Pinocembrin 抑制酒精誘發之 tissue transglutaminase 表現於人類肝癌細胞之分子機制研究

The Molecular Mechanisms of Pinocembrin Inhibited Ethanol-induced Tissue Transglutaminase Activity in Human Hepatocellular Carcinoma Cells

賴彥錞(Yann-Chwen Lai)

指導教授 ： 何元順

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2004年)

摘要

tissue transglutaminase (tTG)是一個Ca2+-dependent的酵素，藉由催化protein-protein間cross-linking，而穩定細胞外基質蛋白或造成纖維化的發生。酒精被認為是導致肝纖維化的主因，我們首先研究酒精在人類肝癌細胞株Hep3B、HepG2中調控tTG mRNA 表現的機制。我們發現在酒精刺激下只有Hep3B細胞被誘發tTG mRNA的表現。文獻指出，轉錄因子NF-?B參與tTG的調控表現。接著我們利用NF-kB的抑制劑--TLCK來研究tTG和NF-kB間的相關性，研究發現，酒精誘發的tTG表現會被TLCK所抑制，所以我們認為NF-kB參與tTG表現的調控。另外，在Hep3B細胞中也發現酒精誘發ERK磷酸化的情形，並且NF-kB的活化被MEK的抑制劑--PD98059所抑制。因此，我們推測酒精是透過ERK及NF-kB的訊息傳遞路徑來調控tTG的表現。Pinocembrin是富存於蜂膠及蜂蜜中之flavanone，可作為抗氧化劑。我們發現pinocembrin可抑制由酒精所誘導的tTG表現及活性，並抑制NF-kB p65 subunit 轉位到核裡的能力。另外pinocembrin也抑制酒精所誘發之ERK的磷酸化現象。綜合上述結果，我們證實pinocembrin透過抑制ERK與NF-kB的活化來調控tTG的作用，而具有預防肝臟纖維化的潛力。

關鍵字

Pinocembrin ； tissue transglutaminase

並列摘要

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme which plays an important role in the stabilization of the extracellular matrix (ECM) and formation of fibrotic lesions by catalysing protein-protein cross-linkage. Ethanol is considered as a significant inducing mediator of the liver fibrosis, we firstly investigated the mechanism of how ethanol regulates tTG mRNA expression in the Hep3B and HepG2 cell. We found that only Hep3B cell synthesize tTG mRNA after addition of ethanol. Recent works have shown that transcription factor NF-kB was required to regulate tTG expression. In this study, we used the NF-kB-specific inhibitor, TLCK, to investigate the correlation between tTG and NF-kB and demonstrated that ethanol-induced tTG expression can be suppressed after addition of TLCK, providing evidence that NF-kB may participate the regulation in tTG expression. In addition, phosphorylation of ERK was also induced by ethanol in the Hep3B cell, and activation of NF-kB can be suppressed by adding MEK inhibitor, PD98059. Therefore, ERK and NF-kB are the possible signaling molecules to mediate the tTG activation. Pinocembrin, the most abundant flavanone which was isolated from the various types of propolis and honey as an antioxidant. We found that the pinocembrin suppressed ethanol-induced tTG expression and its activity, along with the inhibition of nuclear translocation of NF-kB subunit p65. Furthermore, pinocembrin also suppressed ethanol-induced activation of ERK. Overall, these data demonstrated that the pinocembrin inhibits the activation of ERK-and NF-kB-mediated tTG expression, which shows that pinocembrin has a potential to prevent liver fibrosis.

並列關鍵字

Pinocembrin ； tissue transglutaminase

參考文獻

Antonyak, M.A., Boehm, J.E. and Cerione, R.A. (2002) Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase. J Biol Chem, 277, 14712-14716.

Balklava, Z., Verderio, E., Collighan, R., Gross, S., Adams, J. and Griffin, M. (2002) Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion. J Biol Chem, 277, 16567-16575.

Cardile, V., Panico, A., Gentile, B., Borrelli, F. and Russo, A. (2003) Effect of propolis on human cartilage and chondrocytes. Life Sci, 73, 1027-1035.

Chen, C.N., Wu, C.L. and Lin, J.K. (2004) Propolin C from propolis induces apoptosis through activating caspases, Bid and cytochrome c release in human melanoma cells. Biochem Pharmacol, 67, 53-66.

Del Rayo Camacho, M., Sanchez, B., Quiroz, H., Contreras, J.L. and Mata, R. (1991) Pinocembrine: a bioactive flavanone from Teloxys graveolens. J Ethnopharmacol, 31, 383-389.