評估靈芝免疫調節功能蛋白抑制肺癌細胞轉移能力

柯俊良
中山醫學大學
松杉靈芝(Ganoderma tsugae)中分離出免疫調節蛋白質 (FIP-gts)，其分子量13 kDa。此蛋白質對於人類周邊血液細胞具有刺激增殖並誘發TNF-a大量表現，可能是促進Th1路徑有關。在動物試驗中已被證實可當作免疫抑制劑。靈芝一直以來被視為健康食品的一員，以細胞和動物實驗證實靈芝具有保護肝臟、抗腫瘤等功能。腫瘤的治療一直是大家關心的事，尤其是許多人著重於促進腫瘤細胞死亡來達到減緩腫瘤的形成。肺癌是一非常惡性的腫瘤，主要是大部分的肺癌細胞容易進行轉移，經由轉移侵入至其他器官部位造成更大傷害且不易進行治療和控制。所以本計畫考量抑制腫瘤細胞的運動性、轉移性及侵入性的方面著手。所以本研究計畫已先期著手建立了一轉移能力相當強的細胞株：將一點突變型p53(H179Y的突變)表現載體轉染入肺癌A549細胞株中作為主要的分析系統，此細胞的轉移能力相當強。惡性細胞則會以輻射狀向四週擴散，並很快且大量的吸取養分、壓迫和和毀滅相鄰的正常細胞，但我們初步實驗證實松杉靈芝蛋白(FIP-gts)會造成肺癌細胞皺縮，是否有抗腫瘤轉移的特性仍有待進一步分析，所以擬分析FIP-gts毒殺腫瘤細胞和抑制腫瘤細胞的運動性、轉移性及侵入性。癌細胞的轉移通常必須伴隨著一些生理變化，其中主要包括細胞外基質的瓦解，改變細胞與細胞基質間的貼附能力以及調控細胞的移動性等來影響細胞的侵入能力。首先，藉由利用Boyden chamber assay 與 cell-matrix adhesion assay，我們將探討FIP-gts抑制A549(H179Y)肺癌細胞p53突變株侵入的能力，並在gelatin zymography與casein zymography assay中分析FIP-gt影響A549的MMP-2及MMP-9的表現能力。並再進一步將A549(H179Y)肺癌細胞打入裸鼠中，觀察經由松杉靈芝蛋白(FIP-gts)處理後，是否在動物實驗中有預防、治療或抑制肺癌細胞的腫瘤形成和轉移作用。期望能建立一套評估減緩肺癌轉移能力的方法，發展出能預防、治療或抑制腫瘤細胞之輔助性中藥。
關鍵字：靈芝免疫調節蛋白；肺癌轉移

Inhibition of lung metastasis by fungal immunomodulatory protein, FIP-gts, from Ganoderma tsugae

Jiunn-Liang Ko
Institute of Medical and Molecular Toxicology, ChungShan Medical University
Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. The protein has been implicated which activates human peripheral blood mononuclear cells. However, the effect of FIP-gts in cancer cells has not clearly been described. Metastasis was one of the most difficult problems in lung cancer therapy. We observed the A549 cells turn into round shape and were not changed cell shapes in normal cell line-BEAS-2B after FIP-gts treatment. MTS anlysis and counting cell numbers decreased the viability of A549 cells. We will investigate cytotoxicity to A549 cells using colony formation assay. FIP-gts induced p53 expression that in terms induced p21 activity, which caused in G1 arrest. In addition, procaspase-3 by western blotting was decreased. Similarly, there was a small amount of subG1 increased after FIP-gts treatment. Metastasis is a multiple step including high moility, high proteinase activity and high pentration activity. Therefore, we will analyze the wound healing assay to determinewhether the level of motility were decreased by the increasing concentration of FIP-gts. To understand the inhibition mechanism of metastasis by FIP-gts, MMP-2 activity would be also measured by gelatin-zymography. In RT-PCR, the level of MMP-2 and TIMP-2 would be investigate after treated with FIP-gts. Alltogether, FIP-gts could not only cause cytotoxicity but also potentially inhibit metastasis.
關鍵字：FIP-gts；Lung metastasis