Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, 114, Taiwan, Republic of China.
Department of Pharmacy, Taichung Veterans General Hospital, Taichung, 407, Taiwan, Republic of China.
J Biomed Sci. 2017 Aug 15;24(1):55. doi: 10.1186/s12929-017-0363-7.
Epigallocatechin gallate (EGCG) is the major ingredient of sinecatechins ointment, approved for the treatment of external genital and perianal warts. However, the molecular mechanism for EGCG's effect on warts resulting from the human papillomavirus (HPV) infection of keratinocytes is not well understood. HPV may survive in proliferative keratinocytes and may be involved in cell cycle regulation and progression. The objective of this study was to investigate the mechanism underlying EGCG's treatment on external genital warts of HPV infection through the cultured keratinocyte cells from the HaCaT cell line.
MTT and flow cytometry assays were used to measure cell viability and the cell cycle profile, with and without EGCG treatment, for HaCaT keratinocyte cells cultured in a calcium-free medium and 1.8 mM calcium which induced proliferative and differentiated keratinocytes, respectively, for 24 h. The expression levels of cytotoxic proteins and factors were evaluated with the RT-PCR and western blotting analysis.
EGCG influenced the proliferation stage but not the differentiation stage of keratinocytes. We suggest that apoptosis and autophagy might be the possible mechanism for the EGCG's effect on the proliferative HaCaT cells. Furthermore, we found that EGCG reduced the protein levels of cyclin D1 and Zac1 (a zinc-finger protein which regulates apoptosis and cell cycle arrest 1) dose-dependently in proliferative as compared to differentiated keratinocytes. It also induced the expression of p21 and DEC1 (differentiated embryo-chondrocyte expressed gene 1), and promoted G1 arrest of cell cycle in proliferative keratinocytes.
These results help clarify the mechanisms of EGCG treatment of HPV-infected keratinocytes and may contribute to new targets, such as Zac1 and DEC1 for external genital and perianal warts.
表没食子儿茶素没食子酸酯（EGCG）是喜树碱软膏的主要成分，已被批准用于治疗外阴和肛周生殖器疣。然而，EGCG 对人乳头瘤病毒（HPV）感染角质形成细胞引起的疣的作用机制尚不清楚。HPV 可能在增殖的角质形成细胞中存活，并可能参与细胞周期的调节和进展。本研究旨在通过培养的 HaCaT 角质形成细胞系，探讨 EGCG 治疗 HPV 感染外阴生殖器疣的机制。
采用 MTT 和流式细胞术检测 EGCG 处理 HaCaT 角质形成细胞（在无钙和 1.8 mM 钙培养基中培养 24 h，分别诱导增殖和分化的角质形成细胞）后细胞活力和细胞周期谱的变化。采用 RT-PCR 和 Western blot 分析评估细胞毒性蛋白和因子的表达水平。
EGCG 影响角质形成细胞的增殖阶段，但不影响分化阶段。我们推测细胞凋亡和自噬可能是 EGCG 对增殖的 HaCaT 细胞作用的可能机制。此外，我们发现与分化的角质形成细胞相比，EGCG 可剂量依赖性地降低增殖的角质形成细胞中环蛋白 D1 和 Zac1（一种调节细胞凋亡和细胞周期阻滞 1 的锌指蛋白）的蛋白水平。它还诱导 p21 和 DEC1（分化胚胎软骨细胞表达基因 1）的表达，并促进增殖的角质形成细胞中细胞周期 G1 期阻滞。
这些结果有助于阐明 EGCG 治疗 HPV 感染角质形成细胞的机制，并可能为外阴和肛周生殖器疣提供新的靶点，如 Zac1 和 DEC1。
