Green tea prevents down-regulation of gap junction intercellular communication in human keratinocytes treated with PMA.
It has been suggested that connexin (Cx) gap junction proteins act as tumor suppressors and green tea has a potential to prevent tumor development, however, the studies on their association with human keratinocytes were rare. We evaluated the effects of a tumor promoter, phorbol-12-myristate-13-acetate (PMA), on the expression of Cxs and gap junction intercellular communication (GJIC) in human keratinocytes (HaCaT cells) and explored the preventive effects of green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG). We performed neutral red dye uptake assay to determine the optimal concentrations of PMA, EC, and EGCG for this study and confirmed the expression of Cx mRNAs using RT-PCR. We evaluated GJIC quantitatively using the 'scrape-loading dye transfer (SLDT)' technique after 24-h culture of HaCaT cells treated with agents. To analyze the expression change of Cxs, we also performed Western blot and immunocytochemistry. HaCaT cells were found to express Cx26, Cx30, Cx31, and Cx43, but not Cx29. In 'scrape-loading dye transfer' for functional study for GJIC, EC and EGCG significantly prevented PMA-induced down-regulation of GJIC. Western blot analyses revealed that EC and EGCG prevented down-regulation of Cx26 and Cx43 proteins in HaCaT cells treated with PMA. Immunocytochemistry showed decreased expression and abnormal location of Cx26 and Cx43 in HaCaT cells when treated with PMA, and EC and EGCG inhibited its effect. These results suggest an important role of GJIC played in carcinogenesis involving human keratinocytes and green tea as a useful anticancer diet.
Department of Otolaryngology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. yhc@ajou.ac.kr
Yun-Hoon
YH
Choung
Seong Jun
SJ
Choi
Jung Sook
JS
Joo
Jong Bin
JB
Lee
Hae Kyung
HK
Lee
Seung Joo
SJ
Lee
D015153:Blotting, Western; D002450:Cell Communication / Q000187:drug effects* / Q000235:genetics; D000072259:Connexin 26; D017630:Connexins / Q000096:biosynthesis / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics*; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D007822:Laryngeal Neoplasms / Q000473:pathology / Q000517:prevention & control; D010703:Phorbol Esters / Q000494:pharmacology*; D010936:Plant Extracts / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013662:Tea; D014407:Tumor Cells, Cultured
D017630:Connexins; C000607042:GJB2 protein, human; D010703:Phorbol Esters; D010936:Plant Extracts; D012333:RNA, Messenger; D013662:Tea; D000072259:Connexin 26; C032974:12-O-retinoylphorbol-13-acetate