Skip to main content
SpringerLink
Log in

Prooxidative effects of green tea polyphenol (−)-epigallocatethin-3-gallate on the HIT-T15 pancreatic beta cell line

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic constituent in green tea and is believed to function as an antioxidant. However, increasing evidence indicates that EGCG produces reactive oxygen species (ROS) and subsequent cell death. In this study, we investigated the prooxidative effects of EGCG on the HIT-T15 pancreatic beta cell line. Dose-dependent cell viability was monitored with the cell counting kit-8 assay, while the induction of apoptosis was analyzed by a cell death ELISA kit and comet assay. Extracellular H2O2 was determined using the Amplex Red Hydrogen Peroxide Assay Kit. Intracellular oxidative stress was measured by fluorometric analysis of 2′,7′-dichlorofluorescin (DCFH) oxidation using DCFH diacetate (DA) as the probe. Treatment with EGCG (5–100 µM) decreased the viability of pancreatic beta cells, caused concomitant increases in apoptotic cell death, and increased the production of H2O2 and ROS. Catalase, the iron-chelating agent diethylenetriaminepentaacetic acid, and the Fe(II)-specific chelator o-phenanthroline all suppressed the effects of EGCG, indicating the involvement of both H2O2 and Fe(II) in the mechanism of action of EGCG. The antioxidant N-acetyl-cysteine and alpha-lipoic acid also suppressed the effects of EGCG. Furthermore, EGCG did not scavenge exogenous H2O2, but rather, it synergistically increased H2O2-induced oxidative cell damage in pancreatic beta cells. Together, these findings suggest that in the HIT-T15 pancreatic beta cell line, EGCG mediated the generation of H2O2, triggering Fe(II)-dependent formation of a highly toxic radical that in turn induced oxidative cell damage.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Baek WK, Jang BC, Lim JH, Kwon TK, Lee HY, Cho CH, et al. Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (−)-epigallocatechin-3-gallate. Biochem Pharmacol. 2005;70:1560–7.

    Article  CAS  PubMed  Google Scholar 

  • Elbling L, Weiss RM, Teufelhofer O, Uhl M, Knasmueller S, Schulte-Hermann R, et al. Green tea extract and (−)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities. FASEB J. 2005;19:807–9.

    CAS  PubMed  Google Scholar 

  • Fu Y, Koo MW. EGCG protects HT-22 cells against glutamate-induced oxidative stress. Neurotox Res. 2006;10:23–30.

    Article  CAS  PubMed  Google Scholar 

  • Glei M, Pool-Zobel BL. The main catechin of green tea, (−)-epigallocatechin-3-gallate (EGCG), reduces bleomycin-induced DNA damage in human leucocytes. Toxicol In Vitro. 2006;20:295–300.

    Article  CAS  PubMed  Google Scholar 

  • Gorogawa S, Kajimoto Y, Umayahara Y, Kaneto H, Watada H, Kuroda A, et al. Probucol preserves pancreatic beta-cell function through reduction of oxidative stress in type 2 diabetes. Diabetes Res Clin Pract. 2002;57:1–10.

    Article  CAS  PubMed  Google Scholar 

  • Han MK. Epigallocatechin gallate, a constituent of green tea, suppresses cytokine-induced pancreatic beta-cell damage. Exp Mol Med. 2003;35:136–9.

    CAS  PubMed  Google Scholar 

  • Hara Y, Fujino M, Takeuchi M, Li XK. Green-tea polyphenol (−)-epigallocatechin-3-gallate provides resistance to apoptosis in isolated islets. J Hepatobiliary Pancreat Surg. 2007;14:493–7.

    Article  PubMed  Google Scholar 

  • Heikkila RE, Cabbat FS. The prevention of alloxan-induced diabetes in mice by the iron-chelator detapac: suggestion of a role for iron in the cytotoxic process. Experientia. 1982;38:378–9.

    Article  CAS  PubMed  Google Scholar 

  • Hyon SH, Kim DH. Long-term preservation of rat pancreatic islets under physiological conditions. J Biotechnol. 2001;85:241–6.

