褐藻醣膠誘發人類腎臟癌細胞週期停滯及程式死亡之分子相關機制

Fucoidan; a Brown Algae Sulfated Polysaccharide; Induced Cell Cycle Arrest and Cell Apoptosis in Human Renal Cell Carcinoma

林暐棟(Wei-Tung Lin)

指導教授 ： 邱慧芬

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2018年)

摘要

在台灣連續十年之十大死因和最新2017年行政院衛生福利部公告第一名均為惡性腫瘤（癌症）。肺癌、肝癌分居國人癌症死因前兩位。至於腎臟癌雖盛行率較低，但目前臨床上的腎臟癌藥物化療與放射療法普遍反應不佳，因此對於腎臟癌治療有效的藥物之開發，也是目前重要議題和研究之方向。
雖然臨床上對固體癌症治療用藥近十年雖有很大進展有許多標靶藥物出現，但腎臟癌之有效藥卻寥寥無幾而目前之用藥其副作用也可能讓病患身體遭受二次傷害。今若能由海洋中之天然食物中尋找有效抗癌活性成份，不僅有效且可避免不必要的副作用。
海藻為日常飲食常見食材且中國古代藥典神農百草經及李時珍本草綱目均記載海藻可有治療腫瘤效果。褐藻醣膠(fucoidan, Fu)為一硫酸化多醣體 (sulfated polysaccharide)，多醣體骨架主要由岩藻醣(fucose)構成為存在於褐藻綱 (Phaeophyceae) 海藻表面之黏滑液成份。
此外海中棘皮動物(海蔘和海膽)亦含此成份。褐藻醣膠(Fu)其分子量分佈廣泛根據不同的萃取方法約為20,000至200,000 daltons，或者是分子量400至5,000 daltons稱為低分子量褐藻醣膠，亦有分子量從200,000至500,000 daltons為高分子量褐藻醣膠。Fu可溶於水及酸性溶液。
褐藻醣膠對癌細胞的抗癌活性之研究日益增加，如對人類乳癌細胞 (MCF及MDA-MB-231 cell)、人類膀胱癌細胞 (5637 and T-24 cell)、人類肺癌細胞 (A549 cell)、人類肝癌細胞 (HepG2)、人類大腸直腸癌細胞 (HCT 116 cell)及人類攝護腺癌細胞(PC-3 cell)均有效用。
我們以XTT 及methylene blue二種方法檢測 Fucoidan (Fu) 之抗癌毒殺活性，對於人類肺腺癌細胞 (A549)、人類肝癌細胞 (HepG2)、人類近端腎小管亮細胞腎細胞癌細胞株 (786-O)、人類腎細胞癌細胞株 (A-498)等癌細胞之毒殺活性，實驗結果顯示Fu對各癌細胞均具明顯地毒殺活性，該抗癌活性呈現劑量-依存反應相關現象(dose-dependent response phenomenon)。
許多研究顯示Fucoidan能誘導癌細胞之凋亡。因細胞週期與凋亡作用是調控細胞生死的過程，於是我們發現Fucoidan對細胞週期具調控作用，會造成細胞週期G1期停滯及週期相關之訊息蛋白質如：細胞週期素(cyclins, D1/ E) 以及其所相關聯的cyclin-dependent kinases (CDK)如CDK2/ 4/ 6等都觀察到被Fucoidan之抑制作用。
先以 PI 染 DNA 再以流式細胞儀 (flow cytometry)分析細胞週期分佈結果顯示：Fucoidan 48小時後處理後會造成786-O及A-498二種癌細胞停滯於 G1 期的細胞比率增加，且呈現劑量反應依存的正相關。
採用西方墨點法 (WB)分析證實Fucoidan處理後會造成細胞G1期相關之訊息蛋白的表現改變，G1 期 的cyclin D/E及CDK 2/4/6的均顯示表現量下降的現象，P21呈現上升的現象。
以流式細胞儀採 Annexin V/propidium iodide (PI) 雙染分析顯示 Fu會誘導癌細胞凋亡。由西方墨點法(WB)分析在細胞凋亡路徑調控上顯示出Fu具有促進細胞凋亡作用。
包括1. 外在路徑訊息蛋白如 Fas, Fas ligand等表現增加2. 內在路徑中抑制抗凋亡之 Bcl-2蛋白表現下降3.程式死亡訊息之傳遞路徑caspases 7/8/9/3活化造成終究走向細胞凋亡作用。
我們的結果證明Fu對癌症細胞其抗癌活性之作用機轉包括經由抑制癌細胞增生、抑制轉移、細胞週期 G1 期停滯以及誘發細胞凋亡和抑制細胞移行和血管新生等作用機轉而成。
在Fu對癌細胞調控基因的研究中，我們分別分析了與癌生成相關的轉錄因子STAT3 (Signal transducer and activator of transcription 3)、NFκB (nuclear factor-κB)以及HDAC (Histone deacetylase)對這三個因子的表現作用的調控活性。
STAT家族，負責傳導細胞膜信號到細胞核。特別是STAT3會造成促進腫瘤的形成與進展成發炎微環境。因此，調節STAT3的活性對於腫瘤的抑制作用會是一個很關鍵的關鍵。

