榭皮酮影響trichostatin A抗癌效果及副作用的研究

Studies of quercetin on the antitumor and the side effects of trichostatin A

詹淑婷(Shu-Ting Chan)

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/博士(2015年)

https://doi.org/10.6834/CSMU.2015.00051

摘要

Trichostatin A (TSA)是一種組蛋白去乙醯酶抑制劑，具有治療癌症的潛力。TSA會藉由活化死亡接受器或粒線體調節路徑誘發肺癌細胞凋亡。Quercetin是一種類黃酮，廣泛存在於蔬果當中，quercetin被人體攝取後會快速轉換成接合的代謝產物，研究指出quercetin及其代謝物具有多種的生物活性，包括抗氧化、抗發炎或調節訊號傳遞路徑。此外，quercetin可能具有增加化療藥物抗腫瘤的效果，且能降低藥物造成的副作用，然而，quercetin是否會影響TSA的抗腫瘤效果及副作用情形並不清楚的。

首先，利用A549細胞 (正常表現p53蛋白) 及H1299細胞 (不表現p53蛋白)以TSA (25 ng/mL; 82.5 nM)單獨或quercetin(5μM)共同處理，我們發現quercetin透過調節p53依賴性的粒線體訊號路徑顯著的增加TSA所誘發A549細胞生長停滯及細胞凋亡的情形。Quercetin也會增加TSA所誘發H1299細胞凋亡，然而這樣的效果遠低於在A549細胞的效果。此外，我們也證實以腹腔注射方式給予腫瘤小鼠quercetin(10 mg/kg body weight，每周三次) 會增加TSA抗腫瘤效果，且在腫瘤組織中會增加p53蛋白質的表現。

進一步的，我們探討quercetin經由管餵(20 and 100 mg/kg body weight，每周三次)方式是否也會增加TSA抗腫瘤的效果。我們發現quercetin以這樣的劑量經由管餵方式給予無法增加TSA的抗癌效果，這可能與quercetin的代謝轉換有關，體外實驗證明，quercetin的代謝產物quercetin-3-glucuronide增加TSA抗腫瘤作用的效果較quercetin差，且其併入細胞的效果亦較差。然而，quercetin經由管餵或是腹腔注射方式給予腫瘤小鼠皆能降低TSA所誘發淋巴細胞DNA的傷害及血漿中脂質過氧化的情形。
在第一個研究中，我們發現在A549或H1299細胞中，quercetin透過非p53依賴的機制增加TSA所誘發的組蛋白H3及H4的乙醯化作用(這可能會調節與細胞凋亡相關基因的表現)，然而，其機制及重要性不清楚。因此我們進一步探討，在H1299細胞中，quercetin增強TSA (或vorinostat，另一種組蛋白去乙醯酶抑制劑) 增加組蛋白H3及H4乙醯化對其促進細胞凋亡的角色。Quercetin藉由增加DR5 (一種死亡接受器) 的表現而增強TSA造成H1299細胞的凋亡，quercetin合併TSA會顯著的增加p300蛋白 (組蛋白乙醯轉移酶) 的表現，但TSA本身則否，而將H1299細胞中的p300RNAsilencing後，顯著的消減quercetin增強TSA誘發的組蛋白H3及H4乙醯化、DR5蛋白的表現及細胞凋亡的情形。在H1299細胞當中quercetin合併vorinostat的效果及機制與quercetin合併TSA是相似的。

我們推測管餵quercetin無法增加TSA的抗腫瘤作用是由於劑量過低所導致，因此，我們進一步的探討增加quercetin劑量 (藉由給予動物含0.1% 及1% quercetin飼料)，是否會增加TSA在腫瘤小鼠中的抗腫瘤效果。此外，我們也探討是否給予含quercetin的飲食會降低TSA造成的肌肉萎縮的情形及其可能的機制。結果顯示，在腫瘤小鼠中給予1%quercetin飲食顯著的增強TSA的抗癌效果。給予腫瘤小鼠quercetin飲食，尤其是含1%quercetin飲食，會降TSA誘發的淋巴細胞的DNA傷害且會增加白血球數目、體重、附睪脂肪及肌肉重量。而1%quercetin飲食的作用與腹腔注射quercetin效果是相似的。更進一步的我們發現給予quercetin會降低TSA造成肌肉當中與肌肉降解相關的蛋白質Forkhead BoxO1 (FOXO1)/atrophy gene-1 (Atrogen1)/muscle ring-fingerprotein-1 (MuRF1)的表現，且會回復TSA造成myosin heavy chain(MyHC) 蛋白質降低的表現。我們的結果顯示quercetin補充消減TSA所誘發肌肉萎縮的情形可能是透過調節促發炎激素(TNF-α and IL-1β)的表現量有關。

綜合以上結果顯示，本論文研究證實在體內及體外研究中，quercetin可能透過調控p53依賴性及p53非依賴性的機制增加TSA抗肺癌細胞的效果。Quercetin以口服方式增強的效果較腹腔注射方式效果差，這與quercetin代謝產物的轉換有關。然而，不論是以口服或腹腔注射方式給予quercetin皆能降低TSA所誘發淋巴細胞DNA的傷害及脂質過氧化的情形，更進一步的，腹腔注射quercetin與給予1% 及0.1% quercetin飲食皆能消減TSA誘發腫瘤小鼠肌肉萎縮的情形。

