- 學位論文
褐藻糖膠，一個PDL-1跟EGFR的調節者，誘導肝癌細胞凋亡及細胞週期停滯經由轉錄作用調節機制
Fucoidan, a PDL-1 and EGFR Regulator, Induces Apoptosis and Cell Cycle Arrest through Transcription Modulation in Human Hepatoma Cells
摘要
非傳染性疾病已成為全球主要死亡原因，其中又以癌症影響最鉅。而癌症死亡率排名中，肝癌更是躋身前十名。然而，肝癌對目前臨床醫學治療的反應非常差。 近年來，癌症細胞的PD-L1與免疫細胞的PD1之相互作用已被視為免疫調節的重要關鍵點，更是當今癌症治療藥物開發的焦點。避開了現有抗癌藥物的副作用，有關PD-L1的免疫調節藥物研發更是廣泛地引起了專家學者們的密切關注。 褐藻糖膠是一種複雜的硫酸化多醣，多醣骨架主要由?m喃葡萄糖組成。在抗癌活性上，已有研究證實褐藻糖膠能對於多種癌細胞有增生抑制作用，但詳細作用機轉目前仍未清楚。因此，本研究評估了褐藻糖膠在人類肝癌細胞（HepG2），人類肺腺癌細胞（A549），人類乳腺癌細胞（MCF-7）中的抗癌細胞毒性。 在癌細胞毒殺活性研究，XTT與methylene blue結果顯示褐藻糖膠具劑量依賴性地對肝癌細胞造成毒殺反應。利用流式細胞儀分析細胞週期發現褐藻糖膠能使肝癌細胞週期停滯於G1期。進一步用西方墨點法偵測顯示褐藻糖膠減少肝癌細胞蛋白（Cyclin D1 / E）及細胞週期蛋白依賴性激酶（CDK2 / 4）蛋白質表現。 流式細胞儀更用於分析褐藻糖膠對於肝癌細胞凋亡之調控，褐藻糖膠作用48小時後，褐藻糖膠能造成肝癌細胞凋亡比例呈現劑量依賴性;在細胞凋亡內源性途徑中。褐藻糖膠降低抗凋亡蛋白Bcl-2表現及增加死亡信號蛋白Fas、FasL、Caspase 3和8表現。研究結果顯示褐藻糖膠之細胞毒殺作用可能是透過細胞週期G1阻滯和細胞凋亡誘導作為抗癌機制。 NF-κB和STAT-3是癌症發展重要的轉錄因子。兩者能促進癌症在發炎的微環境中發展、血管生成和遠端轉移發生。褐藻糖膠能降低NF-κB和STAT-3由細胞質進入細胞核現象，更能抑制NF-κB和STAT-3蛋白質表現。 癌症免疫抑制蛋白PD-L1與表皮細胞生長因子EGFR的調節是近年來重要的抗癌研究標的。西方點墨法數據顯示褐藻糖膠能抑制肝癌細胞細胞膜上之PD-L1及 EGFR蛋白質表現，進而促進了肝癌細胞死亡。 本研究是第一個針對褐藻糖膠針對人類肝癌細胞分子機制探討。褐藻糖膠透過對抑制肝癌細胞膜PD-L1和EGFR，細胞週期調控，凋亡誘導作用、轉錄因子STAT3和NF-κB調節作用，褐藻糖膠未來可能是一種潛在的新型肝癌之天然抗癌化合物。
並列摘要
Worldwide, non-communicable diseases claim the majority of death, where cancer is leading among them. The human liver cancer account among top ten. Though, the response to medical treatment is very poor. The interaction of the PD-L1of cancer cells and PD1 of immune cells is identified as the key point of immune regulation and recently has been the focus of new cancer treatment drug development. Considering the side effects of existing cancer drug, modulator of PD-L1 and immune stimulant has attracted attention of scholars. Fucoidan is a complex sulfated polysaccharide and the polysaccharide backbone is mainly composed of fucopyranose. Bioactivity of fucoidan on different cancer cells lines has liaised global attention. The present study evaluated anticancer cytotoxicity of fucoidan in human lung adenocarcinoma cells (A549), human hepatoma cells (HepG2), human breast cancer cells (MCF7). Fucoidan exhibited a dose-dependent cytotoxic response. Furthermore, we evaluated the implication of cell cycle and apoptosis as key modulators of cell life and death. In addition, curiously, we analyzed the modulation of fucoidan on immune system. We have found that fucoidan modulate the expression of different proteins, including as cyclins (Cyclin D1/E) and its associated cyclin-dependent kinases (CDK2/4). Those proteins regulate cell cycle, which flow cytometry elucidated the arrest in G1 phase. The expression of G1 phase inhibitory protein expression was else proved by western blot. In the intrinsic pathway anti-apoptotic Bcl-2 proteins and extrinsic pathways signal proteins Fas, Fas ligand these deaths related signals and caspases 3 and 8 were upregulated by fucoidan and lead to cells apoptosis. Our results have proved the cytotoxic effect of fucoidan and elucidated the cell cycle G1 arrest and extrinsic pathway of apoptosis as underlying mechanism. NF-B and STAT3 are transcriptions factors. They command oncogene expression in proliferation, invasion, angiogenesis and metastasis. STAT3 and NF-B synergistically stimulate cancer development through the activation of related oncogenes. The regulation and expression of membrane PD-L1 is associated by the prognosis of the cancer. The copy of PD-L1 vary largely depending on transcription factors, including STAT3 and NF-B. The activation of transcription factors and oncogene correlates with the expression. In particular, STAT3 allows tumors to develop and progress in an inflamed microenvironment. Therefore, regulation of STAT3 activity is an important key factor in tumor inhibition. The present research analyzed the anticancer effect of fucoidan on both membrane PD-L1 and Epidermal Growth Factor Receptors (EGFR), cell cycle regulation, apoptosis-inducing effects and nucleus transcription factors modulation in human hepatoma cancer cells in order to elucidate the molecular modulation of fucoidan in human hepatoma. It is the first report of fucoidan on human hepatoma cell carcinoma, our data have demonstrated that fucoidan may be a potential novel anti-cancer compound for hematoma cell carcinoma.