Preventive Effect of Green Tea Extracts on the Inhibition of Connexin Expression Induced by Carcinogen H2O2

Abstract

Background & Objectives: Carcinogens result in the impairment of intercellular communication as well as intracellular communication of normal cells. Connexin (Cx) is a main constituent protein of gap junctions that let messengers such as ions communicate between cells. We evaluated the effect of carcinogen H2O2 on the expression of Cxs and gap junction intercellular communication (GJIC) in the human keratinocyte cell line (HaCaT) and analyzed the prevention effect of green tea extracts against H2O2.

Materials & Method: We performed neutral red dye uptake tests to determine the optimal concentrations of H2O2, green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG) in this study. To analyze the expression change of Cxs, we performed RT-PCR, Western blot, and immunocytochemistry after 24-hour culture of HaCaT cells treated with agents. We also evaluated GJIC quantitatively using the ’scrape loading dye transfer (SLDT)’ technique.

Results: Cx26, Cx30, Cx31, Cx43, but not Cx29 were expressed in the HaCaT cells. H2O2 (250 uM) down-regulated Cx26 and Cx43 proteins. In HaCaT cells treated with H2O2, EC (175 uM) up-regulated Cx26 and Cx43 proteins, but EGCG (50 uM) up-regulated only Cx43 protein. Immunocytochemistry showed the decreased expression and abnormal location of Cx26 and Cx43 under H2O2, and EC and EGCG (5 uM) inhibited the effect of H2O2, showing similar staining in the control. In SLDT, H2O2 down-regulated GJIC, while EC and EGCG significantly prevented HaCaT cells from the H2O2-induced, down-regulation of GJIC.

Conclusions: The carcinogen, H2O2, inhibits GJIC in the keratinocyte cell line. Green tea extracts, such as EC and EGCG, prevent GJIC inhibition in the keratinocyte cell line treated with H2O2, suggesting they have a potential anti-cancer properties.