2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38804Metastasis of cancer cells, a primary cause of cancer death and a multiple and intricate processes, may complicate the clinical management and lead to a poor prognosis for cancer patients and has tremendous physical or economical impact to patients or communities. In general, metastasis of cancer cells involves multiple processes and various cytophysiological changes, including changed adhesive capability between cells and extracellular matrix (ECM) and damaged intercellular interaction. Degradation of ECM by cancer cells via protease, such as serine proteinase, matrix metalloproteinases (MMPs), cathepsins, and plasminogen activator (PA), may lead to the separation of intercellular matrix to promote the mobility of cancer cells and eventually lead to metastasis. Among these involved proteases, MMP-2, MMP-9 and u-PA are the most vital ones for degradation of base membrane and therefore deeply involved in cancer invasion and metastasis. Flavonoids are a group of polyphonic compounds that widely distributed in dietary foods of vegetables and fruits. Documented biologic effects of dietary flavonoids include anti-inflammatory, anti-allergic, antioxidant, capillary strengthening, and anti-cancer potency and modulation of enzyme activities, among others. In the antioxidant aspect, several previous studies indicated that flavones possess an effective inhibitory effect on NO activation induced by LPA or LTA, and the number of hydroxyl (OH) substitutions was a critical factor in the reactive oxygen species (ROS) scavenging activity. In the anti-cancer potency aspect, flavones might be able to influence processes that are dysregulated during cancer development, and possess the propensity to anti-proliferation and induce apoptosis. Compared to the abovementioned aspects, studies on the inhibitory effect of flavones on cancer cell invasion behavior have been relatively less and warrant a further study. In our preliminary study, a renal carcinoma cell 786-0 has been treated with flavones and then subjected to assays for cell viability, invasion and migration. The results showed that some flavones significantly inhibited invasive and migration potential through a decrease of MMP-2 and MMP-9 expressions in 786-0 cell. Therefore, in the first year of this study, we propose to (1) analyze and compare the effects of different flavones on cell viability of different renal carcinoma cells (Caki-1, Caki-2 and 786-0) using MTT assay and trypan blue dye exclusion assay; (2) demonstrate the mechanism involved in reduced invasion by lower doses (not to induce cell apoptosis or cytotoxicity) of different flavones, including changed adhesive capability and invasive ability and ECM degraded proteinases (MMPs and u-PA) activities.; (3) investigate the changes in the cell cycle of different renal carcinoma cells (Caki-1, Caki-2 and 786-0) after different flavones treatment using flow cytometry; (4) exam the colony formation of renal carcinoma cells (Caki-1, Caki-2 