Studies on Inhibition of the Transcriptional Activity of Human Hepatitis B virus X Protein by Ursolic Acid and Oleanolic Acid

• Main Authors: Hong-Yin Wu, 吳鴻音
• Other Authors: Wen-Ling Shih, Ph. D.
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/21263676054128207990

碩士 === 國立屏東科技大學 === 生物科技研究所 === 98 === Abstract The hepatitis B virus X protein (HBx) contains 154 amino acids, encoded a 17 kDa protein. It plays an important role in the development of liver cancer. HBx is a multifunctional regulator that modulates transcriptional activation, signal transduction, cell proliferation, apoptosis, cancer cell migration, and invasion. Many of transcription factors are known to be activated by HBx through the modulation of intracellular signalings. Flavonoids and triterpenes had been confirmed their anti-cancer activities through the inhibition of cancer cell proliferation and apoptosis induction. This research attempts to investigate whether tangeretin、nobiletin、ursolic acid (UA) and oleanolic acid (OA) could inhibit the transactivation function of HBx and to study the involved molecular mechanisms. Utilizing the luciferase reporter assay, HBx could activate Sp-1, Smad3/4 and NF-B ranging from 5 to 20 folds in Huh7 hepatoma cells. In normal hepatocyte FL83B, HBx could activate Sp-1, Smad3/4 ranging from 2 to 4 folds. MTT analysis was used to evaluate the cytotoxicity of tested compounds. Under the non-cytotoxic conditions, UA and OA selectively inhibited the HBx-induced Sp-1 and Smad3/4 transcriptional activation, however, tangeretin and nobiletin did not have this effect. SB203580 and p38 siRNA releases the UA and OA-mediated inhibitory function, however, SP600125 did not affect this phenomenon. These results suggested the important role of p38.Western blotting analysis revealed the UA and OA enhance the phosphorylation level of MKK3/6, p38 and ATF with different kinetics. Ras dominant negative mutant, but not Csk, suppressed the UA and OA-induced p38 activation. Thus, UA and OA activate the p38 signaling through Ras-dependent mechanism. All of UA, OA and a well-known anti-tumor compound silymarin, could effectively prevent HBV-containing hepatoma cells 2.2.15-induced tumor formation in Nu/Nu mice. Key word: UA、OA、HBx、Sp-1、Smad3/4