11/09/2025
研究生: 邱婷鈺
研究生(外文): CHIU, TING-YU
論文名稱: 褐藻醣膠誘發人類肺癌細胞之抗癌活性及 DNA表現調控之分子作用機轉
論文名稱(外文): Fucoidan induced Anti-cancer Activity and DNA Expression Modulation in Human Lung Cancer Cells
指導教授: 邱慧芬
指導教授(外文): CHIU, HUI-FEN
學位類別: 碩士
校院名稱: 高雄醫學大學
系所名稱: 醫學研究所碩士班
論文出版年: 2021
論文摘要
根據台灣衛生福利部公布的2019年十大死因中，惡性腫瘤（癌症）依舊位居榜首，而在癌症死因中，肺癌、肝癌分別位居台灣癌症死因前兩位，近幾年針對癌症的新藥及治療方法不斷問世，讓癌症患者得以延續生命，然而如化學療法帶來的強烈副作用，以及昂貴的自費用，對患者的身體及經濟無疑是沉重的負擔，若能從自然界中尋求出抗癌成分，或許可以有效且減少嚴重副作用對患者身體帶來的傷害。
海洋蘊含著豐富的資源，而海藻就是其中之一。而海藻分四種而其中的褐藻其表面黏液中含有褐藻醣膠 (Fucoidan)，這個硫化多醣體 (sulfated polysaccharide)化合物，在細胞及動物研究中，越來越多研究證實褐藻醣膠具有抗癌功效，如對乳癌細胞 (MCF-7、MDA-MB-231)、肺癌細胞 (A549) 、肝癌細胞 (HepG2)、大腸直腸癌細胞 (HCT 116)及前列腺癌細胞 (DU-145) 均有抗癌活性。
在本論文中，我們以Methylene blue檢測 Fucoidan 對癌細胞的毒殺效果，分別使用人類肺腺癌細胞 (A549)、人類肝癌細胞 (HepG2)、人類乳癌細胞 (MCF-7) 這三種癌細胞進行測試，實驗結果顯示Fucoidan對這三種癌細胞均有明顯的毒殺效果，而其抗癌活性呈現劑量反應 (dose-dependent) 之現象。
在抗癌活性的相關研究中，有越來越多研究證明Fucoidan能促使癌細胞凋亡作用及停滯細胞週期作用，促凋亡蛋白表現量呈現上升趨勢，而在細胞週期方面則是會造成G1期停滯，及其細胞週期素(cyclins, D1/ E) 以及cyclin-dependent kinases (CDK) 如CDK2/ 4，都觀察到被抑制之現象。
先以Fucoidan處理癌細胞 48小時後，再用 PI 染細胞DNA，以流式細胞儀 (flow cytometry) 分析細胞週期之分佈現象，結果顯示：以Fucoidan處理肺癌細胞後會使癌細胞停滯於 G1 期，且呈現正相關的劑量反應現象。
在蛋白質表現量方面則採用西方墨點法 (Western blot) 進行分析，在以Fucoidan處理48小時後，會導致癌細胞G1期相關的蛋白質，G1 期 的cyclin D/E及CDK 2/4的表現量呈現下降趨勢。
分析癌細胞凋亡現象，則是以Annexin V/propidium iodide (PI) 雙染，並使用流式細胞儀分析，顯示 Fucoidan會導致癌細胞凋亡。由西方墨點法 (Western blot) 分析在細胞凋亡相關路徑蛋白質表現量，顯示出Fucoidan具有促進癌細胞凋亡作用的效果。
在蛋白質表現量方面同樣採用西方墨點法 (Western blot) 分析，發現Fucoidan會導致促凋亡蛋白Fas、FasL、Caspase8、Caspase3、Bad表現量上升，並減少抑制凋亡蛋白Bcl-xL、Bcl-2之表現量。本論文的實驗結果證明Fucoidan可經由促進癌細胞凋亡、在細胞週期 G1 期停滯以及抑制細胞移行和血管新生等作用機轉進而呈現抗癌之活性。
還有位於癌細胞膜的PD-L1 (Programmed cell death 1 ligand 1) 及EGFR (Epidermal growth factor receptor, EGFR) 在Fucoidan的作用下的蛋白質表現量。位於癌細胞膜上的PD-L1 (Programmed cell death 1 ligand 1) 會與免疫系統的T細胞上的PD-1 (Programmed cell death protein 1) 結合，使得T細胞無法發揮作用，進而使癌細胞得以存活。
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 （Histone acetyltransferase, HAT）和組蛋白去乙醯酶（Histone deacetylase, HDAC）二種酵素調控，進而影響基因表現。而HDAC藉由移除組蛋白之乙醯基，進而改變癌細胞之致癌基因的表現。
以核質分離之技術並由西方墨點法 (Western blot) 分析法研究癌症的轉錄因子STAT3 (Signal transducer and activator of transcription 3, STAT3）、pSTAT3 (phospho-STAT3) 和NFκB (Nuclear factor-kappa B, NF-κB)、pNFκB (phospho-NF-κB) 以及細胞核內之組蛋白去乙醯酶包括HDAC1、HDAC2、HDAC4、HDAC11之活性。
STAT家族為細胞質轉錄因子，而STAT3主要是將信號從質膜傳導到細胞核中，在癌細胞中，STAT3會有過度活化的現象。而NFκB主要為負責人體內關於生存、發炎等重要生理反應的轉錄因子，其過度活化會造成癌細胞的增生、轉移、侵襲等作用。而Fucoidan可以同時抑制STAT3、pSTAT3以及NFκB、pNFκB進而達到抑制癌細胞增生的作用。
本篇論文藉由研究Fucoidan對於人類癌細胞的免疫方面的相關機轉，細胞膜上的PD-L1及EGFR之活性、細胞凋亡、細胞週期調控和細胞質、核內轉錄因子STAT3、NFκB、pSTAT3、pNFκB及細胞核內一系列組蛋白去乙醯化酶 (HDAC)，了解Fucoidan在調控基因表現之活性，結果顯示Fucoidan對於肺癌細胞，將會是具有潛力之抗肺癌功效的天然海藻成分。
論文外文摘要
According to Taiwan’s Ministry of Health and Welfare reports, the top one mortality disease of 2020 is cancer. The top two common are lung and liver cancers. Therefore, new drugs development and new therapy are still urgently needed.
