在先前的研究中，我們已經證實在肝癌細胞中高表現的p27Kip1 以及低表現的CKS1B 蛋白質表現量是兩個獨立的預後指標 。然而，過度表現的CKS1B 蛋白質在臨床上會使肝癌細胞的侵犯性複雜化，但不會影響p27Kip1 的蛋白質表現量。因此在本研究中我們探討一個具有毒殺性的化合物，goniothalamin (GTN)，處理肝癌衍生的細胞中後如何去穩定p27Kip1 的蛋白質表現量和調降CKS1B的mRNA和蛋白質表現量。首先，我們分別在TP53-negative 的Hep-3B細胞中以及TP53-positive 的SK-Hep1 細胞中處理GTN 48 小時候測得兩株細胞的IC50 分別為25 和10 μM。在兩株細胞中處理GTN 後，皆會造成DNA 損害、γH2AFX foci 形成並且提升γH2AFX 蛋白質表現量，這些結果都會隨著GTN 的劑量增加而增加。除此之外，GTN 分別造成Hep-3B 細胞的細胞週期停滯在G2/M 期但在SK-Hep1 細胞則是停留在G1/S 期。許多調節細胞週期的分子在處理GTN後包括Hep-3B 細胞中的p27Kip1 以及SK-Hep1 細胞中的p21Cip1都有顯著性的上升。在Hep-3B 細胞中，GTN 會引起p27Kip1/CCND1，p27Kip/CDK2，
p27Kip1/CCNB1，p27Kip1/CDK1 的相互作用，但會抑制p27Kip1/STMN1 的互相作用。反觀，在SK-Hep1 細胞中，GTN 會增加p21Cip1/CCND1 和p21Cip1/CDK2 互相作用但抑制p21Cip1/SKP2以及p21Cip1/CKS1B 的複合物產生。GTN 調控CKS1B 轉錄的層面進而促進CKS1B 蛋白質下降。然而在Hep-3B 細胞中，GTN 會增加p27Kip1 蛋白質的穩定性，尤其是在細胞質中的p27Kip1蛋白質。GTN 和MG132，proteasome 抑制劑，皆會造成p27Kip1 的ubqutination 而且ubqutination的p27Kip1 是因為添加物而增加，而不是受到CKS1B 的調控，我們推論在Hep-3B 細胞中GTN藉由另一個ubquitin-complex 調控p27Kip1 蛋白質。根據即時定量反轉錄聚合酶連鎖反應，在SK-Hep1 細胞中GTN 會調控p21Cip1 的mRNA 上升，更進一步，利用專一的引子以及定量染色質免疫沉澱技術偵測p21Cip1 的啟動子上有兩個專一區域。綜合以上結果，在TP53-negative的Hep-3B 細胞中，GTN 是經由調控p27Kip1 造成細胞週期停滯然而在TP53-positive 的SK-Hep1 細胞會降低CKS1B 的mRNA 含量。CKS1B 的轉錄受到GTN 抑制進而造成細胞週期停滯，但在Hep-3B 細胞中並不會影響p27Kip1 的表現量。

Our previous study identified both high p27kip1 and low CKS1B protein levels were independent prognosis markers in hepatocelular carcinomas (HCC), however, CKS1B overexpression implicated clinical aggressiveness of HCC but not p27Kip1 protein turnover. In this study, we demonstrated a cytotoxic compound, goniothalamin (GTN), that stabilized p27kip1 protein and downregulated CKS1B mRNA as well as protein levels in HCC-derived cell lines. The IC50 of TP53-negative Hep-3B and TP53-positive SK-Hep1 after treatment with GTN for 48 h were detected as 25 and 10 μM, respectively. The GTN induced DNA damages, formation of γH2AFX foci, and upregulation of γH2AFX protein levels in dose-dependent manners in both cell lines. In addition, GTN arrested Hep-3B and SK-Hep1 cells in G2/M and G1 phases, respectively. Protein levels of several cell cycle regulators, including p27Kip1 in Hep-3B and, p21Cip1 and CKS1B in SK-Hep1 cells have been noticeable upregulated after treatment with GTN. In Hep-3B cells, GTN induced the configuration of p27Kip1/CCND1, p27Kip1/CDK2; p27Kip1/CCNB1 and p27Kip1/CDK1, however, repressed the p27Kip1/STMN1 complexes. On the other hand, GTN evidently induced the configuration of p21Cip1/CDK2 and p21Cip1/CCNE1, but repressed the p21Cip1/SKP2 and p21Cip1/CKS1B complexes, in SK-Hep1 cells. The CKS1B protein abundance that was induced by GTN was stemmed from the CKS1B transactivity. However, GTN-induced p27Kip protein profusion in Hep-3B cell line was, instead, due to the stabilization of p27Kip1 protein, mainly in the cytosol. Both GTN and the proteasome inhibitor, MG132, induced p27Kip1 ubiquitination and p27Kip1 protein profusion with an additive effect, regardless of CKS1B protein level, suggesting that the p27Kip1 protein might be regulated by GTN through an alternative ubiquitin-complex in Hep-3B cells. In addition to the quantitative reverse transcription -polymerase chain reaction, GTN-induced p21Cip1 protein level in SK-Hep1 cells could be traced back to mRNA level, by evidence of quantitative immunoprecipitation/chromatin immunoprecipitation assay with primer sets specific to two regions of the p21Cip1 proximal promoter. Taken together, GTN is able to induce the cell cycle arrest via stabilization of p27Kip1 protein and downregulation of CSK1B mRNA and subsequent protein levels in TP53-negative Hep-3B and TP53-positive SK-Hep1 cells, respectively. The GTN-repressed CKS1B transcription and thereafter, cell cycle arrest, was irrelevant to the p27Kip1 protein stability in Hep-3B cells.

摘要………………………………………………………………… I
Abstract………………………………………………………… III
Introduction………………………………………………………1
Materials and Methods………………………………………………….11
Results…………………………………………………………20
Figures………………………………………………………….26
Summary……………………………………………………….45
Discussions………………………………………………… ..46
References…………………………………………………… 49
Abbreviations……………………………………………………V

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