and 786-0) after different flavones treatment and (5) confirm the apoptotic effect of different flavones on different renal carcinoma cells (Caki-1, Caki-2 and 786-0) using DNA fragmentation assay, DAPI stain, TUNEL, and Annexin V-conjugated Alexafluor 488. Then, in the second year of this study, we aim to (1) analyze and compare cell cycle related kinases cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs) after different flavones treatment in different renal carcinoma cells (Caki-1, Caki-2 and 786-0); (2) investigate the binding ability between CDKs and CDKIs in different renal carcinoma cells (Caki-1, Caki-2 and 786-0) after different flavones treatment using immunoprecipitated assay; (3) demonstrate the effect of different flavones on apoptosis related proteins in different renal carcinoma cells (Caki-1, Caki-2 and 786-0); (4) determine the effect of the lower doses of flavones on migration, invasion and adhesion related genes expression; (5) further detect the lower doses of different flavones not to induce the apoptosis or cytotoxicity in different renal carcinoma cells (Caki-1, Caki-2 and 786-0) on the signal transduction pathways of adhesion, invasion and migration; and (6) test the lower doses of flavones not to induce the apoptosis or cytotoxicity in different renal carcinoma cells (Caki-1, Caki-2 and 786-0) on the regulation of transcription factors via western blotting and EMSA assay to determine the transactivity of these transcription factors and binding activity with its target binding sequence. Finally, in the third year of this study, we aim to (1) clone the promoter region of MMP-2, -9, u-PA, TIMPs, PAIs and create promoter constructs driving luciferase to detect the effect of different flavones treatment on regulation of selected gene expression via luciferase assay; (2) propose to determine the effect of flavones on enhance cytotoxic of taxol, vinblastine or 5-fluorouracil of renal carcinoma cells. (3) analyze the effect of different flavones on angiogenesis process in vivo, (4) investigate tumor growth of different renal carcinoma cells (Caki-1, Caki-2 and 786-0) in vivo via cancer cell xenografted nude mice model, and (5) finally analyze the effect of different flavones on migration/invasion related genes via a BALB/c nude mice model by immunohistochemistry assay.癌細胞轉移（metastasis）是癌症導致死亡以及治療複雜度提昇的主要原因; 而 癌細胞轉移已知與多種細胞生理改變密切相關，如改變癌細胞與胞外基質 （extracellular matrix，ECM）間及基底膜（basement membrane）的貼附（adhesion） 能力，破壞細胞間交互作用（intercellular interaction）的力量等。癌細胞之所以能 轉移主要是透過癌細胞分泌蛋白分解酵素， 如： serine proteinase 、matrix metalloproteinases (MMPs)、cathepsins 以及plasminogen activator（PA）分解胞外 基質，最後導致intercellular matrix 分離和基底膜的分解，進而使癌細胞migration 及invasion 提昇。而在這些水解酵素中，MMP-2、MMP-9 及urokinase-type PA （u-PA）在癌細胞migration 及invasion 過程中扮演著最重要的角色。 類黃酮(flavonoids)屬於天然多酚類(polyphenol)廣泛存在於日常生活蔬果之 中。具有抗過敏、抗發炎、抗氧化、強化毛細血管、抗癌化以及調節酵素活性等 生物活性。黃酮化合物(flavones)是類黃酮的主要基本結構，在自然界中具有許多 不同取代基之衍生物，其中以氫氧基(-OH)、甲基(-CH3)及醣苷基(-glycoside, -rutinoside)的取代最為常見。在抗氧化的研究中，有文獻指出黃酮化合物可有效抑 制LPS 或LTA 所誘導之NO 活性，且結構上之氫氧基數目越多其抗氧化能力越 強。