關鍵字

quercetin ； trichostatin A ； p53 ； p300 ； 肌肉 ； 細胞激素

並列摘要

TrichostatinA (TSA), a histone deacetylase inhibitor, is a potential therapy for cancers. Itinduces apoptosis of cancer cells, including lung cancer cells,through activating thedeath receptor- and mitochondria-mediated pathways. Quercetin, a flavonoid, is ubiquitously found in various vegetarian foods.It is converted to conjugated metabolites quickly after intake.Studies suggest that quercetin or its metabolites may possess various bioactivities, including antioxidation, anti-inflammation and modulation of signaling pathways.In addition, quercetinmay enhance the antitumor effect of some chemotherapy drugs and reducesthe cytotoxic sideeffects of these drugs. However, the effects of quercetin on the antitumor and side effects of TSA are unclear.

First, using A549 cells (expressing wild-type p53) and H1299 cells(a p53 null mutant) treated with TSA (25 ng/mL; 82.5 nM) alone or in combination with quercetin (5 μM), we found that quercetin significantly increased the growth-arrest and apoptosisinduced by TSAthrough the p53-dependent mitochondrial signaling pathwayin A549 cells. Quercetin also increased the apoptosisin TSA-exposed H1299cells,however, the effect wasmuch lower than that in A549 cells. In addition, we also demonstrated that quercetin administrated intraperitoneally(10 mg/kgbody weight, 3 times/week) enhanced the antitumor effect of TSA in tumor-bearing mice, companying with the increase of p53 expression in tumor tissues.

Furthermore, we investigated whether quercetin administered by gavage(20 and 100 mg/kg body weight, 3 times/week)enhancesthe antitumor effect of TSA. We found that quercetin given orallyat these doses failed to enhance the antitumor effect of TSAbecause of its metabolic conversion.The in vitro study demonstrated that the enhancing effect and the incorporation of quercetin metabolite, quercetin-3-glucuronide, was less than that of quercetin in A549 cells exposed to TSA. However, oral and intraperitoneal administration of quercetin similarly decreased TSA-induced lymphocyte DNA damage and plasma lipid peroxidation in tumor-bearing mice.

In the first study, we found that quercetin increased the acetylation of histones H3 and H4, which may modulate apoptosis-associated genes expression, induced by TSA in both A549 and H1299 cells through p53-independent mechanisms. However, the mechanism and the significance are unclear. Thus, we further investigated the role of increasing acetylation of histones H3 and H4 by quercetin in the apoptosis in H1299 cells exposed to TSA (or vorinostat, another histone deacetylase inhibitor).Quercetin enhanced TSA-induced apoptosis in H1299 cells through increasing DR5, a death receptor, expression. Quercetin+TSA rather than TSA alone alsosignificantly increased p300, a histoneacetyltransferase,expression.Transfection of p300siRNA significantly diminished the increase ofhistones H3/H4 acetylation, DR5 protein expression, and apoptosis induced by quercetin in H1299 cells exposed to TSA. The combined effects and the mechanisms of quercetin+vorinostat on the apoptosis in H1299 cells were similar to those of quercetin+TSA.

We speculatedthat oral administration of quercetin failed to enhance the antitumor effect may be due to the low doses used. Thus, we furtherinvestigated whether increasing the oral doses of quercetin by given 0.1% or 1% quercetin diet affect the antitumor effect of TSA in tumor-bearing mice. In addition, wealso investigated whether the quercetin containing diet decreases the muscle atrophy induced by TSA and the possible mechanisms. The results showed that 1% quercetin diet significantly enhanced the antitumor effect of TSA in tumor-bearing nude mice.Quercetin containing diet, 1% diet especially,decreased the lymphocyte DNA damage and increasedwhite blood cell numbers and the weight of body, epididymal adipose and muscle in tumor bearing mice exposed to TSA. These effects of 1% quercetin diet were similar to the quercetin administrated intraperitoneally. Furthermore, we found that all quercetinadministration decreased the TSA-inducedForkhead BoxO1 (FOXO1)/atrophy gene-1 (Atrogen1)/muscle ring-fingerprotein-1 (MuRF1) protein expression, which are associated with muscle degradation, and recovered the decrease of myosin heavy chain(MyHC) induced by TSA in the muscle. Our results suggest that the mechanisms by which quercetin supplementation attenuated TSA-induced muscle atrophy were associated withthe down-regulationof pro-inflammatory cytokine (TNF-α and IL-1β) levels.

In summary, this dissertation research demonstrates that quercetin enhancesthe antitumor effect of TSA in vitro and in vivo may throughp53-dependent and p53-independent mechanisms in lung cancer cells. The enhancing effect of quercetinadministration orally is poor than that of intraperitoneal administration because of metabolic conversion. However, both quercetin administrated orally andintraperitoneally similarly decreases TSA-induced lymphocyte DNA damage and lipid peroxidation. In addition, similar to intraperitonealquercetin, 1% and 0.1% quercetin diet also attenuate TSA-induced muscle atrophy in tumor-bearing mice.

並列關鍵字

quercetin ； trichostatin A ； p53 ； p300 ； muscle ； cytokines

參考文獻

Dietary flavonol and flavone intakes and their major food

Flavonol and flavone intakes in US health professionals.

J Am Diet Assoc. 102:1414-1420, 2002.

20:21-29, 1993.

implication of its intestinal absorption and metabolism.