Goniothalamin (GTN) 和 styryl-lactones 衍生物小分子被發現對癌細胞具有毒殺性，且會使許多癌細胞，包含乳癌細胞 MCF-7、子宮頸癌細胞 HeLa 和血癌細胞HL-60 趨向細胞凋亡。在血癌細胞 HL-60 中已被證實 GTN 會誘發粒線體受損、caspase 活化甚至細胞凋亡。
在本實驗中證實 GTN 會誘導肝癌細胞株 (SK-Hep1 和 Hep-3B) 中的phorbol-12-myristate-13-induced 1 (PMAIP1) 蛋白質無論是否透過 TP53 的活化表現量均會
增加，使得粒線體膜電位改變，進而導致細胞凋亡。GTN 會誘導 DNA 受損、reactive oxygen species (ROS) 生成以及 CASP8、CASP9、CASP3 和 poly (ADP-ribose) polymerase 1 的活化斷裂，然而沒有發現 BH3 interacting domain death (BID) 蛋白質有斷裂的現象，所以在 GTN的處理之下應是透過內部訊息傳遞途徑。利用 caspase 的抑制劑 (z-VAD-FMK) 會抑制 GTN
所誘導的細胞凋亡，藉此證明 GTN 誘導的細胞凋亡是受活化的一系列 caspase 所調控。在SK-Hep1 細胞中 ROS 的生成引發 DNA 受損，並活化gamma H2A histone family member X(γH2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2
checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15)，但在 Hep-3B中則是相反的，而且只活化 γH2AFX/pCHEK2 (Thr68)。在五種鑲嵌在粒線體外膜的蛋白質(無論是阻止或導致細胞凋亡) 中，兩種細胞株在處理 GTN 4 到6 小時之後，PMAIP1 的蛋白質以及 mRNA 表現量均增加；而轉染 shPMAIP1 不只降低 PMAIP1 蛋白質和 mRNA的表現量，也會減少 GTN 誘導的細胞凋亡比例。同樣的在 SK-Hep1 細胞中，轉染 shTP53會降低 TP53 和 PMAIP1 的 mRNA，以及TP53、pTP53、PMAIP1 和 cleaved PARP1 蛋白質表現量，也會減少 GTN 誘導的細胞凋亡比例。但是處理 TP53 抑制劑 (PFT-α) 或是轉染dominant negative TP53 plasmid (pTP53mt135)，雖然會影響 TP53、pTP53 和 PMAIP1 的
mRNA 和蛋白質表現量，然而卻明顯增加GTN 誘導的細胞凋亡比例。粒線體分離的實驗中，可知 SK-Hep1 細胞在處理 GTN 不同時間點之後，TP53 會轉換位置到粒線體上。總結，我們的實驗證明無論是TP53-dependent 或 -independent transactivation pathways，GTN 均會導
致 PMAIP1 表現量增加進而造成細胞凋亡。其中在有TP53 的 SK-Hep1 細胞中，PMAIP1和 TP53 有共伴效用的結果。

The goniothalamin (GTN) and related styryl-lactones were found to be cytotoxic and apoptotic
to a variety of tumor cell lines including breast cancer MCF-7, cervical cancer HeLa and leukemia
HL60. In HL60 cells, GTN triggers mitochondria dysfunction, caspase activation and eventually,
apoptosis. In this study, we demonstrated that GTN was able to induce apoptosis in hepatocellular
carcinoma derived cells, SK-Hep1 and Hep-3B cells via upregulation of the phorbol-12-myristate-13-induced 1 (PMAIP1) protein , alternation of mitochondrial membrane potentials (P < 0.05) via TP53-dependent and -independent transactivations. Treatment with GTN induced DNA damage, formation of reactive oxygen species (ROS, P < 0.05) and activated cleaved CASP8, CASP9, CASP3 and poly (ADP-ribose) polymerase 1 proteins. However, cleaved BH3 interacting domain (BID) death agonist protein was not identified, suggesting that an intrinsic cellular stress was produced after GTN treatments in both cell lines. A pan caspase inhibitor, z-VAD-FMK, suppressed GTN-induced apoptotic cell percentages (P < 0.05), demonstrating that GTN-induced apoptosis was mediated by the activation of the caspase cascade protein. The GTN induced ROS formation prior to DNA damage in SK-Hep1, yet in reverse order in Hep-3B cells.
Moreover, GTN induced DNA damages through activation of the gamma H2A histone family
member X (γH2AFX, Ser139)/phospho-CHK1 checkpoint homolog (pCHEK1, Ser345)/phospho-CHK2 checkpoint homolog (pCHEK2, Thr68)/phosphos-tumor protein p53 (pTP53, Ser15), and γH2AFX/pCHEK2 (Thr68), in SK-Hep1 and Hep-3B cells,respectively.
Among five integral outer mitochondrial membrane proteins that blocks or induces apoptotic death,
PMAIP1 protein and PMAIP1 mRNA levels were upregulated after GTN treatments for 4 to 6 h in
both cell lines (P < 0.05). Transfection of shRNA interference plasmids targeting PMAIP1 gene
downregulated PMAIP1 mRNA (P < 0.05) and PMAIP1 protein (P < 0.05) levels, as well as GTN-induced apoptotic cell percentages (P < 0.05) in both cell lines. In parallel, transfection of the
shRNA interference plasmid targeting TP53 gene in SK-Hep1 cells, downregulated TP53 and PMAIP1 mRNA (P < 0.05) and TP53, pTP53, PMAIP1, cleaved PARP1 protein levels and apoptotic cell percentages (P < 0.05). Treatment with the TP53 inhibitor, PFT-α or transfection of a dominant negative TP53 plasmid, pTP53mt135, repressed TP53, pTP53 and PMAIP1 protein and/or TP53, PMAIP1 mRNA levels (P < 0.05), however, significantly augmented GTN-induced apoptotic cell percentages (P < 0.05). Cytosol/mitochondria fractionation identified that TP53, along with PMAIP1 proteins, were translocated to mitochondria after GTN treatment in time-dependent manners. Taken together, our studies demonstrated that GTN induced apoptosis via PMAIP1 via both TP53-dependent and -independent transactivation pathways. In TP53-positive SK-Hep1 cells, PMAIP1 and TP53 proteins conducted synergic effects.

Contents
Abstract ……………………………………. Ⅱ
Chinese …………………………………… Ⅱ
English ……………………………………. Ⅲ
Abbreviations …………………………….. Ⅴ
Literature reviews ……………………….. 1
Introduction ………………………………. 6
Materials and Methods …………………. 10
Results …………………………………… 22
Figures …………………………………… 28
Discussion ………………………………. 44
Supplementary Data ……………………. 47
References ………………………………. 49
Appendix ………………………………….. 53

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