NFκB也是一個重要的轉錄因子及發炎相關之重要因子，其活化之調控會造成癌細胞之增殖、侵犯、血管新生及轉移作用。經由誘導NFκB 和STAT3會相互協同促進癌症發展。而fucoidan卻可以同時抑制此二個因子而達到抑制腎臟癌細胞生長的效用。
組蛋白乙醯化是轉譯後組蛋白再修飾，由組蛋白乙醯轉移酶 （HAT）和組蛋白去乙醯酶（HDAC）二種酵素調控，會影響DNA之基因表現活性。HDAC經由去除染色質DNA之乙醯基而改變細胞致癌基因和抑癌基因的轉錄表現。

本篇論文藉由研究褐藻醣膠(Fu)對於人類腎癌細胞的細胞週期調控、細胞程式死亡和細胞核內轉錄因子STAT3、NFκB和上基因DNA表現相關之組蛋白去乙醯酶(HDAC)之調控活性，進一步了解fucoidan在抗癌活性的基因表現機轉。因我們的研究為褐藻醣膠(Fu)目前對抗腎臟細胞癌細胞之首篇研究，結果也顯示Fu對於腎臟細胞癌會是一個具抗癌潛力活性的新藥成份。

關鍵字

褐藻醣膠 ； 腎臟細胞癌 ； 細胞週期 ； 程式死亡

並列摘要

Cancer has now taken the lead among the ten leading causes of death in Taiwan. The two most common ones are lung cancer and hepatoma. Though renal cell carcinoma is not so prevalent as these two malignancies, the response to medical treatment is very poor.

Therefore, the new effective drugs development for renal cell cancer treatment become very important. Recent advances in the clinical new target drugs use for cancer treatment is much more progress, the side effects may cause secondary injuries to the patients. It is more safte to obtain effective anti-cancer compound from daily edible natural products .

Fucoidan (Fu), is a sulfated polysaccharide and the polysaccharide backbone is mainly composed of fucopyranose. They are mainly distributed as a mucovesicuous fluid on the surface of brown algae.

In addition, the sea echinoderm also contains this component. According to different extraction methods, the molecular weight of fucoidan products can range from 200,000 to 2,000,000 (large) 20,000 to 200,000 (middle) or 400 to 5,000 (small) daltons, which is called low-molecular-weight fucoidan (LMF), soluble in water and acidic solutions.

The anti-cancer activity of fucoidan on cancer cells has caused worldwide attention such as in breast cancer cell (MCF and MDA-MB-231 cells), human bladder cancer cells (5637 and T-24 cells) and human prostate cancer cell (PC-3 cell).

We have previously reported fucoidan on the proliferation of cytotoxic activity in different cancer cells. Present study we continued to explore the molecular related pharmacological mechanism and its anti-cancer epigenetic regulation on gene expression.

We conducted the anti-cancer cytotoxicity of Fucoidan (Fu) with XTT and methylene blue methods at different concentrations (0,20,40,60,80,100 μg/ml) in human lung adenocarcinoma cells (A549) , humans hepatoma cells (HepG2) , humans breast cancer cells (MCF7) , human clear cell renal cell carcinoma (786-O) and renal cell carcinoma (A498).

Fu possessed cytotoxicity to five cancer cell lines . It have demonstrated as a dose-dependent response phenomenon. Since cell cycle and apoptosis modulation are necessary to regulate cell life and death, we examined the cell cycle regulation role of fucoidan.

It is shown that Fu cause cell cycle arrest in G1 phase and cycle-associated regulatory proteins such as cyclins (Cyclin D1/E) and its associated cyclin-dependent kinases (CDK; CDK2/4/6) were all observed have significant changes. Western blotting (WB) analysis also confirmed the the promote expression of G1 phase inhibitory P21 protein expression .

In the intrinsic pathway proapoptotic anti-apoptotic Bcl-2 proteins and extrinsic pathways signal proteins Fas, Fas ligand these death related signals and caspases were modulated by fucoidan and lead to cells apoptosis. Our results demonstrate that anti-cancer activity of fucoidan be attributed to cytotoxic effect, cell cycle G1 arrest and apoptosis induction and inhibition of metastasis.

To study the epigenetic regulation of cancer cell genes expression, we analyzed the signal transducer and activator of transcription 3 (STAT3) , nuclear factor-κB (NFκB) and HDAC (Histone deacetylase) which are involved in the processes of carcinogenesis.

The STAT family is responsible for conducting cell membrane signals to the nucleus. In particular, STAT3 causes the tumor cell to proliferate and malignancy progresses to be in an inflammatoary microenvironment where it develops and progresses.

NFκB is also an important transcription factor. It controls oncogene expression in proliferation, invasion, angiogenesis, and metastasis. NFκB and STAT3 synergistically promote cancer development through the induction of the oncogenes.

HDAC cause post-translational histone acetylation by removing
acetyl group of chromatin and diversify the transcription of oncogenes and tumor suppressor genes.

In this study, we investigate and compare anti-cancer activity of fucoidan with its cell cycle regulation, apoptosis-inducing effects, and nucleus transcription factors STAT3, NFκB and HDAC in human renal cancer cells to elucidate the molecular modulation of fucoidan in gene expression of anti-cancer modulation.

It is the first report of fucoidan on human renal cell carcinoma, our data have demonstrated that fucoidan may be a potential novel anti-cancer compound for renal cell carcinoma.

並列關鍵字

fucoidan ； renal cell carcinoma ； cell cycle ； apoptosis

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