2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38684According to the announcement of Department of Health, cancer is the highest death rate among the top ten causes in Taiwan. Cancer cells are not able to go through the normal apoptosis pathway and have abnormal proliferation ability. Moreover, metastasis may complicate the clinical management and lead to a poor prognosis for cancer patients. Hence, the chemotherapy is usually employed in the adjuvant situation to improve the prognosis and long-term survival. So far, the most important thing for finding new cancer drug is urgent. However, their values were limited by toxicity or lack of efficacy. Osteosarcoma is the most common primary malignant tumor of bone, especially in children. The principles of treatment of osteosarcoma have undergone dramatic changes in the past 20 years. Until recently, 5-year survival of 20% with surgical treatment alone was considered acceptable. This outcome suggested that 80% of the patients had pulmonary metastasis (perhaps undetectable) at the time of presentation. Paclitaxel (Taxol) was isolated from the bark of the Pacific yew and was reported to have cytotoxic activity against a broad range of murine leukemia and solid tumors. Those solid tumors include chemotherapy-resistant epithelial ovarian cancer, advanced breast cancer, small cell and non-small cell lung cancer, and head and neck cancer. We have demonstrated that paclitaxel inhibited N-acetyltransferase activity, DNA adduct formation and gene expression as well as induced apoptosis via caspase-3 activation in U2OS and HL-60 cells. Thus far, little is known regarding the mechanisms of apoptotic-inducing effect of paclitaxel on U2OS cells. We also demonstrated that silibinin significantly inhibited invasive and adhesive potential through a change of cell morphology and a decrease of FAK production to inhibition of ERK 1/2 and c-Jun/AP-1 signaling to reduce the u-PA and MMP-2 expressions in MG-63 cells. In in vitro and in vivo studies, we found that leutolin could increase the apoptotic-inducing effect of paclitaxel on SCC-4 cells. Therefore, in the first year of this study, we propose to (1) analyze and compare the effects of paclitaxel on cell viability of different osteosarcoma cells (MG-63, U2OS and HOS) using MTT assay and trypan blue dye exclusion assay; (2) exam the colony formation of osteosarcoma cells (MG-63, U2OS and HOS) after paclitaxel treatment; (3) investigate the changes in the cell cycle of different osteosarcoma cells (MG-63, U2OS and HOS) after paclitaxel treatment using flow cytometry; (4) confirm the apoptotic effect of paclitaxel on different osteosarcoma cells (MG-63, U2OS and HOS) using DNA fragmentation assay, DAPI stain, TUNEL, and Annexin V-conjugated Alexafluor 488; and (5) further demonstrate the effect of silibinin and leutolin involved in paclitaxel-induced apoptosis and cell cycle changes in different osteosarcoma cells (MG-63, U2OS and HOS) using MTT assay, trypan blue dye exclusion assay and flow cytometry. Then, in the second year of this study, we aim to (1) analyze and compare cell cycle related kinases and cyclin-dependent kinase inhibitors (CDKIs) after paclitaxel treatment in different osteosarcoma cells (MG-63, U2OS and HOS); (2) investigate the binding ability between CDKs and CDKIs of different osteosarcoma cells (MG-63, U2OS and HOS) after paclitaxel treatment using immunoprecipitated (IP) assay; (3) demonstrate the effect of paclitaxel on apoptosis related proteins in different osteosarcoma cells (MG-63, U2OS and HOS); (4) determine the effect