| Abstract: | 人類肝細胞癌是全世界最普遍的惡性腫瘤之一，尤其在亞洲和非洲的比例最高，平均約有百分之八十的癌症病人因癌細胞之轉移而致命，故若能有效抑制癌細胞的轉移及入侵，將可大幅減低癌症的死亡率。因此發展新穎天然藥物來當作癌症預防或是治療藥物是目前最重要的課題。甘草查爾酮A(Licochalcone A)是甘草成分中最具有活性的化合物，且本身也是estrogenic 且屬於黃酮類分子。目前Licochalcone A 被發現的生物功能中包括：抗轉移、抗癌、抗發炎、抗氧化和細胞凋亡等多重功用。但是，目前Licochalcone A 對於人類肝細胞癌的作用機制，至今仍然未知。最近我們的初步結果以MTT 方式測試不同濃度Licochalcone A 處理六株人類肝癌細胞株(HA22T/VGH、SK-Hep-1、Huh-7、PLC/PRF/5、Hep3B 和HepG2)和正常人類肝細胞(Chang liver)分別作用24 和48 小時證實Licochalcone A 不會抑制六株人類肝癌細胞株和正常肝癌細胞的生長。另外，我們利用傷口癒合和細胞移動實驗證實Licochalcone A 會抑制HA22T/VGH 和SK-Hep-1 細胞的侵襲和移動。未來我們將以細胞和動物實驗證實Licochalcone A 抑制細胞侵襲和上皮細胞間質轉化的分子機制。針對這些課題我們將以三年計畫分述各個階段的目標與策略: 第一年目標：以Licochalcone A 處理HA22T/VGH 和SK-Hep-1 細胞分別以wound healing、migration 和invasion assay 證明Licochalcone A 抑制細胞侵襲，進一步利用訊息傳遞路徑的抑制劑和siRNA-MAPK 方式， 分別探討 Licochalcone A 對肝癌細胞的相關訊息傳遞路徑及其蛋白變化。我們採用西方墨點法、免疫螢光染色法和RT-PCR 方法初步證實Licochalcone A 會抑制u-PA的mRNA和蛋白質表現，同時採用Luciferase 方法、ChIP、EMSA 和RNAi 技術釐清Licochalcone A 抑制u-PA 是否透過其它轉錄因子的調控。第二年目標：闡釋TGF-對肝癌上皮細胞(HA22T/VGH 和SK-Hep-1)誘發EMT 現象及其相關的基因表現;了解Licochalcone A 抑制TGF-誘導肝癌上皮細胞 HA22T/VGH 和SK-Hep-1 細胞的E-cadherin、fibronectin、slug 和snail 等與EMT 有關基因的表現? 釐清Licochalcone A 抑制TGF-啟動EMT 之可能途徑及其訊息傳遞，分別以抑制劑或拮抗劑或RNA interference 方式逐一探討:(1) 以EMSA 和Luciferase 方式了解是何種轉錄因子結合；是否透過轉錄因子調控E-cadherin promoter 的專一性區位：(2) Licochalcone A 是否透過抑制其他途徑如MAPK,等調控其下游轉錄因子抑制E-cadherin，增加vimentin 表現。第三年目標：建立動物模式釐清Licochalcone A 對於腫瘤侵襲能力，我們也利用不同訊息傳遞的生物晶片來尋找Licochalcone A 抑制侵襲的相關基因。如果找到特定基因，利用RNAi 和過度表現的方法來證實Licochalcone A 影響特定基因的功能。最後，利用於細胞及動物模式中使用一些臨床抗癌藥物、抑制劑或化療方式搭配Licochalcone A 藉以減緩肝癌侵襲能力。綜合以上的三年的研究將證實Licochalcone A 在肝癌方面當作抗侵襲藥物的發展上深具潛力。
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and especially prevalent in parts of Asia and Africa. The metastasis (invasion) of cancer cells leads 80% patients died, and inhibition of cancer cells metastasis will markly decrease the death rate. Therefore, development of novel nature drug or drug therapy was important topic. Licochalcone A is the main active compound of the licorice species Glycyrrhiza inflate, and is an estrogenic flavonoid with anti-metastatic, anti-cacinogenic, anti-inflammatory, anti-angiogenic, anti-oxidant and apoptosis effects. However, Licochalcone A effect molecular mechanism of human hepatocellular carcinoma is presently unknown. Recently, we have obtained preliminary data from MTT assay studies on test the different concentration of Licochalcone A to treatment with six HCC cells (HA22T/VGH, SK-Hep-1, Huh-7, PLC/PRF/5, Hep3B and HepG2) and normal liver cells (Chang liver) for 24 or 48 hrs. We found that the growth inhibitions of Licochalcone A on these HCC cells were not affect the cell growth by MTT assay.We found that the Licochalcone A inhibited the migrate and invasive on HA22T/VGH and SK-Hep-1 cells were observed by wound healing assay, invasion assay and migration assay after treatment for 24 h. Further we will investigation the molecular mechanism of Licochalcone A inhibition of invasion on human HCC cells in vitro and in vivo. Therefore, three years grant proposal aims to study whether treatment of Licochalcone A would inhibit cell invasion via mechanism(s) involved in vitro and in vivo models. In the first year study: we will focus on studying whether Licochalcone A inhibited cell migration and invasion in HA22T/VGH and SK-Hep-1 cells, and investigation the signal pathways for Licochalcone A affect human HCC cancer cell by MAPK inhibitor or siRNA-MAPK assay. In addition, our preliminary data from western blotting, immunofluorescence assay and RT-PCR assay suggest that Licochalcone A inhibited the mRNA and protein levels of u-PA in dose dependent. Next, therefore we will focus on studying whether Licochalcone A inhibit transcription activity of u-PA, and what kinds of transcription factor to regulate u-PA transcription activity. Using Luciferase assay, ChIP, EMSA assay, and RNA interference techniques to clarify which mechanisms of Licochalcone A effect u-PA in association with transcriptional activators or transcription repressors in vitro. In the second year study: We utilized real-time PCR, western blot and confocal microscope to confirm that TGF- induce epithelial-to-mesenchymal transition phenotype and related gene expression and to determine Licochalcone A whether inhibit THG-induced EMT gene (E-cadherin, fibronectin,slug and snail) in HA22T/VGH and SK-Hep-1 cells？To confirm that Licochalcone A inhibit TGF-induced EMT pathways and other signaling pathway？To evaluate some specific inhibitors, blockers and RNA interference to alleviate TGF--induced EMT pathway in human HCC cells. (1) Using EMSA and Luciferase assay to confirm what kinds of transcription factor binding, and whether through transcription factor to regulate of specific regions of E-cadherin promoter: (2) Licochalcone A whether inhibit other signaling pathways, such as MAPK, AKT, et al to suppress of E-cadherin and increase of vimentin expression. In the third year study: we will establish animal model to confirm that effect of Licochalcone A on growth and invasion of HCC cancer cell inoculated mice. In addition, we found that Licochalcone A inhibit cell invasion related target gene by different signaling cDNA microarray. If any candidate genes were identified, RNAi and gene overexpression will be applied on these genes for studying the involvement of these genes on Licochalcone A effects. Finally, to evaluate some clinical anti-cancer drugs, inhibitor or IR was combined Licochalcone A to alleviate invasion in human HCC cells. These study supports that Licochalcone A may have potential as anti-cancer and anti-invasion agents in human hepatocellular carcinoma cells. |
