junctional intercellular communication (GJIC) has been associated with chemical carcinogenesis, where both chemical tumor promoters and chemopreventive agents (CPAs) are known to conversely modulate GJIC. The aim of this study was to investigate whether attenuation of chemically inhibited GJIC represents a common outcome induced by different CPAs, which could be effectively evaluated using in vitro methods. Rat liver epithelial cells WB-F344 were pretreated with a CPA for either 30 min or 24 h, and then exposed to GJIC-inhibiting concentration of a selected tumor promoter or environmental toxicant [12-O-tetradecanoylphorbol-13-acetate (TPA), lindane, fluoranthene, 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), perfluorooctanoic acid (PFOA), or pentachlorophenol]. Out of nine CPAs tested, quercetin and silibinin elicited the most pronounced effects, preventing the dysregulation of GJIC by all the GJIC inhibitors, but DDT. Metformin and curcumin attenuated the effects of three GJIC inhibitors, whereas the other CPAs prevented the effects of two (diallyl sulfide, emodin) or one (indole-3-carbinol, thymoquinone) GJIC inhibitor. Significant attenuation of chemically induced inhibition of GJIC was observed in 27 (50%) out of 54 possible combinations of nine CPAs and six GJIC inhibitors. Our data demonstrate that in vitro evaluation of GJIC can be used as an effective screening tool for identification of chemicals with potential chemopreventive activity.

b RECETOX Research Centre for Toxic Compounds in the Environment Faculty of Science Masaryk University Brno Czech Republic

c Department of Pediatrics and Human Development and Institute for Integrative Toxicology Michigan State University Michigan USA

Department of Experimental Phycology and Ecotoxicology Institute of Botany of the ASCR Brno Czech Republic

Zobrazit více v PubMed

Persano L, Zagoura D, Louisse J, Pistollato F. Role of environmental chemicals, processed food derivatives and nutrients in the induction of carcinogenesis. Stem Cells Dev. 2015 PubMed

Oliveira PA, Colaco A, Chaves R, Guedes-Pinto H, De-La-Cruz LF, et al. Chemical carcinogenesis. Anais Da Academia Brasileira De Ciencias. 2007;79:593–616. PubMed

Cohen SM, Arnold LL. Chemical Carcinogenesis. Toxicological Sciences. 2011;120:S76–S92. PubMed

Sporn MB, Dunlop NM, Newton DL, Smith JM. Prevention of Chemical Carcinogenesis by Vitamin-a and Its Synthetic Analogs (Retinoids) Federation Proceedings. 1976;35:1332–1338. PubMed

Mehta RG, Murillo G, Naithani R, Peng XJ. Cancer Chemoprevention by Natural Products: How Far Have We Come? Pharmaceutical Research. 2011;27:950–961. PubMed

Shu LM, Cheung KL, Khor TO, Chen C, Kong AN. Phytochemicals: cancer chemoprevention and suppression of tumor onset and metastasis. Cancer and Metastasis Reviews. 2010;29:483–502. PubMed

Gonzalez-Vallinas M, Gonzalez-Castejon M, Rodriguez-Casado A, Ramirez de Molina A. Dietary phytochemicals in cancer prevention and therapy: a complementary approach with promising perspectives. Nutr Rev. 2013;71:585–599. PubMed

Trosko JE, Chang CC, Upham BL, Tai MH. Ignored hallmarks of carcinogenesis: Stem cells and cell-cell communication. Annals of the New York Academy of Sciences. 2004;1028:192–201. PubMed

Leone A, Longo C, Trosko JE. The chemopreventive role of dietary phytochemicals through gap junctional intercellular communication. Phytochemistry Reviews. 2012

Trosko JE, Chang CC. Mechanism of up-regulated gap junctional intercellular communication during chemoprevention and chemotherapy of cancer. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis. 2001;480:219–229. PubMed

Trosko JE, Ruch RJ. Gap junctions as targets for cancer chemoprevention and chemotherapy. Current Drug Targets. 2002;3:465–482. PubMed

