人類癌症基因體以驗證探討抗癌中草藥-子計畫六、中草藥調節抑癌基因對抗肝癌機制的探討

呂紹俊
國立台灣大學
肝癌是世界上最普遍的癌症，常發生在慢性病毒性肝炎、黃麴毒素暴露或是過量飲酒，一旦發展成肝癌預後都不是很好。癌症的形成往往是因細胞正常生長的調節機制受損；常因不正常的訊息傳遞、導致細胞不正常的增生或無法以程式性的細胞凋亡清除異常的細胞。林榮耀院士於卓越計畫研究肝癌組織基因表現的結果顯示在肝癌組織中有不少基因有異常表現的情形。其中不少基因是曾被報導與癌症發生有關，這些基因可分類為致癌基因、生長因子及其接受器之基因、血管新生相關基因、細胞定期凋亡基因、癌細胞轉移及浸潤相關之基因及控制細胞週期之基因等。於後基因時代，為提升基因體中醫藥研究，本計畫將對我國高罹患率之肝癌進行「證據基礎」(Evidenced Based) 之探討，由林院士在國人肝癌組織中所找到的異常基因且分佈於六大類與肝癌相關基因之抑癌基因(tumor suppressor genes)為標的，分析供應可能具有抗癌保肝相關作用之中藥、草藥或方劑共61種之萃取液尋找能提升抑癌基因(phosphatase and tensin homologous on chromosome 10 gene (PTEN)、Kruppel-like factor 6 (KLF6)及deleted in liver cancer-1(DLC-1))或影響細胞週期(CDK2, CCND1, CCND3, CCNE, CDC2, CDK4, PLK1, p73, DZTS1, HIC1, RIZ等)及細胞凋亡(BCL2, BAX, STAT1, CAP1, ANXA2等)等相關基因的藥材或成分，並以動物實驗證實該中草藥萃取液抗癌作用之活性。 此計畫將分一年五個月進行，各年將執行之分析分述如下：第一年度擬以特定轉錄因子調控之報導質體分析 (reporter assay)如AP1-Luciferase、NF-αB-Luciferase等，篩選中草藥之萃取液對肝癌細胞內細胞凋亡基因、細胞週期基因、抑癌基因、訊息傳遞基因、細胞黏著基因及其他的影響，尋找具有抑癌功能的中草藥表現的影響，進一步用反轉錄-即時定量聚合酵素鏈鎖反應(reverse transcription-quantitative real-time polymerase chain reaction; RT-qPCR)定量基因表現的變化並以Western blot analysis驗證。第二年度於細胞層次以肝癌細胞株分析第一年篩選出有效果的中草藥萃取液對癌細胞的影響，如細胞群落形成 (colony formation analysis)、細胞移動(migration)，細胞增生，凋亡及細胞週期等的影響，由細胞層次驗證中草藥萃取液的抗癌功能，並計畫以含腫瘤實驗動物，以「證據基礎」證實中草藥之抗癌功能。對有效之中草藥萃取液更進一步用TLC、HPLC及LCM/MS等分析其有效成份，以供新藥的開發。
關鍵字：中草藥；肝癌基因；抑癌基因

Evidenced-base studies on anticancer of Chinese herbs by genomic medicine-subproject 6 -Studies on the anticancer activity of Chinese herbs:regulation of tumor suppressor-related genes

Shao-Chun Lu
National Taiwan University
Hepatocellular carcinoma (HCC) is one of the most common cancers in the world and in Taiwan as well. It usually develops in patients with hepatitis virus infection, aflatoxin exposure and/or excessive alcohol use. Most patients with HCC present with advanced disease and have a poor prognosis. Like most cancers, HCC is develpoded due to dysregulation of genes that control of cell growth. According to the results of full-length cDNA sequencing of HCC cDNA liberaries, done in Dr. JY Lin’s laboratory, genes dysregulated in HCC can be divided into 7 categories: (a) oncogenes, (b) tumor suppressor genes, and genes related to (c) growth factors and their receptors, (d) angiogenesis, (e) apoptosis, (f) metastasis and (g) cell cycles. The aims of this study are to identify Chinese herbs that can be used treatment of chronic hepatitis or prevention of HCC by evidence-based methods. In this sub-grated program, in the first and second year, over 61 herbs and Chinese medicines will be screened for that can inhibit activation of AP1 and NF-αB by Luciferase reporter assay systems. The results will be further evaluated by qRT-PCR and Western blot analyses of the endogenous genes; furthermore, their effects on cancer cell will be investigated by colony forming, cell migration, cell proliferation and apoptosis methods. In the third year, the anticancer activity of the selected herbs will be further evaluated with nude mice bearing tumor cells.
關鍵字：Hepatocellular carcinoma (HCC)；Chinese herbs；tumor suppressor genes