However, the strong side effects caused by chemotherapy, and high spending of treatments were heavy burdens on the patient’s body and economy. If we can obtain anti-cancer compound from nature ingredients, it will reduce the damage of serious side effects.
The seaweed is one of the abundant nature resources in the ocean. There are four kinds of seaweeds. The surface mucus of brown algae contains fucoidan, a sulfurized polysaccharide compound. Many studies have confirmed that fucoidan has anti-proliferative effects, such as breast cancer cells (MCF-7) , lung cancer cell (A549) , liver cancer cell (HepG2) , colorectal cancer cell (HCT 116) and prostate cancer cell (DU-145) are all possessed significant anticancer effects.
In this study, we used Methylene blue and XTT assay to assess the anricancer effects of fucoidan on different cancer cell lines: human lung adenocarcinoma cells (A549) , human liver cancer cells (HepG2) , and human breast cancer cells (MCF-7) . Cancer cells were treated with fucoidan, our results showed that fucoidan has significant toxic effects on these three cancer cell lines in a dose dependent manner.
Our results showed the cell cycle was arrested at G1 phase. Then, we check G1 phase proteins by Western-blotting analysis, it was shown that cyclins (D/E)、cyclin-dependent kinases 2/4 (CDK2/4) were inhibited by fucoidan.
To analyze the apoptosis of lung cancer cells, we used double staining with Annexin V/propidium iodide (PI) and analyzed by flow cytometry, result showed fucoidan can induce lung cancer cell apoptosis. We also checked the apoptosis-related protein expression, it were shown Fas, FasL, Caspase8, Caspase3, Bad were increased, Bcl-xL and Bcl-2 were all decreased. So fucoidan has the effect of increasing the apoptosis phenomenon in lung cancer cells.
PD-L1 (Programmed cell death 1 ligand 1) and EGFR (Epidermal growth factor receptor, EGFR) proteins were located on the cancer cell membrane. PD-L1 (Programmed cell death 1 ligand 1) combined with PD-1 (Programmed cell death protein 1) , so the T cells can’t kill the cancer cell, both PD-L1 and EGFR were decreased by fucoidan after 48 hr addition.
Histone acetylation is the re-modification effect of histones after gene translation. It was regulated by histone acetyltransferase (HAT) and histone deacetylase (HDAC) , which affected gene performance. HDAC removes the acetyl group of histones, changing the expression of oncogenes in cancer cells.
We used nuclear and cytoplasm separation method analyzed STAT3, pSTAT3, NFκB, pNFκB, and HDAC1, HDAC2, HDAC4, HDAC11 protein expression in the nucleus by Western-blotting. STAT family was a cytoplasmic transcription factor, which mainly transmitted signals from the plasma membrane to the nucleus. STAT3 was over-activated in cancer cells.
NFκB was mainly a transcription factor, which responsible for important physiological responses, such as survival and inflammation in the human body. Its over-activation can cause cancer cell proliferation, metastasis, invasion and other effects. Fucoidan can simultaneously inhibit STAT3, pSTAT3, NFκB, and pNFκB to achieve the effect of inhibiting the proliferation of cancer cells.
To understand the activity of fucoidan in regulating gene expression, the results showed that fucoidan, the brown seaweed ingredient, which have potential anti-lung cancer proliferative effect, through the inhibition of cell cycle on G1 phase and cell apoptosis induction.
Moreover, fucoidan also possessed modulation on histone deacetylase (HDACs), STAT3 and NFκB. It is demonstrated that fucoidan inhibited HDAC1,2,4,11, STAT3,pSTAT3 and NFκB transcription factors to regulated the down stream protein expression.
This study proved fucoidan modulated immune mechanism on human lung cancer cells, the inhibition of PD-L1 and EGFR on the cell membrane, nuclear transcription factors STAT3, NFκB, pSTAT3, pNFκB, and a series of histone deacetylases (HDACs) in the lung cancer cell nucleus.
