| 摘要: | Part-I
已知肝癌成因於許多相關基因改變的累積，包括致癌基因的活化，或因B型肝炎病毒所造成之c-jun和NF-κB轉錄的活化；亦包括抑癌基因如p53受到抑制或突變。然而，目前除在臨床診斷上還沒有很好的預警標誌，可以用來判斷肝癌發生甚至判斷其嚴重程度外，更無法掌握肝癌轉移之時機；因此，希望透過比較不同分化程度肝癌細胞株之癌症相關基因表現差異，並以肝癌組織相對照，來尋找診斷肝癌轉移之因子，以利肝癌之治療。我們之前的實驗使用RT-PCR/PCR和immunohistochemistry來比較肝癌區與鄰近非肝癌之β-catenin的表現，結果發現肝細胞癌組織的β-catenin量要比肝細胞癌鄰近正常組織來的高，且有統計上的顯著差異；另外在肝癌分期上，β-catenin則多發生於肝癌末期的病人。顯然β-catenin的表現增加也與肝癌的分化不良有正相關，而在分化不良之肝細胞癌中，更發現β-catenin的高度轉位和表現伴隨著其下游IL-8基因的表現上升，說明了β-catenin極可能是造成血管新生、遷移和轉移的主要原因，扮演著metastasis factor的角色。我們因此使用不同分化程度肝癌細胞株（HA22T, PLC, Hep3B, HepG2, and normal chang liver cells分別代表高度增生至分化良好的細胞），以RT-PCR/PCR、 western blot、 MTT、 wound healing、 migration and invasion assays的方式來更進一步驗證我們之前肝癌組織的的結果。如我們除發現肝癌細胞株的結果與肝癌組織一致，並在我們使用β-catenin antisense oligos來阻斷其蛋白質生成時，確實發現細胞遷徙及轉移的能力明顯下降。綜合這些結果似乎也說明了β-catenin確實是扮演一個肝癌細胞metastasis factor的重要角色，將有利肝癌轉移抑制藥物之開發。
Part-II
肝癌在中國南部、東南亞還有南非一直是一個高發生率的疾病，其中也包括了台灣。然而目前對於肝癌的治療成效及早期診斷方式並不近人意，因而從分子生物醫學的角度尋找有利肝癌治療或診斷的機制，是重要且需積極進行的課題。根據性別而論，全世界男女罹患肝癌的比率大約是4：1，這似乎也暗示著，雌性激素及其受體可能再肝臟組織保護及肝癌防護之作用上扮演一定角色。我們實驗室先前實驗也發現E2， ER-α及ER-β而最近文獻亦指出，在肝癌中，時常發現有高頻率的β-catenin突變。已知β-catenin 是穩定細胞adhesion及junction之重要蛋白，free型式的β-catenin則為一個轉錄因子，可以調控細胞基因的表現並誘發oncogenesis 和metastasis。我們的實驗亦發現β-catenin扮演HA22T cell之轉移因子角色。有趣地，最近的報導亦指出在in vivo中ER-α和β-catenin有物理性的交互作用，並在我們之前的研究指出雌性素接受體-α (ER-α)在肝癌患者鄰近非癌區的表現量遠大於癌區而β-catenin則呈相反現象。因此我們採用的ER-α stable clone HA22T（當處理doxycycline時會持續表現ER-α）的肝癌細胞株來觀察ER-α是否具調控β-catenin之作用？結果發現不論在RT-PCR/PCR或西方點墨法實驗中， ER-α過度表現則down-regulated β-catenin 之mRNA之表現以及蛋白質的含量，且隨著時間及doxycycline劑量的增加，β-catenin有更明顯逐漸下降的趨勢，同時有抑制β-catenin核轉位之作用。而當我們處理雌性素或雌性素接受體-α時，亦發現細胞的轉移能力明顯下降。所有證據顯示，ER-α可透過抑制β-catenin基因表現及核轉位能力進而抑制肝細胞癌HA22T細胞的轉移作用。
Part-I
The occurrence of HCC could be resulted from several factors associated with genetic alterations, such as the activation of oncogenes, the enhancement of c-jun and
NF-κB caused by HBV, the inhibition or mutation of antitumor gene, p53. However, in the present, there is no good markers for diagnosis and metastasis which can be used as treatment for HCC. Therefore, analyzing the gene expressions in differently differentiated HCC cell lines, and in HCC tissues become necessary to find the markers for the diagnosis and anti-metastasis of HCC. Our previous studies showed that β-catenin level is significantly higher in tumor areas than in non-tumor areas, and appeared more significant at the late stages of HCC. In addition, the translocation level of β-catenin into nucleus was closely correlated with the development of tumor. Particularly, in the poorly differentiated tumor area, the increases of β-catenin translocation and its downstream, IL-8, expression might play the roles of enhancing neovasculization, migration, and tumor invasion, which promote the HCC metastasis.
In the present studies, we further confirm the findings in human HCC tissues by RT-PCR/PCR, western blot, MTT, wound healing, migration and invasion assays in differently differentiated HCC cell lines (HA22T, PLC, Hep3B, HepG2, and normal chang liver cells). Similarly, β-catenin were found more highly expressed in HA22T the most proliferative HCC cell lines than the other more differentiated cell lines. Furthermore, we apply the β-catenin antisense oligonucleotides to knock down β-catenin gene, resulting in the inhibition of cell migration and invasion activities in HA22T cells. All the findings suggest that β-catenin played the key role of metastasis effects in human HCC.
Part-II
Hepatocellular carcinoma (HCC) is one of the most common cancers in south China, sub-Saharan Africa, and southeastern Asia, including Taiwan. Today, the improvement of HCC diagnosis and the treatment outcome are still needed. Therefore, efforts have been made for finding the metastasis markers and biological mechanism of HCC development to solve these problems. Thus far, the male to female ratio of HCC incidence is around 4 :1 in the world. Therefore, estrogen and estrogen receptors are suggested to play roles to protect the liver tissue. Our previous results also showed ERs induced HCC cell apoptosis and cell cycles arrest. Recently, many published data also showed that β-catenin mutations are observed in human hepatocellular carcinomas. β-catenin is a component of stable cell adherent complexes, whereas its free form functions as a transcription factor that regulates genes involved in oncogenesis and metastasis. Interestingly, the physical association as well as transcriptional and genetic interaction between ER-α and β-catenin in vivo have been reported recently. Moreover, our previous study found the gene expression of ER-α was higher in non-tumor tissue than in tumor tissue, but the data of β-catenin expression showed the contrary results. Therefore, in the present study, we further identify the negative regulation of ER-α and β-catenin each other in the highly proliferative HA22T cells. Firstly, HA22T cells were stablely expressed wt-hER-α induced by the addition of doxycycline to investigate the change of β-catenin gene expression and nuclear translocation. Results showed that the ER-α not only down-regulated β-catenin mRNA and protein levels in a time- and dose-dependent manner, but also inhibit the nuclear translocation of β-catenin, resulting in the anti-metastatic effect in HA22T cells. All these results suggest that ER-α inhibit the metastasis through the down-regulation of β-catenin transactivation and its nuclear translocation in HA22T cells. |
