Pin1在細胞週期中，扮演重要的調控角色。亦有文獻報導指出，Pin1在乳癌形成的過程中，有過度表現的情形且扮演著重要的角色。然而，Pin1與食道癌的關係，特別是國人常罹患的鱗狀細胞癌中，所扮演的角色並不清楚。本研究的目的主要在探討Pin1與食道鱗狀細胞癌之間的關係以及尋找可預防或治療食道鱗狀細胞癌的新藥。在研究中，藉由西方墨點法以及免疫組織染色法，發現超過65%的食道鱗狀細胞癌病人當中，有Pin1蛋白過度表現的情況，且Pin1與其下游的β-catenin and cyclin-D1蛋白的表現，與食道鱗狀細胞癌形成過程中，扮演重要的角色。再者，藉由RT-PCR所得的結果發現，食道鱗狀細胞癌中Pin1蛋白過度表現的結果，主要是因為Pin1 mRNA 量增加所致。重要的是經由統計分析的結果，我們發現Pin1蛋白的過度表現，與食道鱗狀細胞癌病人存活率間有正相關性(p<0.001)。因此，我們認為Pin1可當作食道鱗狀細胞癌預後的指標。此外，也藉由 Pin1 shRNA技術來證明Pin1 suppression可以負向調控β-catenin and cyclin-D1的表現且可以有效的抑制食道鱗狀細胞癌細胞的生長。由以上結果可推論，Pin1 可以當成食道鱗狀細胞癌化學治療的標的。從實驗室建立的小分子chemical library中，我們利用cell proliferation assay，篩選出兼具有抑制Pin-1-β-catenin and cyclin-D1及能抑制細胞分裂的化合物。在化合物篩選過程中，發現其中一個化合物VGHKS-040不但可以有效的抑制食道鱗狀細胞癌細胞的生長(IC50=4.8±0.6μM)，也可以影響食道鱗狀細胞癌細胞中Pin1- β-catenin and cyclin-D1蛋白質的表現量。最後，利用colony formation assay以及在SCID小鼠或裸鼠中所建立的人類腫瘤動物模式證明，不管是在in vitro或 in vivo中，VGHKS-040皆可有效的抑制食道鱗狀細胞癌腫瘤的生長。在我們的研究過程中，成功的篩選出了一個可以抑制Pin1的小分子化合物，並可以應用於發展治療食道鱗狀細胞癌新藥的基礎上。

It has been demonstrated that overexpression of Pin1, a novel cell cycle regulator, in human breast cancer and might play an important role in tumorigenesis. However, the role of Pin1 and whether Pin1 is overexpressed in esophageal squamous cell carcinoma (ESCC) remained unclear. There are two specific aims in this study. The first specific aim is to study the correlation between Pin1 and human ESCC cancer. The second specific aim is to discover drugs that can intervent the Pin1 mediated signaling and provide a novel chemotherapy for human ESCC. In this dissertation, we have demonstrated that Pin1 was overexpressed in more than 65% of clinical esophageal cancer tissues and its level correlated with β-catenin and Cyclin D1 expression by western blotting and immunohistochemistry analyses. The RT-PCR results suggested that Pin1 overexpression maybe due to the up-regulation of Pin1 transcription. By statistic analysis, we have demonstrated that ESCC patients with Pin1 overexpression had significantly poorer survival rate than those non-Pin1 overexpression patients (p< 0.001). Pin1 therefore can be used as a prognosis marker for ESCC. In addition, we have demonstrated that Pin1 suppression by shRNA could downregulate β-catenin and Cyclin D1 expression and inhibit ESCC cell proliferation efficiently. Taken together, these results suggest that Pin1-β-catenin and Cyclin D1 could be used as a potential chemotherapy target for ESCC. In order to discover novel drugs in the chemoprevention or therapy of human ESCC, we screened a chemical library using cell proliferation assay. Among them, compound VGHKS-040 not only effectively inhibited the cell proliferation rate in ESCC cell lines with IC50 value of 4.8±0.6μM but also abated protein levels of Pin1, β-catenin, and cyclin D1. By colony forming assay as well as the Xenograft SCID and nude mice model, we have demonstrated that VGHKS-040 can inhibit the tumor growth in vitro and in vivo. These results suggested that VGHKS-040 provide a new avenue for chemotherapy of esophageal squamous cell carcinoma.

Contents

Abstract
Chinese．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． I
English． ．．．．．．．．．． ．．．．．．．．．．．．．．．．．．．．．．．．．．．．． III
Abbreviations．．．．．． ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．V
Introduction．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 1
Materials and Methods．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 15
Results ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．27
Conclusions and Discussions．．．．．．．．．．．．．．．．．．．．．．．．．．．． 41
Figures ．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 47
References．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 66
Appendix．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．．． 71

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