纖維母細胞生長因子九為纖維母細胞生長因子的成員，這類因子對於胚胎及組織發育皆具有重要作用，且纖維母細胞生長因子九在性別發育成熟具有重要功能。然而，高表達的纖維母細胞生長因子九與幾種人類癌症如肺癌、卵巢癌和前列腺癌的發展及預後息息相關。先前，我們已經證實纖維母細胞生長因子九可誘導MA-10小鼠萊氏腫瘤細胞和TM3小鼠萊氏細胞中的細胞增殖，並在動物實驗中纖維母細胞生長因子九可促進MA-10細胞腫瘤的生長，顯示這可能與睾丸癌的發展相關。此外，蟲草素是一種具有抗腫瘤作用的真菌冬蟲夏草萃取物，由我們的微陣列分析顯示，在蟲草素處理後會影響MA-10細胞中纖維母細胞生長因子九和纖維母細胞生長因子受體的基因表達。因此，我們提出的論點為蟲草素可以抑制FGF9所誘導的睾丸癌腫瘤的生長。在本研究中，我們先利用MEK抑製劑（PD98059）闡明纖維母細胞生長因子九是通過活化ERK 1/2蛋白的方式促進細胞週期相關蛋白的表現。其次，蟲草素處理後可顯著降低纖維母細胞生長因子九對MA-10和TM3細胞的促進細胞增生的作用，並會隨著蟲草素劑量的增加，效果越來越顯著。由西方墨點法的結果顯示，蟲草素亦可抑制纖維母細胞生長因子九處理的MA-10細胞中pRb、E2F1、cyclin A、cyclin E1、cyclin B、CDK4、CDK2和CDK1蛋白的表達，另外也會降低纖維母細胞生長因子九所促進表現的FGFR1-4的蛋白表現量。此外，透過C57BL / 6J小鼠皮下注射MA-10腫瘤細胞的移植研究，結果顯示，蟲草素處理組能夠明顯的抑制纖維母細胞生長因子九所促進腫瘤生長的現象。進一步利用免疫組織化學染色法觀察小鼠的腫瘤切片，顯示與單獨FGF9處理組相比，纖維母細胞生長因子九/蟲草素共同處理組可顯著降低我們外源給予的纖維母細胞生長因子九蛋白的表達。蟲草素也可降低纖維母細胞生長因子九所誘導的FGFR1、FGFR2、FGFR3和FGFR4蛋白表現量。根據以上結果顯示，蟲草素可以抑制MA-10和TM3細胞增殖，並且通過降低MA-10細胞中細胞週期相關蛋白的表達。於小鼠活體試驗中，蟲草素也能有效的抑制纖維母細胞生長因子九所刺激的睾丸腫瘤發生。因此，蟲草素可能為潛在的作為治療睾丸或其他癌症的新型抗癌藥物。

Fibroblast growth factor 9 (FGF9), a member of fibroblast growth factors, exhibits important roles for tissue developments. However, high expression of FGF9 is associated with several human cancers such as lung, ovarian and prostate cancers. Previously, we have shown FGF9 could induce cell proliferation in MA-10 mouse Leydig tumor cells and TM3 mouse Leydig progenitor cells in vitro and in vivo, respectively, which could be associated with testicular cancer developments. It has been shown that Cordycepin, an extract from the fungus Cordyceps sinensis with anti-tumor effect, can reduce MA-10 and TM3 cell proliferations related to tumorigenesis. Our microarray analysis shows that Cordycepin could down-regulate gene expressions of FGF9 and fibroblast growth factor receptors (FGFRs) in MA-10 cells. Thus, we hypothesized that Cordycepin could inhibit FGF9-induced testicular tumorigenesis, and the possible mechanisms were investigated. First, we found that FGF9 could upregulate cell cycle related proteins through the activation of ERK1/2 pathway, which was clarified by using MEK inhibitor (PD98059). Second, cell proliferation rates on FGF9-treated MA-10 and TM3 cells were significantly decreased by Cordycepin treatment in a dose-dependent manner. Western blotting results showed that Cordycepin could possibly reverse pRb, cyclin E1, cyclin A, cyclin B, CDK4, CDK2 and CDK1 protein expression on FGF9-treated MA-10 cells. Furthermore, an allograft study was performed by subcutaneous injection of MA-10 tumor cells to C57BL/6J mice. The results showed that the tumor volumes and tumor weights in FGF9-treated group were significantly higher than that in control groups. FGF9-induced tumor growth in Cordycepin-treated group was significantly smaller than that in FGF9 groups, indicating Cordycepin could inhibit FGF9-induced MA-10 tumor growth. Immunohistochemical staining further revealed that FGF9/Cordycepin group could significantly reduce FGF9 protein expressions compared to the FGF9 alone treatment group. Moreover, Cordycepin could decrease FGF9-induced FGFR1, FGFR2, FGFR3 and FGFR4 protein expression. In summary, Cordycepin could suppress MA-10 and TM3 cell proliferation, and probably by reducing the expressions of cell cycle related proteins in MA-10 cells. Cordycepin also inhibited FGF9-stimulated testicular tumorigenesis in MA-10 cell induced allograft mouse model.

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