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|標題:||Inhibition and mechanism of the invasion and metastasis in human hepatoma cells by Ganoderma lucidum and its bioactive components in cells and animal models
|關鍵字:||靈芝;Ganoderma lucidum;侵入;赤芝酸;訊息蛋白;轉移;基質金屬蛋白酶;-9;免疫不全裸鼠;invasion;lucidenic acid;MAPK;metastasis;MMP-9;SCID nude mice||出版社:||食品暨應用生物科技學系||摘要:||
靈芝 (Ganoderma lucidum) 是一種具有多種藥理效果的食用性菇蕈類，已被廣泛使用於維持健康與長壽的用途，並用來預防或治療肝臟疾病以及降低癌細胞的侵入與轉移。縱然靈芝的水/酒精萃取物已知具有效抗癌細胞轉移活性，但對於抑制人類肝細胞癌 (hepatocellular carcinoma, HCC) 的侵入性、作用機制與活性成分仍未十分清楚。因此，本研究由一株本土靈芝 (G. lucidum YK-02) 中製備靈芝萃取物 (G. lucidum extract, GLE)，並分離出赤芝酸 (lucidenic acid) A, B, C 與 N 等三萜類 (triterpenoid) 成分，主要探討靈芝萃取物對人類肝癌細胞侵入性與轉移性之抑制效應與分子機制，並確認赤芝酸為抗侵入性活性成分。研究內容共分四大部分：(一) G. lucidum YK-02 靈芝萃取物對肝癌細胞侵入性抑制功效之評估，(二) G. lucidum YK-02 靈芝萃取物在免疫不全裸鼠模式中對肝癌細胞生長與轉移之抑制功效， (三) G. lucidum YK-02 靈芝中赤芝酸抗氧化力與抑制肝癌細胞侵入性效應評估，(四) 靈芝赤芝酸透過調控 MAPK/ERK 訊息路徑，降低 NF-kB 及 AP-1 對 DNA 結合活性，抑制 PMA 誘發肝癌細胞侵入性。
研究結果顯示：(一) 此靈芝萃取物對 PMA (phorbol-12-myristate-13-acetate) 誘發的肝癌細胞侵入性與基質金屬蛋白酶 (matrix metalloproteinase, MMP)-9 活性表現的抑制作用，是藉由抑制訊息路徑中 ERK1/2 (extracellular signal-regulated kinase) 與 Akt (protein kinase B) 發生磷酸化，使其維持在不活性化狀態，進而促使HepG2 細胞核內轉錄因子AP-1 (activator protein-1) 及 NF-kB (nuclear factor-kappa B) 的含量下降所導致的結果。可見該靈芝萃取物對肝癌細胞侵入性具有效抑制效能。(二) 為驗證此靈芝萃取物抑制體內肝癌生長或轉移的實際功效，本研究採用 T 細胞免疫不全裸鼠為實驗動物，以皮下接種與尾靜脈注射 HepG2 細胞作為肝癌細胞生長與轉移的體內模式。結果發現，裸鼠每日管灌 200 mg/kg b.w. 以上劑量之靈芝萃取物，對裸鼠皮下肝癌細胞的生長具有顯著抑制作用；而管灌 50 mg/kg b.w. 以上劑量之靈芝萃取物，亦可有效降低裸鼠體內肝癌細胞之轉移程度。另外，經管灌靈芝萃取物之裸鼠，其血液樣品與腫瘤組織中基質金屬蛋白酶-9 活性與表現量亦有明顯下降趨勢，可見管灌靈芝萃取物對抑制裸鼠體內肝癌細胞轉移與基質金屬蛋白酶-9 的活性及表現量有關。由此動物模式驗證此靈芝萃取物的攝取確實可降低裸鼠體內基質金屬蛋白酶-9 的活性及表現量，並進而具有抑制腫瘤生長與轉移之功效。(三)目前靈芝所具備的藥理與保健功效，雖然多半認定是多醣體或三萜類化合物多種混合成分的共同作用所致，但靈芝萃取物中所含抑制癌細胞侵入與轉移的活性成分之相關試驗資料仍相當有限。靈芝中的三萜類成分在各種體內或體外試驗模式中驗證，具有抗癌、抗腫瘤及抗轉移功效，因此本研究推論，靈芝三萜類成分之ㄧ的赤芝酸可能與抑制肝癌細胞的侵入與轉移有關。為釐清該靈芝萃取物與傳統靈芝萃取物成分上的不同，以及探討其抗侵入性之活性成分，本研究以半製備逆向高效液相層析技術 (reverse-phase HPLC)，由該靈芝萃取物中分離出赤芝酸 A, B, C 與 N 等四種三萜類成分，且其含量比傳統靈芝高達十倍左右。經試驗後發現，赤芝酸 A, B, C 與 N 對 HepG2 細胞經 PMA 誘發之侵入性皆具有抑制效應，可見赤芝酸應為抗侵入性之有效活性成分，其中以赤芝酸 B 作用較為顯著。然而，這種抑制作用卻不與赤芝酸的抗氧化力成正相關。(四) 最後經由赤芝酸 B 對細胞抗侵入性作用機制的探討，並與靈芝萃取物相互比較，發現兩者抑制肝癌細胞侵入性的功效都是透過抑制 ERK1/2 蛋白磷酸化發生，並且降低 AP-1 及 NF-kB 對 DNA 的結合活性，進而降低基質金屬蛋白酶-9 表現量所致。
本研究經由體外細胞模式及體內動物模式的驗證，除證明 G. lucidum YK-02為富含赤芝酸之靈芝，其萃取物具有抗腫瘤生長與轉移之功效外，更發現赤芝酸可能是靈芝中預防肝癌細胞侵入發生的有效活性成分。本研究的完成除使靈芝的保健與藥理效能更加清楚外，亦可提供業界未來開發靈芝相關保健產品之參考。
Ganoderma lucidum is a well-known edible mushroom with various pharmacological effects for health and longevity purposes as well as for reducing the likelihood of invasion and metastasis. Although aqueous/ethanol extract of G. lucidum possessed potential anti-invasive activity, but little is known regarding the anti-invasive bioactive component and underlying mechanisms of G. lucidum extract (GLE) on hepatocellular carcinoma (HCC). In this study, we first prepared the extract from the fruiting body of a strain of G. lucidum (YK-02). Four triterpenoid components, including lucidenic acid A, B, C and N, in an ethanol extract of G. lucidum YK-02 were separated by means of a semi-preparative reverse-phase HPLC. The anti-invasive and anti-metastatic effects and underlying mechanisms of GLE on HCC as well as the bioactive component in GLE were investigated by in vitro and in vivo models. There were four topics in this study: (1) the anti-invasive effect of G. lucidum YK-02 extract on human hepatoma cells; (2) inhibitory effect of GLE on human hepatoma cells growth and metastasis in immunodeficient nude mice model; (3) anti-invasive and antioxidant effects of lucidenic acids isolated from G. lucidum YK-02; (4) lucidenic acid inhibits PMA-induced invasion of human hepatoma cells through inactivating MAPK/ERK signal transduction pathway and reducing binding activities of NF-kB and AP-1.
