Clausenidin及其結構類似物對於人類肝癌細胞株Hep G2/A2與Hep3B/T2作用的研究Study the effects of clausenidin and it's analogue on human hepatoma cell lines Hep G2/A2 and Hep3B/T2蘇俊圖Su, Chun-Tu生物藥學研究所關鍵詞:生物科技;藥學;肝癌細胞株;Clausenidin;Keyword:BIOTECHNOLOGY;PHARMACOLOGY;摘要:B型肝炎是全球性的流行疾病，尤其在亞非地區最為盛行。
由 流行病學上的研究顯示，B型肝炎帶原者罹患肝癌的機率遠大於正常人，但至今仍無有效藥物可供治療之用，因此這方面的新藥開發是一重要課題。
本論文以帶有B型肝炎病毒基因且能表現B型肝炎病毒表面抗原的人類肝癌細胞株Hep G2/A2、Hep 3B/T2作為藥物篩選模式，並對所篩得之化合物其抑制表面抗原產生及對細胞生長之調節機制做探討。
在本論文中，發現到在一些pyranocoumarin的結構類似物中，Clausenidin對於抑制 B型肝炎病毒表面抗原的能力最強，其中Clausenidin之抑制濃度EC(50)為1.0μg/ml。
由RT-PCR分析，發現表面抗原基因之mRNA表現量會受影響。
在另一株無HBV基因但有高效率轉染HBV基因之人類肝癌細胞株HuH-7細胞，發現Clausenidin處理後，其HBV表面抗原的promoter-SPII，以及HBV基因會受到影響。
經對細胞預先處理TPA以去除蛋自激活酵素C(PKC)的實驗，得知Clausenidin抑制細胞產生表面抗原的作用與PKC活化途徑無關。
人類肝癌細胞株HepG2/A2經Clausenidin處理其抑制B型肝炎病毒表面抗原的能力具有可逆性，但處理Clausenidin長時期(6天)對HepG2/A2細胞會抑制細胞生長；但在另一肝癌細胞株 Hep3B/T2，細胞生長則不被抑制。
經由細胞週期及DNA複製分析發現經Clausenidin處理，使HepG2/A2之細胞週期進行停止，DNA複製能力減弱。
在兩種不同人類肝癌細胞 Hep G2/A2，Hep3B/T2經lausenidin長期處理後對於另一肝癌細胞指標Ñ甲型胎兒蛋白(AFP)，在Hep G2/A2 細胞中AFP分泌量增加，但在Hep 3B/T2細胞中分泌量卻減少 。
在藥物篩選中同時發現另一pyranocoumarinÑClausarin，會造成細胞死亡的效果。
經由細胞外型、去氧核糖核酸分析，以及用螢光顯微鏡染色，觀察細胞核，認為細胞之死亡其應是細胞程序性死亡(apoptosis)。
一些pyranocoumarin的結構類似物，對於抑制表面抗原之結構-活性間之關係(SAR)，以及Clausenidin如何影響細胞生長、甲型胎兒蛋白在不同細胞的作用差異，與 Clausarin如何造成細胞死亡，都值得做更進一步的探討。
Hepatitis B virus (HBV) infection is a world-wide public health and epidemiology problem, especially in Asia and Africa. Epidemic studies have clearly shown that the chronic carriers of HBV can cause higher percentage of liver diseases such as hepatocellular carcinoma (HCC) and cirrhosis than normal people. To date, no truly effective drugs against HBV are available, therefore it is important to develop anti-HBV drugs. In these studies we demonstrated that human hepatoma, Hep G2/A2 and Hep 3B/T2 cells, cell line containing HBV genomes and by transfection HBVgenomes respectively, which continually secrete HBsAg into culture medium, can serve as a quick assay system for drug screening for anti- HBV activity. Furthermore the regulatory mechanism of suppression of production of HBsAg and cell growth were also investigated. In this studies from some pyranocoumarins, Clausenidin strongly suppressed the production of HBsAg. Clausenidin suppressed the HBsAg production by both Hep G2/A2 and Hep 3B/T2 cells withEC5o 1.0μg/ml. RT-PCRanalysis reveals that the suppression of HBsAg gene expression by Clausenidin is mainly at the mRNA level. Clausenidin not only suppress the endogeneously expressed HBsAg in the Hep G2/A2 cells but also suppresses the HBsAg, HBeAg producedeither from transfected HBV DNA in another human hepatoma cell line HuH-7 which carry no endogenous HBV genome. Also the promoter activity of SPII were also suppressed using a CAT assay in transfected HuH-7 cells. The effect of suppression of HBsAg production by Clausenidin was abolished in the protein kinase C (PKC) down regulation. It suggests that thesignal pathway of suppression of HBsAg production was not mediated through PKC. In human hepatoma Hep G2/A2 cells, whenClausenidin treatment removed, the suppression of HBsAg production was reversible. However, the cell proliferation after long term treatment of Clausenidin was arrested in Hep G2/A2 cells in contrast to the continuous cell growth on Hep 3B/T2 cells. Flow cytometric analysis of cell cycle showed that cell cycle progression stopped on Clausenidin treated HepG2/A2 cells. Additionally, after long term treatment of Clausenidin in Hep G2/A2 and Hep 3B/T2 cells, the secretion ofa-fetoprotein, another marker of human hepatoma cells, increased on Hep G2/A2 cells, however decreasing amount in Hep 3B/T2 cells. During the process of drug screening, another pyranocoumarm Clausarm caused cell death on both Hep G2/A2 andHep 3B/T2 cells. Clausarin treatment to human hepatoma cells made the cells go through the program of cell death (apoptosis) by observation of morphological changes, DNA fragmentation and nuclear staining pattern. There are many interestingpoints, such as the structure-activity relationships in some pyranocoumarm possessing suppression of HBsAg production,the regulatory action of cell growth and cc-fetoprotein secretion by Clausenidin treatment and how Clausarin caused celldeath, to be worthy of further studies.Abstract:none。