    Article  CAS  PubMed  Google Scholar 

  • Igarashi K, Honma K, Yoshinari O, Nanjo F, Hara Y. Effects of dietary catechins on glucose tolerance, blood pressure and oxidative status in Goto–Kakizaki rats. J Nutr Sci Vitaminol (Tokyo). 2007;53:496–500.

    Article  CAS  Google Scholar 

  • Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull. 1996;19:1518–20.

    CAS  PubMed  Google Scholar 

  • Jabbar SA, Twentyman PR, Watson JV. The MTT assay underestimates the growth inhibitory effects of interferons. Br J Cancer. 1989;60:523–8.

    CAS  PubMed  Google Scholar 

  • Kanadzu M, Lu Y, Morimoto K. Dual function of (−)-epigallocatechin gallate (EGCG) in healthy human lymphocytes. Cancer Lett. 2006;241:250–5.

    Article  CAS  PubMed  Google Scholar 

  • Kaneto H, Kajimoto Y, Miyagawa J, Matsuoka T, Fujitani Y, Umayahara Y, et al. Beneficial effects of antioxidants in diabetes: possible protection of pancreatic beta-cells against glucose toxicity. Diabetes. 1999;48:2398–406.

    Article  CAS  PubMed  Google Scholar 

  • Kaneto H, Xu G, Song KH, Suzuma K, Bonner-Weir S, Sharma A, et al. Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress. J Biol Chem. 2001;276:31099–104.

    Article  CAS  PubMed  Google Scholar 

  • Koh G, Suh KS, Chon S, Oh S, Woo JT, Kim SW, et al. Elevated cAMP level attenuates 2-deoxy-d-ribose-induced oxidative damage in pancreatic beta-cells. Arch Biochem Biophys. 2005;438:70–9.

    CAS  PubMed  Google Scholar 

  • Landis-Piwowar KR, Huo C, Chen D, Milacic V, Shi G, Chan TH, et al. A novel prodrug of the green tea polyphenol (−)-epigallocatechin-3-gallate as a potential anticancer agent. Cancer Res. 2007;67:4303–10.

    Article  CAS  PubMed  Google Scholar 

  • Maritim AC, Sanders RA, Watkins JB 3rd. Effects of alpha-lipoic acid on biomarkers of oxidative stress in streptozotocin-induced diabetic rats. J Nutr Biochem. 2003;14:288–94.

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa H, Wachi M, Woo JT, Kato M, Kasai S, Takahashi F, et al. Fenton reaction is primarily involved in a mechanism of (−)-epigallocatechin-3-gallate to induce osteoclastic cell death. Biochem Biophys Res Commun. 2002;292:94–101.

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa H, Hasumi K, Woo JT, Nagai K, Wachi M. Generation of hydrogen peroxide primarily contributes to the induction of Fe(II)-dependent apoptosis in Jurkat cells by (−)-epigallocatechin gallate. Carcinogenesis. 2004;25:1567–74.

    Article  CAS  PubMed  Google Scholar 

  • Pozzolini M, Scarfi S, Benatti U, Giovine M. Interference in MTT cell viability assay in activated macrophage cell line. Anal Biochem. 2003;313:338–41.

    Article  CAS  PubMed  Google Scholar 

  • Rahimi R, Nikfar S, Larijani B, Abdollahi M. A review on the role of antioxidants in the management of diabetes and its complications. Biomed Pharmacother. 2005;59:365–73.

    Article  CAS  PubMed  Google Scholar 

  • Rehman A, Nourooz-Zadeh J, Moller W, Tritschler H, Pereira P, Halliwell B. Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus. FEBS Lett. 1999;448:120–2.

    Article  CAS  PubMed  Google Scholar 

  • Robertson RP. Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J Biol Chem. 2004;279:42351–4.

    Article  CAS  PubMed  Google Scholar 

  • Robertson RP, Harmon JS. Diabetes, glucose toxicity, and oxidative stress: a case of double jeopardy for the pancreatic islet beta cell. Free Radic Biol Med. 2006;41:177–84.

