Green tea prevents down-regulation of gap junction intercellular communication in human keratinocytes treated with
Yun-Hoon Choung1, Seong Jun Choi, Jung Sook Joo
1Department of Otolaryngology, Ajou University School of Medicine, San 5, Wonchon-dong, Yeongtong-gu, Suwon 443-721, Republic of Korea. yhc@ajou.ac.kr
Abstract:
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.
Insights
Green tea compounds epicatechin (EC) and epigallocatechin-3-gallate (EGCG) protect human keratinocytes from tumor promoter effects. These compounds maintain connexin (Cx) protein levels and gap junction intercellular communication (GJIC), suggesting a role in cancer prevention.
Area of Science:
- Cell Biology
- Oncology
- Dermatology
Background:
- Connexin (Cx) proteins form gap junctions, potentially acting as tumor suppressors.
- Green tea components like epicatechin (EC) and epigallocatechin-3-gallate (EGCG) show promise in cancer prevention.
- Limited research exists on Cx proteins and green tea's effects in human keratinocytes.
Purpose of the Study:
- To investigate the impact of phorbol-12-myristate-13-acetate (PMA) on Cx expression and gap junction intercellular communication (GJIC) in HaCaT cells.
- To explore the potential preventive effects of green tea extracts (EC and EGCG) against PMA-induced changes.
Main Methods:
- HaCaT cells were treated with PMA, EC, and EGCG.
- Neutral red dye uptake assay determined optimal concentrations.
- Cx mRNA expression was confirmed via RT-PCR.
- GJIC was quantified using scrape-loading dye transfer (SLDT).
- Cx protein levels and localization were analyzed by Western blot and immunocytochemistry.
Main Results:
- HaCaT cells express Cx26, Cx30, Cx31, and Cx43.
- EC and EGCG significantly prevented PMA-induced down-regulation of GJIC.
- EC and EGCG inhibited PMA's effect on Cx26 and Cx43 protein expression and localization.
- PMA treatment led to decreased expression and abnormal localization of Cx26 and Cx43.
Conclusions:
- GJIC plays a significant role in human keratinocyte carcinogenesis.
- Green tea extracts (EC and EGCG) demonstrate protective effects against tumor promotion in keratinocytes.
- Green tea may be a beneficial dietary component for cancer prevention.
