近年來國人的十大死因以惡性腫瘤為首，而癌細胞的轉移往往是造成癌症患者致命的原因之一。Inotilone 是桑黃( Phellinus linteus )的主要成分之一，文獻指出桑黃具有抗氧化、抗發炎及抗癌等作用，但目前 inotilone 對於肺癌細胞轉移是否有抑制作用，尚無相關文獻探討，因此本實驗就以Inotilone 來探討對於人類非小細胞肺癌細胞( A549 )及小鼠 Lewis 肺臟惡性腫瘤細胞( LLC )之轉移的抑制作用及其機制的探討。
由細胞移行( Cell migration )及細胞侵襲( Cell invasion )試驗可看到 inotilone 對於 A549 及 LLC 細胞的爬行及入侵能力均有抑制的效果。由明膠蛋白酵素電泳法顯示 inotilone 可減少 MMP-2 和 MMP-9 及 u-PA 的活性；而同樣的也可由西方墨點分析來探討 inotilone 抑制 MMPs 的分泌及促進 TIMPs 的分泌。接著探討inotilone 在 A549 細胞中抑制轉移的相關路徑時則是推測可能與減少和發炎相關蛋白表現量如： iNOS、 COX-2 及抑制 MAPK 及 WNT 途徑。
此外也可從動物實驗中看出 inotilone 抑制癌細胞轉移的能力，以皮下注射方式給予 LLC 細胞，三天後確定誘導成功產生原位癌後即可開始給予 inotilone 治療21天，犧牲後發現在血清中 MMP-2 和 MMP-9 的活性都有顯著的被抑制之外也抑制了發炎因子 TNF-α 的活性而降低 iNOS 及 COX-2 的活性，而 iNOS 活性降低則也連帶的使 NO 含量下降；在動物體內除了抑制了肺臟組織中 MMP-9 和增加 TIMP-2 的活性之外其抗氧化酵素 catalase、 SOD 及 GPx 的活性也因為 inotilone 的治療而提升，如此也可證實 inotilone 的抑制轉移可能也和減少體內過多的氧化壓力有關。
綜合以上的實驗結果， inotilone 應該是具有開發成抑制癌細胞轉移的藥物潛力，期望未來能將其開發為臨床藥物，以利癌症的治療。
According to the statistic analysis the most common death cause is cancer. Metastasis in cancer patients is often fatal cancer caused by one of the reasons. Inotilone is a major component from Phellinus Linteus, one of the traditional Chinese medical herbs, which have been proved to have such effects as anti-oxidation and anti-inflammation. However, the mechanism of inotilone on the anti-metastasis is still unclear.
In this study, inotilone was used to evaluate growth inhibition, the inhibitory effects of inotilone on the growth of tumor cells were determined by MTT assay. Inotilone showed a inhibit effect on cell migration and invasion by transwell chamber assay. In addition, we found that inotilone can reduce the MMP-2, MMP-9 and uPA activity by gelatin and casein-plasminogen zymography. Western blot analysis is then to confer the inhibition of the MMP secretion and the promotion of TIMP secretion. Inotilone in A549 cell inhibited cancer metastasis pathway may reduce the protein expressions of inflammation-related such as iNOS and COX-2…etc, and inhibit the MAPK and WNT pathway.
In addition, the ability of inotilone in inhibition of cancer metastasis can also be seen in animal experiments. C57BL6 mice were implanted subcutaneously with LLC cells and three days later determined the tumor was induced successful then start to give inotilone as treatment. After sacrificing the animals, it was found that inotilone not only reduced the activity of MMP-2 and MMP-9, but also inhibited TNF-α activity and reduced NO content. In LLC-bearing mice, inotilone reduced the MMP-9 and increased the TIMP-2 protein expression in lung tissues and the antioxidant enzymes in lung tissues such as catalase, SOD and GPx activity was improved. So the results also confirm that the anti-metastatic efficacy of inotilone may be related to the antioxidant activities.
The results indicated that inotilone exerted anti-metastatic efficacy associated with not only MAPK and WNT signaling pathways but also related to inflammation-related factors and antioxidant activities.
