Phytochemicals促進trichostatin A抑制A549細胞生長之效果

The enhanced effect of phytochemicals on the growth inhibition of trichostatin A in A549 cells

陳開湧(Kai-Yong Chen)

指導教授 ： 葉姝蘭

中山醫學大學/健康管理學院/營養學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00096

摘要

Trichostatin A (TSA)是一個與組蛋白乙醯化與去乙醯化修飾有關的藥物，研究已證實在許多癌細胞中，TSA會造成癌細胞生長停滯與細胞凋亡，但藥物本身可能造成的副作用，也限制了它在癌症治療上的使用。本研究利用人類肺癌細胞株A549 細胞，篩選可能增加TSA對細胞生長抑制的phytochemicals或營養素，包括10 μM的genistein（G）、daidzein（D）、β-carotene（BC）、retinoic acid以及α-tocopherol，這些都是對癌症可能具有化學預防效果的物質。如預期的，TSA在誘發細胞生長停滯與死亡上具有劑量效應。由於TSA在100 ng/mL顯著導致細胞死亡，我們將選擇較低劑量TSA測試它與受試物質的合併效果。當TSA在與各種phytochemicals共同培養72小時後，G、D及BC均可顯著增加50 ng/mL TSA（TSA50）對細胞生長上的抑制作用，其作用大小依序為G≒BC > D，而在缺乏TSA下，並不會對細胞生長有任何影響。至於 retinoic acid以及α-tocopherol合併則對TSA的生長抑制作用沒有影響。進一步以流式細胞儀分析細胞凋亡情形，結果顯示在TSA50合併G或BC處理下，確能增加A549細胞的凋亡，至於D僅有輕微但不顯著的增加TSA所誘發的細胞凋亡。培養24小時，G、BC及D均顯著的增加TSA誘發的histone H3蛋白乙醯化作用。另外，對於會調控pro-apoptosis相關基因表現的p53蛋白，合併G與BC培養12小時即顯著增加p53表現。在caspase-3活性上，只有G合併TSA50在24 小時顯著較TSA單獨時高。這些結果顯示G，BC及 D增強TSA抑制細胞生長的機制可能與調控histone H3乙醯化、p53表現及caspase-3活性有關，但它們的機制應不盡相同。不過G，BC及 D增強TSA抑制細胞生長確實的機制及在活體內的重要性仍須進一步研究。

關鍵字

Trichostatin A ； genistein ； β-胡蘿蔔素 ； daidzein ； 細胞程式凋亡 ； acetyl-histone H3 ； p53 ； csapase-3

並列摘要

Trichostatin A (TSA), a compound interferes with the acetylation/deactylation pattern of histones, is known to cause growth arrest and apoptosis induction of various cancer cells. However, the side-effects may limit its use in anticancer chemotherapy. In the present study, employing a human lung carcinoma cell line, A549 cells, we first examined whether 10μM of genistein,daidzein, β-carotene, retinoic acid or α-tocopherol, which are phytochemicals or nutrients with potential chemopreventive effect, enhance the suppressed effect of TSA on cell growth. As expected, TSA induced A549 cells growth arrest or death in a dose-dependent manner. Since TSA at 100 ng/mL strongly induce cell death, we used lower doses of TSA to examine the combinative effect of TSA with test compounds. Incubated with A549 cells for 72 h, genistein, daidzein, and β-carotene significantly enhanced the growth-suppressed effect of 50 ng/mL TSA (TSA50) in an order G≒ BC > D, while themselves did not affect cell growth. Retinoic acid and α-tocopherol had no effect. Furthermore, flow cytometric analysis revealed that G and BC significantly increased the apoptosis of A549 cells induced by TSA50. D also slightly increased TSA-induced apoptosis, but the effect was not significant. All G, BC and D significantly increased TSA50-induced acetyl-histone H3 expression as incubated with A549 cells for 24 h. In addition, G and BC significantly increased the expression of p53, a transcriptional controller of many pro-apoptotic genes, than TSA alone at 12 h. However, only G+TSA50 significantly increased the activity of caspase-3 at 24 h as compared with TSA50 alone. These results suggest that the mechanisms underling the enhancing effects of G , BC, and D on the cell growth-inhibiting effect of TSA may be associated with the modulation of histone H3 acetylation, the expression of p53, and the activity of caspase-3, however, they should be not completely the same. Further studies are warranted to investigate the precise mechanisms and the significance of these findings in vivo.

並列關鍵字

Trichostatin A ； genistein ； β-carotene ； daidzein ； apoptosis ； acetyl-histone H3 ； p53 ； csapase-3

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