Genistein藉由增加乙醯轉移酶活性強化trichostatin A抑制肺癌細胞生長之效果

Genistein enhances the inhibitory effect of trichostatin A on lung cancer cell growth by upregulation acetyltransferase activation.

黃珮茹(Pei-Ru Huang)

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(2012年)

https://doi.org/10.6834/CSMU.2012.00072

摘要

我們先前的研究顯示genistein (10uM)會增加Trichostatin A ( TSA，一種組蛋白去乙醯酶抑制劑，被視為新的抗癌藥物)所誘發的人類肺腫瘤A549細胞組蛋白乙醯化及細胞凋亡，但對正常的人類肺纖維母細胞則無影響。不過此一合併處理造成組蛋白乙醯化的機制及對其他肺癌細胞是否也有相同的效果，目前並不清楚。因此本研究首先選用三株人類肺癌細胞株A549、NCI-H460(對TSA敏感)及ABC-1(對TSA抗性較大)來比較genistein促進TSA抗腫瘤性的效果，以及影響genistein效果的可能機制。結果發現，Genistein促進TSA誘發的肺癌細胞生長停滯及凋亡的效果大小為：A549，NCI-H460＞ABC-1。在A549及NCI-H460細胞中，Genistein可促進TSA (50 ng/ml)誘發的細胞凋亡及caspase-3活化。然而，TSA(濃度達200 ng/ml)單獨或合併genistein仍無法誘發ABC-1細胞凋亡及caspase-3表現。Genistein在三株細胞中，均使細胞中TSA誘發的組蛋白(H3/H4)乙醯化程度增加，而組蛋白乙醯轉移酶抑制劑-anacardic acid則可抑制genistein的增加效應，Anacardic acid同時抑制genistein促進TSA誘發A549及NCI-H460細胞凋亡的效果。在NCI-H460細胞中，Genistein單獨或合併TSA增加乙醯轉移酶-p300的mRNA表現，但在A549細胞中genistein的效果沒有顯著上升。在A549及NCI-H460細胞中p53基因為wild type，而ABC-1則為mutant，因此我們推測p53的表現可能是造成TSA單獨或合併genistein對三株肺癌細胞有不同效果的原因。我們檢測p53蛋白的表現量，發現在A549細胞中genistein單獨或合併TSA會增加p53的表現量，而NCI-H460細胞在p53蛋白總量並無增加，但會增加乙醯化p53蛋白的表現量。我們轉染siRNA的方式抑制A549及H460細胞內p53的表現，結果發現genistein促進TSA抑制細胞生長及凋亡的效應都被抑制。此外，我們檢測NQO1的蛋白表現，TSA單獨或合併genistein在三株細胞中對NQO1蛋白的表現均無顯著影響。

接著我們檢測TSA單獨或合併genistein對RERF-LC-KJ (另一株對TSA抗性較大的肺癌細胞)細胞生長及凋亡的效果。結果顯示，TSA單獨或合併genistein對其生長抑制的效果與ABC-1細胞相似。然而，TSA (200 ng/ml)可誘發此細胞凋亡，而genistein可促進TSA的效果。Genistein增加TSA誘發的組蛋白乙醯化程度，anacardic acid亦可抑制genistein促進TSA的組蛋白乙醯化程度、細胞生長停滯及細胞凋亡。
綜合以上，本研究結果顯示在不同的肺癌細胞中，Genistein促進TSA的抗腫瘤效果與細胞對TSA敏感性有關。透過增加組蛋白乙醯轉移酶活性，Genistein增加TSA誘發的組蛋白乙醯化程度，進而誘發細胞生長停滯及細胞凋亡。然而，下游蛋白的表現，例如腫瘤抑制因子-p53，可能會影響TSA單獨或合併genistein的抗腫瘤之效果。

