中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/41330
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    Please use this identifier to cite or link to this item: http://ir.cmu.edu.tw/ir/handle/310903500/41330


    Title: AC10 及其主成分抑制小鼠黑色素瘤細胞B16F1 和B16F10 之抗 腫瘤功效：Wnt/β-catenin signaling pathway 的機制調控
    Anti-tumor Activity of AC10 and its major compound Against Murine Melanoma Cells Through the Modulation of Wnt/β-catenin Signaling Pathway
    Authors: 鄒曉彤
    Contributors: 營養學系碩士班
    Keywords: AC-10;Wnt/β-catenin pathway;細胞凋亡;細胞週期;轉移 Antrodia Camphorata;Wnt/β-catenin pathway;melanoma;metastasis;Apoptosis;cell cycle
    Date: 2011-07-27
    Issue Date: 2011-10-17 16:51:58 (UTC+8)
    Publisher: 中國醫藥大學
    Abstract: 黑色素瘤(melanoma)為皮膚惡性腫瘤的一種，惡性度最高，容易轉移，對西方人而言是皮膚疾病中發病率佔第一位的死亡原因，因黑色素瘤對化學治療及放射線治療的效果不佳，又伴隨著高度的轉移性；再者，目前對於治療黑色素瘤 (melanoma) 藥物的研究尚未完善；若能尋找到輔助癌症治療的天然食材，是當前重要的課題。
    Wnt/β-catenin 訊息途徑可調控許多細胞的進程，包括增生(proliferation)、分化(differentiation)、存活(survival)、細胞凋亡(apoptosis)及細胞的移行(motility)。約有30%的黑色素瘤可觀察到Wnt/β-catenin訊息途徑異常活化的現象，因此抑制Wnt/β-catenin 訊息途徑也成為治療黑色素瘤的策略之一。
    樟芝(Antrodia camphorata；AC) 為台灣常見的傳統中藥，這種特有的蕈類生長於牛樟樹上，先前文獻指出樟芝具有抗癌、抗發炎及調節免疫等功效。在第一部分的實驗利用樟芝發酵液(AC-10)，作用在小鼠黑色素瘤細胞B16F1及B16F10，其IC 50皆約為80ug/mL。在細胞凋亡分析上，AC-10具有劑量效應誘發細胞凋亡的能力，並伴隨p53的蛋白表現增加，cytochrome c 釋出至細胞質、BAX/Bcl-2 的ratio 提高以及caspase-3 、caspase-9以及PARP的裂解。在Wnt/β-catenin調控路徑之下，利用免疫螢光染色法發現AC-10可抑制β-catenin入核表現，並且降低核質β-catenin的蛋白表現；再同時給予AC-10及MG132(proteasome inhibitor)，則會使β-catenin的蛋白表現量回復，並利用SB216763(GSK3β抑制劑) 得知AC-10降解β-catenin為GSK3β依賴型(GSK3β-dependent)。但由RT-PCR實驗得知，AC-10 並不改變β-catenin mRNA 表現。而同時給予Cycloheximide (CHX, protein synthesis inhibitor)及AC-10可縮短β-catenin
    蛋白質降解的半衰期，此現象說明了AC-10造成β-catenin的減少原因為加速蛋白質的降解作用。同時也利用Reporter assay觀察到AC-10可抑制β-catenin調控的轉錄活性，導致下游標的基因如c-myc、cyclin D1、survivin、MMP-9及VEGF的減少。Flow cytometry分析AC-10會使黑色素細胞瘤皆停滯在G1期，並連帶的抑制細胞週期蛋白cyclin D1、CDK4；增加p27、p21的表現。在細胞轉移部分上，細胞刮傷試驗(wound healing assay)以及細胞侵襲試驗(invasion assay)測定發現AC-10可降低癌細胞遷移的表現，並連帶的使MMP-9、MMP-2及VEGF表現量減少。
    在第二部分的實驗中，另以AC-10純化後產物AC-0進行體外試驗觀
    察到AC-0也具有抑制β-catenin及促進細胞凋亡表現，由RT-PCR實驗得知，AC-0同樣不會改變β-catenin mRNA表現，而同時給予CHX及AC-0卻無法加速β-catenin蛋白質的降解，因此推測AC-0雖無法加速降解，但令β-catenin減少仍為蛋白降解作用。利用SB216763(GSK3β抑制劑)得知AC-0降解β-catenin為GSK3β非依賴型(GSK3β-independent)；並利用免疫沉澱法(IP)來探討GSK3β與β-catenin之交互作用，同樣也證明了GSK3β的非依賴型。
    又以體內實驗(裸鼠腫瘤模式)證實AC-0確實可以抑制裸鼠植入B16F10誘發之腫瘤大小、重量、及體積，並且經免疫組織染色分析、腫瘤蛋白分析得知β-catenin及其target gene表現也被抑制。
    總結本研究得知AC-10及其主成分AC-0可藉由抑制Wnt/β-catenin訊
    息路徑，而達到抑制腫瘤生長及轉移的效用。
    Melanoma is the most serious form of skin cancer. Aberrant activation of Wnt/β-catenin signaling cascade has been observed in approximately