Trosko JE, Chang CC, Upham BL, Tai MH. The role of human adult stem cells and cell-cell communication in cancer chemoprevention and chemotherapy strategies. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis. 2005;591:187–197. PubMed

Trosko JE. The role of stem cells and gap junctions as targets for cancer chemoprevention and chemotherapy. Biomedicine & Pharmacotherapy. 2005;59:S326–S331. PubMed

Vinken M, Doktorova T, Decrock E, Leybaert L, Vanhaecke T, et al. Gap junctional intercellular communication as a target for liver toxicity and carcinogenicity. Critical Reviews in Biochemistry and Molecular Biology. 2009;44:201–222. PubMed

Sai K, Kanno J, Hasegawa R, Trosko JE, Inoue T. Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol. Carcinogenesis. 2000;21:1671–1676. PubMed

Warngard L, Flodstrom S, Ljungquist S, Ahlborg UG. Interaction between quercetin, TPA and DDT in the V79 metabolic cooperation assay. Carcinogenesis. 1987;8:1201–1205. PubMed

Sigler K, Ruch RJ. Enhancement of Gap Junctional Intercellular Communication in Tumor Promoter-Treated Cells by Components of Green Tea. Cancer Letters. 1993;69:15–19. PubMed

Matesic DF, Blommel ML, Sunman JA, Cutler SJ, Cutler HG. Prevention of organochlorine-induced inhibition of gap junctional communication by chaetoglobosin K in astrocytes. Cell Biology and Toxicology. 2001;17:395–408. PubMed

Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis. 1989;10:1003–1008. PubMed

Takahashi H, Nomata K, Mori K, Matsuo M, Miyaguchi T, et al. The preventive effect of green tea on the gap junction intercellular communication in renal epithelial cells treated with a renal carcinogen. Anticancer Res. 2004;24:3757–3762. PubMed

Zefferino R, Leone A, Piccaluga S, Cincione R, Ambrosi L. Mercury modulates interplay between IL-1beta, TNF-alpha, and gap junctional intercellular communication in keratinocytes: mitigation by lycopene. J Immunotoxicol. 2008;5:353–360. PubMed

Leone A, Zefferino R, Longo C, Leo L, Zacheo G. Supercritical CO2-Extracted Tomato Oleoresins Enhance Gap Junction Intercellular Communications and Recover from Mercury Chloride Inhibition in Keratinocytes. Journal of Agricultural and Food Chemistry. 2010;58:4769–4778. PubMed

Upham BL, Guzvic M, Scott J, Carbone JM, Blaha L, et al. Inhibition of gap junctional intercellular communication and activation of mitogen-activated protein kinase by tumor-promoting organic peroxides and protection by resveratrol. Nutrition and Cancer. 2007;57:38–47. PubMed

Upham BL, Park J-S, Babica P, Sovadinova I, Rummel AM, et al. Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems. Environmental Health Perspectives. 2009;117:545–551. PubMed PMC

Sovadinova I, Babica P, Boke H, Kumar E, Wilke A, et al. Phosphatidylcholine specific PLC-induced dysregulation of gap junctions, a robust cellular response to environmental toxicants, and prevention by resveratrol. PLOS One. 2015;10:e0124454. PubMed PMC

Tsao MS, Smith JD, Nelson KG, Grisham JW. A diploid epithelial-cell line from normal adult-rat liver with phenotypic properties of oval cells. Experimental Cell Research. 1984;154:38–52. PubMed

Kao CY, Oakley CS, Welsch CW, Chang CC. Growth requirements and neoplastic transformation of two types of normal human breast epithelial cells derived from reduction mammoplasty. In Vitro Cellular & Developmental Biology-Animal. 1997;33:282–288. PubMed

El Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer - a rapid and simple technique to study gap junctional intercellular communication. Experimental Cell Research. 1987;168:422–430. PubMed

Upham BL. Role of integrative signaling through gap junctions in toxicology. Current Protocols in Toxicology. 2011;47:1–18. PubMed PMC

Choung YH, Choi SJ, Joo JS, Lee JB, Lee HK, et al. Green tea prevents down-regulation of gap junction intercellular communication in human keratinocytes treated with PMA. Eur Arch Otorhinolaryngol. 2011;268:885–892. PubMed