而在細胞癌化的化學預防方面，發現其可促使癌細胞的凋亡並抑制其增生能 力及抑制正常細胞的癌化。但對於抑制癌瘤細胞侵襲轉移能力方面的研究，則尚 未明瞭。 本實驗室初步以十種不同的黃酮類化合物(flavone, flavonol, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, baicalein, apigenin, luteolin, quercetin 及kaempferol) 處 理腎臟癌細胞株786-0，發現其中有些黃酮化合物可有效降低其細胞存活率；而有 些黃酮化合物不具細胞毒性，但對癌細胞之invasion 及migration 能力則有顯著抑 制。而有些黃酮化合物也會抑制786-0 分泌MMP-9 及MMP-2 的能力。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 ）， 探討不同的黃酮類化合物(flavone, flavonol, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, baicalein, apigenin, luteolin, quercetin 及kaempferol)對不 同腎臟癌細胞株(Caki-1, Caki-2, 786-0) 存活率的影響；【2 】進一步利用 migration/invasion assay、adhesion assay 及zymography 分析方式完成不同的黃酮類 化合物對腎臟癌細胞株(Caki-1, Caki-2, 786-0)，其細胞移動、侵襲、貼附和轉移能 力、基質分解相關蛋白酶活性的影響之分析，包括如：MMPs、TIMPs、plasmin、 u-PA、t-PA、PAIs 及u-PA receptor（u-PAR）等【3】以流式細胞儀（flow cytometry） 測定細胞週期，探討不同的黃酮類化合物對腎臟癌細胞株(Caki-1, Caki-2, 786-0) 其細胞週期的影響；【4】利用colony formation，觀察不同的黃酮類化合物能否抑 制多種不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)形成colony 之能力；以及【5】以 DNA fragmentation assay、DAPI stain、TUNEL、Annexin V-conjugated Alexafluor 488 用來分析不同的黃酮類化合物是否能誘導不同腎臟癌細胞株(Caki-1, Caki-2, 786-0) 之凋謝死亡。本計畫第二年擬進行【1】利用western blot 探討不同的黃酮類化合 物對不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)其與細胞週期相關的cyclin A、cyclin D1、cyclin D2、cyclin B1、cyclin E、CDK1、CDK2、CDK4、CDK6；cyclin-dependent kinase inhibitor（CDKI）: p53、p21、p27、p16 的影響；【2】利用immunoprecipitated （IP），探討不同的黃酮類化合物是否會對腎臟癌細胞株(Caki-1, Caki-2, 786-0)透 過影響與細胞週期相關的CDK1、CDK2、CDK4、CDK6 分別與p21、p27 之結合 情形；【3】以western blot 探討對腎臟癌細胞株(Caki-1, Caki-2, 786-0)細胞凋亡之 相關蛋白表現，包括: caspase 3、caspase 8、caspase 9、PARP、Fas、Fas ligand、 Bcl-xl、Bcl-2、Bax、Bid、t-Bid 及cytochrome C 之release；【4】以RT-PCR 及real-time PCR 分析不同的黃酮類化合物抑制不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)其 migration/invasion 和adhesion 相關基因的影響，包括如：MMPs、TIMPs、plasmin、 u-PA、t-PA、PAIs 及u-PAR 等；【5】進一步探討不同的黃酮類化合物抑制不同腎 臟癌細胞株(Caki-1, Caki-2, 786-0)其細胞移動、侵襲、貼附和轉移能力之蛋白酶分 泌的相關訊息傳遞路徑的蛋白變化，如：與細胞移動和貼附能力有關的訊息傳遞 Rho family、FAK pathway，與影響侵襲能力的基質金屬蛋白水解酶活性有關的訊 息傳遞MAPK family ERK 1/2、p38 及JNK 1/2、phosphatidylinositol 3-kinase（PI3K） /Akt pathway；以及【6】以electrophoretic mobility shift assay（EMSA）、western blot 探討不同的黃酮類化合物是否抑制上述基因的相關轉錄因子表現，或與其target binding sequence 的結合能力，如：MMP-2 的轉錄因子CREB、AP-2、SP-1，MMP-9 的轉錄因子AP-1、NF-κB、c-Jun、c-Fos、SP-1 及u-PA 的轉錄因子Ets-1、NF-κB 等。本計畫第三年擬進行【1】選植基質分解相關蛋白酶（MMP-2、MMP-9、u-PA、 TIMP-1 及PAI-1）的promoter region，構築luciferase/reporter gene plasmid（pGL3）， 送入不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)中，同時處理對目標基因有抑制效果 的黃酮類化合物，以luciferase assay 分析黃酮類化合物對個別基因promoter 的調 控；【2】探討不同的黃酮類化合物對增強抗癌藥(taxol, vinblastine, 5-fluorouracil) 其 誘導腎臟癌細胞凋亡的能力，並進一步探討其中的詳細機制為何？【3】以免疫缺 陷的小鼠（BALB/c nu/nu mice）接種人類不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)，並處理不同的黃酮類化合物，進一步觀察其腫瘤大小、重量及其動物存活 率，分析不同的黃酮類化合物對於接種不同腎臟癌細胞的老鼠其腫瘤大小、重量 及其動物存活率的影響；【4】利用免疫組織染色的方式，分析上述處理不同的黃 酮類化合物其腫瘤組織切片，觀察腫瘤相關因子Ki-67 及一些血管新生因子如： VEGF、PDGF、COX-2、TGF-b等血管新生之相關因子的影響；【5】利用in vivo 實驗將matrigel 混合VEGF 或FGF 處理的不同不同腎臟癌細胞株(Caki-1, Caki-2, 786-0)，再處理不同的黃酮類化合物，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。Project