關鍵字

Genistein ； Trichostatin A ； Histone acetylation

並列摘要

Our previous studies showed that genistein enhanced histone acetylation and apoptosis induced by trichostatin A (TSA, a histone deacetylase inhibitor), a novel anticancer drug, in human lung carcinoma A549 cells; but not in normal human lung fibroblasts. However, the mechanism by which genistein enhances TSA-induced histone acetylation and whether the combined treatment has the same effects on other human lung cancer cells are unclear. In the present study we first compared the enhancing effects of genistein on the antitumor effect of TSA in three human lung cancer cell lines, A549 and NCI-H1640, sensitive to TSA, as well as ABC-1, more resistant to TSA. The results showed that the enhancing effects of genistein on TSA-induced cell-growth-arrest were in an order: A549, NCI-H640 > ABC-1. Genistein enhanced TSA (50 ng/ml)-induced apoptosis and caspase-3 activation in A549 and NCI-H460 cells. However, TSA (even at 200 ng/ml) alone or in combination with genistein did not induce apoptosis and increase caspase-3 activation in ABC-1. Genistein also increased TSA-induced acetylation of histone H3/H4 in three cell lines. The inhibitor of acetyltransferase, anacardic acid, diminished such an effect of genistein in all cells. Anacardic acid also diminished the enhancing effects of genistein on TSA-induced apoptosis in A549 and NCI-H640 cells. Genistein alone or in combination with TSA increased p300, an acetyltransferase, mRNA expression in NCI-H460 cells; such an effect of genistein was not marked in A 549 cells. P53 gene in A549 and NCI-H460 cells are wild type while which in ABC-1 is mutant, suggesting that the p53 expressions may contributed to the different effects of TSA alone or in combination with genistein in lung cancer cells. We determined the levels of p53 protein and found that genistein alone or in combination with TSA increased p53 expression in A549 cells rather than in H460 cells. But the level of acetyl-p53 protein expression was increased when genistein combination with TSA in H460 cells.Using transfection of siRNA to slience the expression of p53, we found that ths enhancing effects of genistein on TSA-induced cell growth arrest and apoptosis in A549 and H460 cells were suppressed. In addition, we determined the expressions of NQO1 in three cell lines induced by TSA alone or in combination with genistein and no significant differences were observed among various treatments in all lung cancer cells.
Secondly, we determined the effects of TSA alone or in combination with genistein on cell growth and apoptosis on RERF-LC-KJ (RERF) cells, another TSA-resistant human lung cancer cells. The results showed that the effects of TSA alone or in combination with genistein were similar to those on ABC-1. However, TSA at 200 ng/ml induced apoptosis and genistein enhanced such an effect of TSA. Genistein increased TSA-induced histone acetylation in RERF cells. Anacardic acid also decreased the enhancing effect of genistein on TSA-induced histone acetylation, cell-growth-arrest and apoptosis in RERF cells.
The results of the present study suggest that the enhancing effect of genistein on the antitumor effects of TSA in various lung cancer cells were TSA-sensitivity dependent. Through increasing acetyltransferase activation, genistein increased TSA-induced histone acetylation, which in turn induced cell growth arrest and apoptosis. However, the down stream protein expression, such as tumor suppressed protein-p53, influencing the antitumor effects of TSA alone or in combination with genistein are required to be elucidated.

並列關鍵字

Genistein ； Trichostatin A ； Histone acetylation

參考文獻

100年國人十大主要死因統計。行政院衛生署, 2011

楊盈芝。藉由增加TNF receptor-1 表現Genistein 促進trichostatin A抑制A549細胞生長之效果。中山醫學大學營養科學研究所碩士學位論文。2010。

陳開湧。Phytochemical 促進trichostatin A 抑制A549細胞生長之效果。中山醫學大學營養科學研究所碩士學位論文。2007。

Asomaning K, Miller DP, Liu G, Wain JC, Lynch TJ, Su L, Christiani DC. Second hand smoke, age of exposure and lung cancer risk. Lung Cancer. 2008, 61: 13-20.

Balasubramanyam K, Swaminathan V, Ranganathan A, Kundu TK. Small molecule Modulators of Histone Acetyltransferase p300. J Biol Chem. 2003, 278: 19134-40.