臺灣博碩士論文加值系統

多醣體是指單醣藉由醣苷鍵連結所形成的巨大分子，臨床上已證實具有抗老化、提升免疫力以及抗癌的能力。上皮－間質轉化過程是一系列的細胞狀態改變，最初是在胚胎發育時所發現，而現今則是著重於癌症轉移的過程中對細胞移動能力所產生的改變。首先我們證實了多醣體，包括靈芝多醣萃取物以及岩藻醣膠皆具有抑制肺癌以及乳癌細胞株生長的效果，更明確的說是藉由阻斷細胞PI3K以及MAPK的訊息傳遞路徑而達成。接著我們以觀察細胞型態的變化來評估多醣體參與上皮－間質轉化過程的可能性，發現細胞株在多醣體刺激後會表現出上皮細胞的各種特性，同時也觀察到細胞的移動能力會有明顯的下降，所以影響上皮－間質轉化過程確實可能是多醣體抑制癌症的其中一種機制。我們同時也發現在多醣體的刺激下，會造成與TGF-β相關的訊息傳遞分子表現的改變，包括藉由ELISA的測定發現TGF-β分泌的下降，以及由西方墨點法觀察到Smad2/3磷酸化、Smad4由細胞質轉移到細胞核的比例都有下降的趨勢。同時多醣體的刺激下可能會藉由減少一些轉錄因子如Snail以及Slug的表現來而造成與上皮－間質轉化過程相關的分子標誌表現量的轉變，包括上皮型態的標誌E-cadherin的上升以及間質型態的標誌N-cadherin的下降。我們也觀察到多醣體在參與TGF-β的訊息傳遞時會從另一個特殊的路徑，稱作非Smad參與路徑，藉由降低p38蛋白的磷酸化使得多醣體在影響上皮－間質轉化過程中扮演一個相當全面性的角色。而考慮到靈芝多醣萃取物以及岩藻醣膠在動物體內的影響力，進行了多醣體對帶有腫瘤的小鼠腫瘤大小是否會有影響的相關研究。我們將乳癌細胞4T1以及肺癌細胞LLC-1分別植入BALB/c以及C57B/6兩種品系的小鼠體內，並且以口腔灌食或是腹腔注射的方式施予靈芝多醣萃取物或岩藻醣膠的治療，結果顯示這兩種多醣體都具有明顯抑制腫瘤大小的能力。總結我們目前的發現，證明了多醣體在體外和體內都有抑制腫瘤生長的能力，並且同時可以藉由阻斷TGF-β的訊息傳遞路徑而影響細胞的上皮－間質轉化過程以達到減緩癌症進程的效果。

Glycan is a giant, complicated molecule composed of glycosidic bond-connected monosaccharides. The clinical studies have proven that the glycan has beneficial to human health, including anti-aging, improvement of immune system and anti-tumor activities. Epithelial-mesenchymal transition (EMT) process is a series of development of characterized cells, which is first discovered in embryogenesis. And now the major issue of EMT is focus on effects of cell mobility during cancer metastasis. At first, we confirmed that the glycan such as the extract of Reishi polysaccharides (EORP) and fucoidan have the anti-proliferation property in lung and breast cancer cells and it caused by blocking the PI3K and MAPK signaling pathway. Then we observed the cell morphology differences which made us speculate the possibility of glycan participate in epithelial-mesenchymal transition and discovered that after glycan treatment, the cell lines turned into epithelial type and the cell mobility had a tremendous decrease, indicated that the interference of EMT could be one of the anti-cancer mechanism performed by glycan. We also found that glycan treatment decreased TGF-β signaling molecules such as TGF secretion (via ELISA), Smad2/3 phosphorylation and translocation of Smad4 from cytoplasm to nucleus (via Western blotting). Treating cells with glycan led to the alteration of EMT biomarkers, for example, glycan increased E-cadherin (epithelial biomarkers), decreased N-cadherin (mesenchymal biomarkers), which probably due to down-regulation of transcription factors Snail and Slug expression. We also observed the glycan participated in other TGF-β pathway, known as non-Smad pathway, by downregulating p38 phosphorylation, making glycan contribute a comprehensive interruption of EMT. Considering the impacts of EORP and fucoidan on tumor in vivo, we studied the effects of the testing glycan on tumor size of tumor-bearing mice in vivo. We inoculated breast cancer cell 4T1 into BALB/c mice or lung cancer cell LLC-1 into C57B/6 mice and followed with oral gavage of EORP or intraperitoneal of fucoidan respectively. We got remarkable results indicated that both glycan derivatives were provided with tumor reducing ability. In summary, we proved that the glycan had an anti-proliferation ability not only in vitro but in vivo and it could block the TGF-β signaling pathways to interrupt the epithelial-mesenchymal transition process, allowing the glycan be a useful medical material to alleviate progression of cancer.

Contents

? Contents ……………………………………………………………………… 1
? Chinese Abstract …………………………………………………………… 3
? English Abstract …………………………………………………………… 4
? Introduction ………………………………………………………………… 6
? Materials and Methods …………………………………………………… 10
? Preparation of EORP ……………………………………………………… 10
? Preparation of fucoidan …………………………………………………… 10
? Cell culture ……………………………………………………………… 10
? Antibodies ………………………………………………………………… 10
? Cell Viability ……………………………………………………………… 11
? Cell extracts ……………………………………………………………… 11
? Western blotting analysis ………………………………………………… 11
? Transwell assay …………………………………………………………… 12
? Determination of cytokines ……………………………………………… 12
? EORP treatment in mice ………………………………………………… 12
? Fucoidan treatment in mice ……………………………………………… 13
? Results ……………………………………………………………………… 14
? Glycan inhibited proliferation of breast and lung cancer cells. …………… 14
? The cell survival signaling was influenced when treated with EORP and fucoidan. ………………………………………………………………… 14
? The treatment of EORP and fucoidan cause the change of cell morphology ……………………………………………………………… 15
? Both EORP and fucoidan can inhibit the cancer cell migration in Transwell Assay ……………………………………………………………………… 15
? Fucoidan treatment decreased the TGF-β secretion and inhibited the TGF-β downstream Smad–dependent pathway ………………………………… 16
? Fucoidan probably participated in TGF-β Smad-independent pathway …… 17
? Oversulfated fucoidan also interfered the EMT process and was more powerful than fucoidan ……………………………………………………………… 17
? EORP and fucoidan inhibited tumor growth in vivo ……………………… 18
? Discussion …………………………………………………………………… 19
? References ………………………………………………………………… 24
? Figure Legands …………………………………………………………… 27

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