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Carcinogenesis Advance Access published online on February 17, 2005
Carcinogenesis, doi:10.1093/carcin/bgi048
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org
Received November 12, 2004
Revised February 1, 2005
Accepted February 6, 2005

MOLECULAR EPIDEMIOLOGY AND CANCER PREVENTION

Human epidermal keratinocytes undergo (-)-epigallocatechin-3-gallate-dependent differentiation but not apoptosis

Sivaprakasam Balasubramanian 1, Michael T. Sturniolo 1, George R. Dubyak 1, and Richard L. Eckert 2*

1 Department of Physiology and Biophysics, Case School of Medicine, Cleveland, Ohio 44106-4970
2 Department of Physiology and Biophysics, Case School of Medicine, Cleveland, Ohio 44106-4970; Department of Dermatology, Case School of Medicine, Cleveland, Ohio 44106-4970; Department of Biochemistry, Case School of Medicine, Cleveland, Ohio 44106-4970; Department of Reproductive Biology, Case School of Medicine, Cleveland, Ohio 44106-4970; Department of Oncology, Case School of Medicine, Cleveland, Ohio 44106-4970

* To whom correspondence should be addressed.
Richard L. Eckert, E-mail: rle2{at}po.cwru.edu


  Abstract

Epigallocatechin-3-gallate (EGCG) is an important chemopreventive agent derived from green tea. We recently reported that EGCG treatment enhances keratinocyte differentiation as evidenced by increased human involucrin (hINV) promoter activity (Balasubramanian et al., J. Biol. Chem., 277, 828-836, 2002). In the present manuscript we extend these findings and show that EGCG also increases expression of other differentiation markers - procaspase 14, and type I transglutaminase. Both type I transglutaminase mRNA and protein level, and activity are increased by treatment with EGCG. Increased type I transglutaminase activity is evidenced by direct transglutaminase assay, and by the ability of EGCG to stimulate covalent incorporation of fluorescein cadaverine substrate into crosslinked intracellular structures. In contrast, type II transglutaminase levels are not altered by EGCG treatment. We also assessed whether EGCG promotes keratinocyte apoptosis. We show that EGCG treatment does not promote cleavage of procaspase-3, -8, -9 or PARP. Moreover, treatment with the pan-caspase inhibitor, Z-VADFMK, does not reverse the EGCG-associated reduction in cell viability. In addition, there is no increase in cells having sub-G1/S DNA content, and no evidence for release of cytochrome c from the mitochondria. These findings confirm, using several endpoints, that EGCG treatment enhances normal keratinocyte differentiation but does not promote apoptosis.

Keywords: EGCG; keratinocyte differentiation; epidermis; transglutaminase; involucrin; green tea polyphenol; antioxidant; apoptosis.
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