    Article  CAS  PubMed  Google Scholar 

  • Robertson R, Zhou H, Zhang T, Harmon JS. Chronic oxidative stress as a mechanism for glucose toxicity of the beta cell in type 2 diabetes. Cell Biochem Biophys. 2007;48:139–46.

    Article  CAS  PubMed  Google Scholar 

  • Sagara M, Satoh J, Wada R, Yagihashi S, Takahashi K, Fukuzawa M, et al. Inhibition of development of peripheral neuropathy in streptozotocin-induced diabetic rats with N-acetylcysteine. Diabetologia. 1996;39:263–9.

    Article  CAS  PubMed  Google Scholar 

  • Saitoh Y, Chun-ping C, Noma K, Ueno H, Mizuta M, Nakazato M. Pioglitazone attenuates fatty acid-induced oxidative stress and apoptosis in pancreatic beta-cells. Diabetes Obes Metab. 2008;10:564–73.

    Article  CAS  PubMed  Google Scholar 

  • Sakuraba H, Mizukami H, Yagihashi N, Wada R, Hanyu C, Yagihashi S. Reduced beta-cell mass and expression of oxidative stress-related DNA damage in the islet of Japanese type II diabetic patients. Diabetologia. 2002;45:85–96.

    Article  CAS  PubMed  Google Scholar 

  • Shin CS, Moon BS, Park KS, Kim SY, Park SJ, Chung MH, et al. Serum 8-hydroxy-guanine levels are increased in diabetic patients. Diabetes Care. 2001;24:733–7.

    Article  CAS  PubMed  Google Scholar 

  • Song EK, Hur H, Han MK. Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin in mice. Arch Pharm Res. 2003;26:559–63.

    Article  CAS  PubMed  Google Scholar 

  • Tanaka Y, Gleason CE, Tran PO, Harmon JS, Robertson RP. Prevention of glucose toxicity in HIT-T15 cells and Zucker diabetic fatty rats by antioxidants. Proc Natl Acad Sci U S A. 1999;96:10857–62.

    Article  CAS  PubMed  Google Scholar 

  • Wolfram S, Raederstorff D, Preller M, Wang Y, Teixeira SR, Riegger C, et al. Epigallocatechin gallate supplementation alleviates diabetes in rodents. J Nutr. 2006;136:2512–8.

    CAS  PubMed  Google Scholar 

  • Yun SY, Kim SP, Song DK. Effects of (−)-epigallocatechin-3-gallate on pancreatic beta-cell damage in streptozotocin-induced diabetic rats. Eur J Pharmacol. 2006;541:115–21.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study is supported by a grant no. R 13-2002-020-01001-0 (2007) from the Korea Science & Engineering Foundation and a research fund from Kyung Hee University Medical Center.

Author information

Authors and Affiliations

  1. Department of Endocrinology and Metabolism, Kyung Hee University School of Medicine, 1 Hoegi-dong, Dongdaemun-gu, Seoul, 130-701, South Korea

    Kwang Sik Suh, Suk Chon, Seungjoon Oh, Sung Woon Kim, Jin-Woo Kim, Young Seol Kim & Jeong-Taek Woo

  2. Research Institute of Endocrinology, Kyung Hee University, Seoul, 130-702, South Korea

    Kwang Sik Suh, Suk Chon, Seungjoon Oh, Sung Woon Kim, Jin-Woo Kim, Young Seol Kim & Jeong-Taek Woo

Authors
  1. Kwang Sik Suh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Suk Chon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seungjoon Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sung Woon Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jin-Woo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Young Seol Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jeong-Taek Woo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jeong-Taek Woo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Suh, K.S., Chon, S., Oh, S. et al. Prooxidative effects of green tea polyphenol (−)-epigallocatethin-3-gallate on the HIT-T15 pancreatic beta cell line. Cell Biol Toxicol 26, 189–199 (2010). https://doi.org/10.1007/s10565-009-9137-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-009-9137-7

Keywords

Search

Navigation