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，我們發現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中也觀察到FIP-gt影響A549的MMP-2及MMP-9的表現能力，且利用RT-PCR的方式觀察MMP的抑制劑TIMP-1和TIMP-2，發現到TIMP-1則受到重組FIP-gts蛋白增加其表現，但TIMP-2則沒有變化，而MMP-2的量同時也看到表現量的減少，因此得知重組FIP-gts蛋白能抑制MMP-2的活性，使得細胞移動性也被抑制。在此時，我們也發現到重組FIP-gts蛋白有誘導Nm23 抑癌蛋白及其mRNA活化的能力，進而導致抑制細胞的轉移能力，且在過度表現Nm23蛋白的A549細胞中，也觀察到細胞的移動能力很明顯下降許多。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。所以本研究闡示松杉靈芝免疫調節蛋白FIP-gts有潛力發展成臨床抑制細胞轉移能力的藥劑。 A large number of polysaccharide and triterpenoids of Ganoderma lucidum has been reported to inhibit the growth of tumors. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. In this study, we expressed and purified the fungal immunomodulatory protein (FIP-gts) in E. coli. However, the effect of FIP-gts on cancer cells has not been characterized clearly. Metastasis is one of the most difficult problems in cancer therapy. In wound healing assay, the level of motility have been decreased by the increasing concentration of FIP-gts in the previous study. In order to confirm the effect of FIP-gts, the Modified Boyden chamber assay was applied. Treatment of A549 cells with up to 0.07 ？H？HM of FIP-gts decreased cell migration by 11% for 8 h. 0.075 ？H？HM of FIP-gts decreased cell invasion significantly. In addition, Nm23 protein and mRNA was increased by reFIP-gts addition on A549 cells. We also observed the inhibitor of cell migration by overexpression of Nm23 protein in A549 cells. The secreted MMP-2 activity was notably inhibited measured by gelatin-zymography. Inhibitioty effect the FIP-gts on the MMP-2 activity of A549 cells in gelatin-zymography assay. In RT-PCR, the level of MMP-2 and MMP-9 were decreased but the MMP inhibitor of TIMP-2 had no effect and TIMP-1 increased after treated with FIP-gts. To further ascertain the antimetastatic activity of FIP-gts, we assessed the counteraction of FIP-gts with monoclonal FIP-gts antibody. Monoclonal FIP-gts antibody could reverse cell migration by 55%. In gelatin-zymography assay, MMP-2 activity was also slightly increased. We also observed different proteins that changed between untreated or treated with FIP-gts in A549 cells using 2-DE gel analysis. We are going to identify these proteins in the future. Our results highlight the potential of FIP-gts in the treatment of clinical cancer metastasis.
