Enediyne衍生物導致人類血癌細胞(HL-60)之細胞凋亡與G2/M期停滯

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDB, in human promyelocytic leukemia (HL-60) cells

盧育章(Yu-Jhang Lu)

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

(Z)-2-(6-(Thieanisyl-2-yl)hexa-3-en-1,5-diynyl) benzenamine (THDB)為本篇主要化合物，屬於Enediynes系列之化合物。Enediynes是一系列從人工合成與天然物鏈黴菌屬（Streptomyces）分離而得,含有烯雙炔結構的化合物，具多樣化生物活性。在先前的研究中，對於其他癌細胞發現具有細胞毒性。我們發現THDB在不同濃度以及不同時間對於血癌細胞HL-60具有生長抑制的效果。在我們加入THDB後的48小時，觀察到藥物影響HL-60細胞週期，使得細胞停滯在G2/M期。我們進一步的探討蛋白質的變化，發現cyclin B1、 cyclin E、 Cdk1 以及Cdc25C沒有變化，而Cdk2及cyclin A都有減少的趨勢。我們也觀察到THDB也會造成HL-60的凋亡，如細胞的斷裂(藉由觀察DNA片斷和Sub G1體的產生)、和PARP的斷裂；還有caspase家族的caspase-3、caspase-8、caspase-9的活化，我們拿caspase家族的抑制劑來證明THDB主要是經由活化caspase的路徑來導致細胞凋亡。另外我們也觀察到THDB造成p-ERK以及p-Akt增加的趨勢，但是分別加入了ERK以及PI3K的抑制劑卻沒有抑制凋亡以及G2/M停滯的效果。由這些結果得知THDB的確會造成細胞凋亡以及G2/M停滯但非經由ERK以及PI3K路徑。

關鍵字

細胞凋亡 ； 細胞週期停滯 ； 血癌

並列摘要

(Z)-2-(6-(Thieanisyl-2-yl)hexa-3-en-1,5-diynyl)benzenamine (THDB), an enediyne compound, was identified in our laboratory as a novel antineoplastic agent with broad spectrum of antitumor activities against many human cancer cells. THDB was found to inhibit the growth of HL-60 cells in a time- and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest in HL-60 cells following 48 h exposure to THDB. Analysis of the cell cycle regulatory proteins demonstrated that THDB did not change the steady-state levels of cyclin B1, cyclin E, Cdk1 and Cdc25C, but decreased the protein levels of Cdk2 and cyclin A. THDB also caused a marked increase in apoptosis, as characterized by DNA fragmentation (DNA ladder and sub G1 formation), and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Moreover, the THDB-induced apoptosis was significantly attenuated in the presence of specific inhibitors of caspase-3, -8, and -9. In addition, we also observe that THDB has increased the protein levels of p-ERK and p-Akt, whereas adding the ERK and PI3K inhibitors did not inhibit apoptosis and G2/M arrest. These molecular alterations provide an insight into THDB-caused growth inhibition, G2/M arrest and apoptotic death of HL-60 cells.

並列關鍵字

apoptosis ； enediyne ； caspase ； cell cycle arrest

參考文獻

1. Nicolaou, K. C., Dai, W. M., Tsay, S. C., Estevez, V. A., and Wrasidlo, W. (1992) Designed enediynes: a new class of DNA-cleaving molecules with potent and selective anticancer activity. Science 256, 1172-8.

2. Hakimelahi, G. H., Gassanov, G., Hsu, M. H., Hwu, J. R., and Hakimelahi, S. (2002) A novel approach towards studying non-genotoxic enediynes as potential anticancer therapeutics. Bioorg Med Chem 10, 1321-8.

3. Yan, C., Mirnics, Z. K., Portugal, C. F., Liang, Y., Nylander, K. D., Rudzinski, M., Zaccaro, C., Saragovi, H. U., and Schor, N. F. (2005) Cholesterol biosynthesis and the pro-apoptotic effects of the p75 nerve growth factor receptor in PC12 pheochromocytoma cells. Brain Res Mol Brain Res 139, 225-34.

4. Kennedy, D. R., and Beerman, T. A. (2006) The radiomimetic enediyne C-1027 induces unusual DNA damage responses to double-strand breaks. Biochemistry 45, 3747-54.

5. Collins, S. J., Gallo, R. C., and Gallagher, R. E. (1977) Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture. Nature 270, 347-9.