單寧酸、香豆素、黃連素加強三氧化二砷抗癌機制之研究

解決癌細胞轉移和抗藥性問題是目前抗癌最重要的課題之一。利用天然物中各種植物素具溫和、無毒與抗腫瘤特性，加強化學治療效果克服抗藥性、抑制癌細胞轉移並保護正常細胞，希望發揮雙重作用。三氧化二砷可有效的引起急性前骨髓性白血病細胞凋亡反應，然而在臨床上用三氧化二砷治療其他急性骨髓性白血病，骨髓分化不良症候群，和多發性骨髓瘤，成效卻不理想，其抗癌机制仍未完全明白。在各種植物素中，單寧酸(tannic acid)是由多個沒食子酸(gallic acid)聚合而成的糖苷體化合物，且已被證實具有抑制細菌、抑制酵素活性及止血收斂的作用。6,7-二羥基香豆素亦具有抗發炎、抗氧化、抗腫瘤及增強化學治療抗癌效果等作用。而黃連素具有多樣化的抗癌活性，像是抑制細胞生長與激酶活性，誘導細胞凋亡與抑制基質金屬蛋白酶以及腫瘤侵犯的能力。本實驗以對三氧化二砷有抗藥性的血癌細胞株HL-60、U937為模式，分別探討單寧酸或香豆素加強三氧化二砷抗癌作用之機轉；利用黃連素加強三氧化二砷抗鼠神經膠質瘤(Rat C6 glioma cell line)和人類神經膠質瘤U87細胞株的轉移侵犯。結果發現，HL-60細胞株在加入單寧酸的處理下，會造成細胞存活率(cell viability)的明顯下降，且下降趨勢具有濃度及時間關性。而在流式細胞儀(Flow cytometry)的分析中，發現sub-G1的細胞群會隨單寧酸的濃度提高而增多，顯示單寧酸具有誘導HL-60細胞凋亡(apoptosis)的效果。進一步以DAPI stain以及DNA電泳的分析，證實單寧酸會造成HL-60細胞染色質(chromosome)濃聚以及DNA斷裂的現象。而在分子機制的探討中發現，單寧酸所誘導的細胞凋亡，主要是透過抑制SOD，提升細胞內superoxide量，進而破壞粒腺體膜電位(mitochondrial membrane potential)進而釋放出cytochrome c，並且活化caspase-8、 caspase-9 以及caspase-3，而造成poly (ADP-ribose) polymerase (PARP)切割的結果。在U937細胞株，香豆素具有抑制MEK/ERK及JNK訊息路徑的特性，合併三氧化二砷一起處理，明顯的藉由GSH下降，增加氧化壓力亦增加caspase活化和Bid切割，引發粒腺體及溶小體傷害，造成細胞凋亡現象，單寧酸與6,7-二羥基香豆素均對於三氧化二砷具有相當不錯的輔助效果。利用Boyden chamber assay、Matrigel invasion assay、Scratch assay及西方墨點法等方法，以不同濃度(0-20 μM) 之黃連素和三氧化二砷，結果發現，透過抑制蛋白激酵素proetein kinase C (PKC) α, ε蛋白活化與轉位，可以影響細胞骨架蛋白actin及酵素MMP-2, MT1-MMP和下游轉錄因子myc, jun等，進而減少癌細胞侵犯能力和轉移。

關鍵字

單寧酸；6,7-二羥基香豆素；黃連素；三氧化二砷；凋亡；神經膠質瘤

並列摘要

Cancer metastasis and drug resistance are major problems in modern cancer treatment. The dual effects of phytochemicals to enhance chemotherapy and protect normal cells are applicable. Arsenic trioxide (As2O3) is widely used in treating human acute promyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As2O3. Tannic acid (TA), a glucoside of gallic acid polymer, or esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of cancer cells by displaying anticancer potential. We find that tannic acid treatment decreased the viability of HL-60 cells in a dose- and time-dependent manner. Flow cytometry analysis revealed that tannic acid significantly induced apoptosis of HL-60 cells as confirmed by a dose-dependent increase of sub-G1 fraction. The further analysis of DAPI stain and DNA integrity also evidenced that tannic acid caused chromosome condensation and DNA fragmentation. The molecular analysis showed that tannic acid-induced apoptosis of HL-60 cells was associated with disruption of mitochondrial membrane potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). These effects were associated with the increase of intracellular superoxide in mitochondrial signal pathway which attributed to the down-regulation of superoxide dismutase (SOD). Esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. The study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As2O3-induced GSH depletion. In addition, esculetin enhanced the effect of As2O3 on caspase activation in U937 cells. The combined esculetin and As2O3 treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As2O3-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As2O3-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal–mitochondrial pathway of apoptosis. The wound scratch and Boyden chamber assays were applied to determine the effect of As2O3 and berberine on the migration capacity and invasiveness of glioma cancer cells. Zymography and Western blot analyses provided information on the effect of As2O3 and berberine on the intracellular translocation and activation of protein kinase C (PKC), and some PKC related downstream factors. The As2O3-mediated reduction in motility and invasion of glioma cells was enhanced upon co-treatment with berberine. Furthermore, it has been reported that PKC isoforms influence the morphology of the actin cytoskeleton, as well as the activation of metalloproteases MT1-MMP and MMP-2, reported to be involved in cancer cell migration. Treatment of glioma cells with As2O3 and berberine significantly decreased the activation of PKC α and ε and led to actin cytoskeleton rearrangements. The levels of two downstream transcription factors, myc and jun, and MT1-MMP and MMP-2 were also significantly reduced. Upon co-treatment of glioma cells with As2O3 and berberine, cancer cell metastasis can be significantly inhibited, most likely by blocking the PKC-mediated signaling pathway involved in cancer cell migration. This study is potentially interesting for the development of novel chemotherapeutic approaches in the treatment of malignant gliomas and cancer development in general.

並列關鍵字

Tannic acid ； Esculetin ； Berberine ； Arsenic trioxide ； Apoptosis ； Glioma

參考文獻

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單寧酸、香豆素、黃連素加強三氧化二砷抗癌機制之研究

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