of paclitaxel on cell cycle, angiogenesis, migration, invasion and adhesion related genes expression using cDNA microarray; (5) confirm the above changes of mRNA levels and their expressions using real time RT-PCR and western blotting; and (6) further test the silibinin and leutolin involved in paclitaxel-induced apoptosis and cell cycle changes in different osteosarcoma cells (MG-63, U2OS and HOS) using western blotting. Finally, in the third year of this study, we aim to (1) determine and compare the effect of paclitaxel with or without silibinin and leutolin on tumor growth via a BALB/c nude mice model, (2) analyze the effect of paclitaxel with or without silibinin and leutolin on angiogenesis process in vivo, (3) investigate tumor growth of different osteosarcoma cells (U2OS、MG-63 及HOS) in vivo via cancer cell xenografted nude mice model, and (4) finally analyze the effect of paclitaxel on migration/invasion related genes via a BALB/c nude mice model by immunohistochemistry assay.根據衛生署公佈之台灣十大死亡原因顯示癌症仍為十大死因之首，居高不下， 如何治療癌症病人，尋找更有效的抗癌藥物，是一個迫切的問題。癌症的形成是透 過許多複雜且多重的過程，其中形成的原因雖然被了解，但治療的效果仍然有限， 且化療對人體具有相當大之副作用與傷害，因此目前許多研究趨向以天然物成份如 多酚類或黃酮類等天然物合併抗癌藥物的使用，期望能加強抗癌藥物的療效，降低 抗癌藥物的濃度，以減少化療對人體造成之傷害，達到輔助治療的效果，抑制癌細 胞的惡化。 早在 1970 年代之前，肢體骨癌（osteogenic sarcoma、osteosarcoma）的治療只 有截肢且預後悲慘。幸運地近20 年來有效的輔助化學療法，使骨癌的預後顯著改善。 如今2/3 的骨癌病人，可以預期達到長期的存活（long-term survival），但仍舊無法完 全控制，甚至於發現時已經80%（或許有些無法偵測到）有肺部轉移（pulmonary metastasis）。太平洋紫杉醇（paclitaxel：taxol）是由太平洋紫杉（Pacific yew）之樹 皮抽取出來的，有很強的抗癌效果，而且被核准可以用來治療轉移性卵巢癌及乳癌。 另外，paclitaxel 用於其它惡性腫瘤的治療如非小細胞及小細胞肺癌、頭頸部鱗狀 癌、膀胱癌、生殖細胞瘤等都有不錯的療效。我們也發現在人類骨癌細胞株（U2OS） 體外及體內實驗，paclitaxel 會抑制癌化相關的芳香胺類乙醯轉移酶之活性及其基因 表現並透過caspase 3 造成細胞的凋亡，在人類血癌細胞株（HL-60）也有類似的情 形，其他則仍無明確報告有關paclitaxel 能否影響人類骨癌細胞株的基因表現及其詳 細的抗癌機轉。另外，我們曾發現類黃酮silibinin 會壓制人類骨癌MG-63 細胞的侵 襲，它是藉由抑制胞外訊息調節蛋白水解酶（ERK）依賴之c-Jun/活酶蛋白-1（AP-1） 所誘導出的MMP-2。在體內及體外試驗，我們也都發現leutolin 會加強paclitaxel 所 引起口腔癌細胞（SCC-4）的毒殺作用。 因此，本計畫第一年擬進行【1】利用MTT assay 與細胞計數實驗（trypan blue dye exclusion assay），探討paclitaxel 對不同骨癌細胞株（U2OS、MG-63 及HOS）存活 率的影響；【2】利用colony formation，觀察paclitaxel 能否抑制多種不同骨癌細胞株 （U2OS、MG-63 及HOS）形成colony 之能力；【3】以流式細胞儀（flow cytometry） 測定細胞週期，探討paclitaxel 對不同骨癌細胞株（U2OS、MG-63 及HOS）其細胞 週期的影響；【4】以DNA fragmentation assay、DAPI stain、TUNEL、Annexin V-conjugated Alexafluor 488 用來分析paclitaxel 是否能誘導不同骨癌細胞株（U2OS、 MG-63 及HOS）之凋謝死亡；以及【5】利用MTT assay 與細胞計數實驗（trypan blue dye exclusion assay）及流式細胞儀（flow cytometry）的方式，探討不同的類黃酮， 如：silibinin、leutolin 對於增強paclitaxel 對不同骨癌細胞株（U2OS、MG-63 及HOS） 其細胞存活率及細胞週期的增強或加乘效果的影響。 本計畫第二年擬進行【1】利用western blot 探討paclitaxel 對不同骨癌細胞株 （U2OS、MG-63 及HOS）其與細胞週期相關的cyclins、cyclin-dependent kinases （CDKs）和CDK inhibitors（CDKIs）的影響；【2】利用immunoprecipitated（IP） 的技術，探討paclitaxel 是否會對不同骨癌細胞株（U2OS、MG-63 及HOS）透過影 響與細胞週期相關的CDKs 分別與CDKIs 之結合情形；【3】以western blot 探討 paclitaxel 對不同骨癌細胞株（U2OS、MG-63 及HOS）細胞凋亡之相關蛋白表現；【4】 以cDNA microarray 探討paclitaxel 對不同骨癌細胞株（U2OS、MG-63 及HOS）其 與細胞週期，血管新生及其migration/invasion 和adhesion 相關的基因變化；【5】進 一步以real time RT-PCR 及western blotting，確認上述有變化的相關基因其mRNA 及 蛋白質的變化；以及【6】利用western blot 的方式，探討不同的類黃酮，如：silibinin、 leutolin 對於paclitaxel 對不同骨癌細胞（U2OS、MG-63 及HOS）其與細胞凋亡之相 關蛋白表現的增強或加乘效果。 本計畫第三年擬進行【1】以免疫缺陷的小鼠（BALB/c nu/nu mice）接種人類 不同骨癌細胞（U2OS、MG-63 及HOS），分析paclitaxel 及合併處理不同類黃酮對 於接種不同骨癌細胞的老鼠其腫瘤大小、重量及其動物存活率的影響；【2】利用免 疫組織染色的方式，分析上述未處理、單獨處理paclitaxel 及合併處理不同類黃酮其 腫瘤組織切片，觀察腫瘤相關因子及一些血管新生之相關因子的影響；【3】利用in vivo 實驗將matrigel 混合VEGF 或FGF 處理的不同骨癌細胞株（U2OS、MG-63 及 HOS），再處理或不處理paclitaxel，種到C57BL/6 老鼠皮下，再將matrigel 取出，觀 察其抑制血管新生之能力；以及【4】利用不同接種方式，將不同骨癌細胞（U2OS、 MG-63 及HOS）接種至免疫缺陷的小鼠（BALB/c nu/nu mice）的尾靜脈，並處理 paclitaxel，分析其轉移至肺部之癌細胞colony 數目及組織切片，利用免疫組織染色 的方式觀察其migration/invasion 相關基因蛋白層次的影響，以評估其抑制細胞轉移 之能力。Project