    one-third of melanomas, indicating that modulation of Wnt/β-catenin activation might be a novel strategy for melanoma treatment. The

    downstream targets of Wnt/β-catenin signaling pathway including c-Myc, cyclinD1, MMPs, and survivin, which are regulating number of cellular

    functions, such as proliferation, differentiation, survival, apoptosis and invasion. Antrodia camphorata, a well known medicinal mushroom in Taiwan that has been used as Chinese folk medicine for many years.

    Previous studies have shown that A. camphorata (AC) possessed greater anti-tumor activity against a variety of tumor cells. However, the anti-tumor efficacy of AC against melanoma was poorly understood. In addition, the currently employing treatment for melanoma is a tough topic, due to the high resistance to radio-and chemotherapy and most the synthetic

    chemotherapeutic drugs are volnarable to non-melanoma skin cells.Therefore, the present study, we aimed to investigate the anti-tumor efficacy

    of fermented culture broth extracts of A. camphorata (AC-10) and it derived pure compound (AC-0) in murine melanoma cells. The first set of experiment, we observed AC-10 treatment significantly decreased murine melanoma B16F1 and B16F10 cell viability with an IC 50

    value of 80ug/mL. The reduction of cell viability is directly correlated with the inhibition of β-catenin and its downstream protein expression.

    Immunofluorescence analysis confirmed that AC-10-treatment markedly reduced β-catenin translocation into the nucleolus, and also downregulates

    ??-catenin-mediated transcriptional activity. Furthermore, MG132 a proteosomal inhibitor that prevent proteosomal degradation of β-catenin,

    conversely, GSK3β inhibitor SB216312 also suppressed β-catenin degradation which strongly suggest that β-catenin degradation is GSK3β dependent. In a similar way, AC-10-treatment attenuate GSK3β expression, suggesting that AC-10 may regulate the proteasomal

    degradation of β-catenin by GSK3β-dependent nmechanism. Furthermore, flow cytometry analysis showed that AC-10-treatment significantly arrest

    G1 to S-phase transition followed by the suppression of cyclin D1 (wnt/β-catenin target gene), CDK4 expression and increased in p27, p21

    levels. Moreover, TUNEL assay revealed that AC-10-treatment induce apoptosis in a dose-dependent manner, followed by the disregulation of

    BAX/BCL-2 ratio, and the down-regulation of pro-caspase-9, pro-caspase-3 and pro-PARP. Migration and invasion assay shows AC-10 could abate melanoma metastatic ability, through the inhibition of MMP-9, MMP-2,

    VEGF expression, which are also wnt/β-catenin target genes. In the second part, AC-0 also found to effectively inhibit Wnt/β-catenin pathway cascades, and suppressed β-catenin-mediated transcriptional

    activity. Similar with AC-10, AC-0 treated cell found to decreased melanoma metastasis and augmented apoptotic induction. MG132 and AC-0

    treatment prevents β-catenin expression, however, SB216312 failed to prevent GSK3β expression, which suggesting that AC-0-treatment may regulate the proteasomal degradation of β-catenin via GSK3β-independent mechanism. This phenomenon also demonstrated with immunoprecipition assay that AC-0 treated cells decreased the interaction between β-catenin and GSK3β.

    In vivo study, AC-0 decreased the growth of B16F10-derived tumors development in the athymic nude mice. The decreased B16F10-derived tumor growth was associated with a down-regulation of Wnt/β-catenin target

    genes such as c-myc, cyclin D1, MMP-9. AC-0 treatment also induced B16F10-derived tumors apoptosis in nude mice. In conclusion, our data demonstrated that AC-10 and its major compound AC-0 appreciably modulate Wnt/β-catenin pathway in melanoma cells.

    Therefore, we believe AC-10/AC-0 might be a potential chemo-preventive agent for melanoma treatment.
    Appears in Collections:[Graduate Institute of Nutrition ] Theses & dissertations

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