Our studies have revealed the following findings: (1) The data of Western blots with cell lysates and nuclear extracts indicated that the GLE inhibited the phorbol-12-myristate-13-acetate (PMA)-induced invasion and matrix metalloproteinase (MMP)-9 expression through inactivating the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and protein kinase B (Akt) as well as reducing activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kB) in nucleus of HepG2 cells. Our findings suggested that the GLE was a potential anti-invasion agent on HCC. (2) To further determine the in vivo efficacy of GLE in treating cancer, T cells immune deficient nude mice implanted with HepG2 cells by subcutaneous and tail vein injections were used as the in vivo model for the anti-growth and anti-metastasis evaluations of GLE on HCC. The growth of subcutaneous tumor was inhibited by daily oral administration of GLE at a dosage higher than 200 mg/kg b.w. The metastasis of tumor was also decreased by daily oral administration of GLE at a dosage higher than 50 mg/kg b.w. In addition, we further discovered that the MMP-9 activity and expression in serum and tumor tissue of nude mice were also decreased by oral administration of GLE. According to the results of this in vivo test, it was demonstrated that the efficacy of GLE on anti-growth and anti-metastasis might be through suppressing the MMP-9 activity and expression in animal. (3) Fungal triterpenoids were found to be one of the biologically active compounds responsible for the pharmaceutical effects of Ganoderma. Nevertheless, the individual bioactive component of triterpenoids corresponding to the anti-invasive effects is unclear. To distinguish the differences of composition between the G. lucidum YK-02 and common G. lucidum strain as well as to find out the anti-invasive bioactive component, the triterpenoid components in an ethanol extract of G. lucidum YK-02 were separated by means of a semi-preparative reverse-phase HPLC. Four triterpenoid components, including lucidenic acids A, B, C and N were isolated and the content of lucidenic acids in G. lucidum YK-02 were ten times higher than those of the common G. lucidum. We further found that the treatments with the lucidenic acids A, B, C and N in the presence of PMA resulted in significant inhibitory effects on PMA-induced invasion of HepG2 cells. Lucidenic acid B was found to have the most significant inhibitory effect. According to the results, the lucidenic acids might be the major anti-invasive bioactive component on HCC. However, the anti-invasive effect of lucidenic acids is not correlated to their antioxidant activity. Moreover, (4) the mechanism underlying the anti-invasive effects of GLE and lucidenic acids on the PMA-induced HepG2 cells both might be through inhibiting the phosphorylation of ERK1/2 and reducing AP-1 and NF-kB DNA binding activities, leading to down-regulation of MMP-9 expression.
In this study, the anti-growth and anti-metastasis activities of GLE were demonstrated via the in vitro cells and in vivo animal models. It was known that G. lucidum YK-02 is a strain of G. lucidum with high content of lucidenic acids. We have demonstrated that the lucidenic acids might be the major bioactive components of anti-invasion in G. lucidum YK-02. The results might provide information for the anti-metastatic effects of lingzhi on hepatoma cells and for the application of GLE in the development of high-value health food.
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