Lee DE, Shin BJ, Hur HJ, Kim JH, Kim J, et al. Quercetin, the active phenolic component in kiwifruit, prevents hydrogen peroxide-induced inhibition of gap-junction intercellular communication. Br J Nutr. 2010;104:164–170. PubMed

Kim YJ, Seo SG, Choi K, Kim JE, Kang H, et al. Recovery effect of onion peel extract against H2 O2 -induced inhibition of gap-junctional intercellular communication is mediated through quercetin. J Food Sci. 2014;79:H1011–H1017. PubMed

Hwang JW, Jung JW, Lee YS, Kang KS. Indole-3-Carbinol Prevents H2O2-Induced Inhibition of Gap Junctional Intercellular Communication by Inactivation of PKB/Akt. Journal of Veterinary Medical Science. 2008;70:1057–1063. PubMed

Li Y, Fu Z, Chen X, Han R. [Effects of curcumin derivatives on the GJIC of normal and tumor cells] Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 1996;18:111–115. PubMed

Pasti G, Kertai P, Adany R. Curcumin does not alter the phorbol ester effect on cell-cell transfer of lucifer yellow CH. Carcinogenesis. 1995;16:1229–1231. PubMed

Jung JW, Park SB, Lee SJ, Seo MS, Trosko JE, et al. Metformin Represses Self-Renewal of the Human Breast Carcinoma Stem Cells via Inhibition of Estrogen Receptor-Mediated OCT4 Expression. PLOS One. 2011;6 PubMed PMC

Anisimov VN. Do metformin a real anticarcinogen? A critical reappraisal of experimental data. Ann Transl Med. 2014;2:60. PubMed PMC

Canovas-Jorda D, Louisse J, Pistollato F, Zagoura D, Bremer S. Regenerative toxicology: the role of stem cells in the development of chronic toxicities. Expert Opin Drug Metab Toxicol. 2014;10:39–50. PubMed

Lee DE, Kang NJ, Lee KM, Lee BK, Kim JH, et al. Cocoa polyphenols attenuate hydrogen peroxide-induced inhibition of gap-junction intercellular communication by blocking phosphorylation of connexin 43 via the MEK/ERK signaling pathway. Journal of Nutritional Biochemistry. 2010;21:680–686. PubMed

Kim JH, Lee BK, Lee KW, Lee HJ. Resveratrol counteracts gallic acid-induced down-regulation of gap-junction intercellular communication. Journal of Nutritional Biochemistry. 2009;20:149–154. PubMed

Kim JH, Choi SH, Kim J, Lee BK, Lee KW, et al. Differential regulation of the hydrogen-peroxide-induced inhibition of gap-junction intercellular communication by resveratrol and butylated hydroxyanisole. Mutat Res. 2009;671:40–44. PubMed

Loch-Caruso R, Upham BL, Harris C, Trosko JE. Divergent roles for glutathione in lindane-induced acute and delayed-onset inhibition of rat myometrial gap junctions. Toxicological Sciences. 2005;85:694–702. PubMed

Upham BL, Trosko JE. Oxidative-Dependent Integration of Signal Transduction with Intercellular Gap Junctional Communication in the Control of Gene Expression. Antioxidants & Redox Signaling. 2009;11:297–307. PubMed PMC

Ruch RJ, Trosko JE, Madhukar BV. Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation. Journal of Cellular Biochemistry. 2001;83:163–169. PubMed

Hasler CM, Frick MA, Bennink MR, Trosko JE. TPA-induced inhibition of gap junctional intercellular communication is not mediated through free radicals. Toxicology and Applied Pharmacology. 1990;103:389–398. PubMed

Upham BL, Kang KS, Cho HY, Trosko JE. Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells. Carcinogenesis. 1997;18:37–42. PubMed

Applicability of Scrape Loading-Dye Transfer Assay for Non-Genotoxic Carcinogen Testing

Improved multiparametric scrape loading-dye transfer assay for a simultaneous high-throughput analysis of gap junctional intercellular communication, cell density and viability