THDA誘使人類血癌細胞K562產生細胞週期停滯與細胞凋亡機制之探討

The Mechanism of THDA induced cell cycle arrest and apoptosis in chronic myeloid leukemia K562 cells

吳政哲(Zchong-Zcho Wu)

指導教授 ： 林信仁

高雄醫學大學/生命科學院/醫藥暨應用化學研究所/碩士(2007年)

摘要

2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline (THDA) 為一個最近被開發的抗癌化合物，屬於Enediynes的衍生物之ㄧ，對人類許多腫瘤細胞具有毒殺作用。在此研究中以THDA處理K562 細胞，並探討其細胞增殖、細胞週期的發展，以及細胞凋亡的進一步歸納。
THDA 實驗發現可以time- and dose-dependent 方式抑制K562 細胞的增生。在細胞週期部份，經由THDA處理過的K562細胞，得知其會被抑制在G2/M phase，並且發現在處理24小時後會有細胞凋亡的產生。
在G2/M 抑制期間, CDKIs中的 p21 和p27 在time dependent有漸增的趨勢。在細胞週期調節蛋白質的分析指出，以THDA處理過後，cyclin B1, cyclin D3 和Cdc25C等蛋白並無改變 , 但Cdk1 , Cdk2 和cyclin A卻有減少的改變。另外在MAPK方面，包括JNK、ERK，甚至是JNK途徑上的c-Jun均有活化，以此THDA導致活化的ERK與JNK，分別加入PD98059 (ERK抑制劑)及JNK-1(JNK抑制劑)後確實能有效降低細胞凋亡，caspase-3,-9、Bax、XIAP 與PARP[poly(ADP-ribose) polymerase]的表現，因此，新型炔類衍生物的THDA能導致K562細胞走向細胞凋亡。藉由THDA誘導JNK與ERK途徑上活化表現增加，反而影響Bax/Bcl-2比率的增加，XIAP減少，caspase-3,-9的活化，以及PARP的斷裂，都顯示著這些均為細胞死亡的標定。我們的結果為首次探討出THDA和其他enediynes衍生物誘導K562細胞產生細胞凋亡的新型機制。

關鍵字

細胞凋亡 ； 細胞週期停滯 ； K562細胞

並列摘要

2-(6-(2-thieanisyl)-3(Z)-hexen-1, 5-diynyl)aniline( THDA ), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A.. THDA-induced apoptosis was associated with upregulation of Bax, downregulation of XIAP as well as activation of caspase-3 -9 and proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, the MAP family kinases, including JNK and ERK kinases, and the transcription factor c-Jun were all activated by phosphorylation exposure to THDA. Phosphorylation of JNK and ERK kinases by THDA was blocked by an ERK inhibitor, PD98059, or a JNK inhibitor, JNK-1, respectively, suggesting that THDA-induced apoptosis in K562 cells is ERK- and JNK-dependent. Moreover, blockage of ERK and JNK also attenuated the modulation of Bax and XIAP, as well as the activation of caspase-3 and caspase-9 induced by THDA. These findings suggest that the activation of JNK and ERK is involved in the THDA-induced apoptosis of K562 cells. Furthermore, our results, for the first time, highlight a novel mechanism by which THDA and possibly other enediynes induced apoptosis in K562 cells.

並列關鍵字

apoptosis ； enediyne ； caspase ； K562 cells ； cell cycle arrest

參考文獻

Adams JM, Cory S. 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281:1322-6

Anderson HJ, Coleman JE, Andersen RJ, Roberge M. 1997. Cytotoxic peptides hemiasterlin, hemiasterlin A and hemiasterlin B induce mitotic arrest and abnormal spindle formation. Cancer Chemother Pharmacol 39: 223-6

Antonsson B, Martinou JC. 2000. The Bcl-2 protein family. Exp Cell Res 256:50-7

Bacus SS, Gudkov AV, Lowe M, Lyass L, Yung Y, et al. 2001. Taxol-induced apoptosis depends on MAP kinase pathways (ERK and p38) and is independent of p53. Oncogene 20: 147-55

Barkett M, Gilmore TD. 1999. Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18: 6910-24