ABT-751是一種新型的阻斷有絲分裂藥物，在先前的臨床前研究中發現它對癌細胞具有毒殺作用，並且能誘發細胞凋亡。本研究擬探討ABT-751對於肝癌細胞株Hep-3B的影響，進而探討ABT-751對Hep-3B的凋亡與自噬之相互關係。首先，我們藉由MTT分析方法得知ABT-751處理Hep-3B細胞在48小時的IC50為0.5 M。而在ABT-751的處理下可以引發肝癌細胞的DNA斷裂、干擾微管的聚合、STMN1表現量下降，並且透過ATM-CHK1-CDC25C訊息傳導路徑導致細胞週期G2/M期停滯。接下來，我們想進一步瞭解ABT-751如何誘導肝癌細胞引發凋亡，以及ABT-751造成的環境壓力是否會影響細胞自噬作用，並且探討細胞凋亡與自噬之間的相關性。由於細胞凋亡與自噬之間的關係是非常複雜的，自噬作用能夠使細胞適應壓力環境以躲避細胞凋亡的機制，另外，自噬作用也可以被認為是一種細胞死亡的途徑。然而，我們卻發現ABT-751處理Hep-3B細胞抑制了其細胞自噬作用。在細胞凋亡的部分，我們觀察到ABT-751處理Hep-3B細胞造成磷脂酰絲氨酸外翻，PARP1裂解但是主要的caspases卻沒有活化，所以我們進一步利用ABT-751與caspase抑制劑(z-VAD-fmk)單獨或共同處理Hep-3B細胞後，結果顯示細胞死亡的現象並沒有因此減少，由此可知細胞凋亡可能是經由caspase-independent pathway。我們更進一步實驗，發現apoptosis-induced factor (AIF)在核內的表現量增加，此現象也再度證實在Hep-3B細胞中，ABT-751所誘導的細胞凋亡主要是透過caspase-independent pathway。總而言之，ABT-751抑制Hep-3B細胞的自噬作用，但會透過caspase-independent pathway誘導細胞凋亡。

The study was to evaluate the effect of ABT-751 on apoptosis and autophagy in Hep-3B cells, a human hepatocellular carcinoma (HCC)-derived cell line. ABT-751 is a novel anti-mitotic agent that exerts cytotoxic effects in preclinical studies. Although ABT-751 induces apoptosis in cancer cells, whether this drug affects autophagy, is unclear. Notably, the functional relationship between apoptosis and autophagy is complex under certain circumstances; autophagy constitutes a stress adaptation that avoids cell death, whereas in other cellular setting, it constitutes an alternation of cell-death pathway. In the present study, we elucidated that ABT-751 reduced the Hep-3B cell viability considerably with an IC50 of 0.50.0 uM, disrupted the polymerization of tubulin into microtubules, caused DNA fragmentation and arrested cell cycle at G2/M phase, through ATM-CHK1-CDC25C checkpoint pathway. However, it is seen to inhibit autophagy after ABT-751 treatments in Hep-3B cells. Phosphatidylserine (PS) externalization on the cell membrane and PARP1 cleavage but no activation of caspases occurred, revealing that apoptotic cell death had begun through caspase-independent pathway, which proved by pan-caspase inhibitor z-VAD-fmk treatments. We observed the nuclear translocation of apoptosis-inducing factor (AIF) in ABT-751-induced apoptosis of Hep-3B cells, which may be responsible for the caspase-independent pathway. Taken together, those results indicated ABT-751 inhibit autophagy, but activated apoptosis by caspase-independent pathway.

Approval page i
Acknowledgements ii
Abstract iii
Chinese iii
English iv
List of figures vi
List of tables vii
Introduction 1
Materials and Methods 10
Results 19
Figures 25
Discussion 35
References 39

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