Zoledronate 為臨床上針對骨骼併發症及預防骨質疏鬆之標準用藥，近年來對於部分癌症之骨轉移治療也有相當療效，但其對於骨肉瘤在癌轉移機制之探討仍有待釐清。我們利用 Zoledronate (0-100 μM)處理四株骨肉瘤細胞株 Saos2、MG-63、HOS 及 U2OS，結果發現，Zoledronate 不影響骨肉瘤細胞的存活率。此外我們發現經 Zoledronate處理後，骨肉瘤細胞其細胞型態明顯改變並且發生細胞骨架的降解，利用 Wound healing assay、Migration assay 及 Invasion assay 發現Zoledronate 抑制骨肉瘤細胞的移動及侵襲作用。但是進一步利用Gelatin zymography 卻 發現 Zoledronate 並不影 響 骨肉 瘤 細 胞 的Matrixmetalloproteinase(mmp)-2 以及 MMP-9 的活性。另外，我們利用 Western blot 方式觀察 Zoledronate 對於骨肉瘤細胞 Epithelial mesenchymal transition (EMT)相關蛋白的影響，結果發現 Zoledronate 增加 E-cadherin 同時抑制 N-cadherin 蛋白表現，尤以 U2OS 細胞最為明顯。接著我們觀察 Zoledronate 對於 U2OS 細胞調控 EMT 之轉錄因子及相關訊息傳遞路徑蛋白影響為何，結果發現 Zoledronate 透過抑制轉錄因子 Slug 和 Twist 來增加 E-cadherin 並抑制 N-cadherin 表現且減少磷酸化 Jun N-terminal kinases (JNK)、磷酸化 p38 以及上游 Focal adhesion kinase (FAK)/Src 訊息傳遞路徑來降低 U2OS 細胞轉移能力。我們更發現 Zoledronate 影響小分子 G 蛋白(Small GTPase protein) Rho A 以及 Cdc42 的 geranylgeranylation 和活化作用進而促使骨架蛋白F-actin 降解。過去研究指出 GGOH (geranylgeraniol)及 FOH (farnesol)能有效回復由 Zoledronate 所造成的抑制現象，因此本篇利用外源性GGOH 及 FOH 來探討其影響機制為何。結果發現 GGOH 而非 FOH，能夠使 Zoledronate 所抑制上述之抗癌轉移能力及細胞貼附作用有回復的現象。綜合以上結果印證 Zoledronate 是透過抑制 Rho A 以及Cdc42 的 geranylgeranylation 來降低 FAK 及其下游 JNK 和 p38 訊息傳 遞路徑而阻斷 EMT，進而抑制人類骨肉瘤細胞株 U2OS 細胞黏附和 細胞與細胞間交互作用、移動及侵襲的能力。未來希望 Zoledronate 能廣泛應用於骨癌轉移及治療上。 Zoledronate is a standard treatment for preventing skeletal complications of osteoporosis and some types of cancer associated with bone metastases, but we little know whether the effect of zoledronate on metastasis of osteosarcoma. Here, we investigated the inhibitory effects of zoledronate on cell viability, motility, migration and invasion of 4 osteosarcoma cell lines (Saos2, MG-63, HOS and U2OS) by affecting cell morphology, epithelial-mesenchymal transition (EMT) and cytoskeletal organization as well as induction of E-cadherin and reduction of N-cadherin with activation of transcription factors Slug and Twist, especially in U2OS cells. Zoledronate decreased JNK and p38 phosphorylation and upper streams of focal adhesion kinase (FAK) and Src to suppress the motility, invasiveness and migration of U2OS cells. In addition to zoledronate-inhibited Rho A and Cdc42 membrane translocation and GTPg S activities, the anti-metastatic effects in U2OS cells including inhibition of adhesion were reversed by geranylgeraniol, but not farnesol. In conclusion, Zoledronate blocks geranylgeranylation not farnesylation to suppress human osteosarcoma U2OS cell-matrix and cell-cell interactions, migration potential, the invasive activity, and the adhesive ability by EMT via Rho A activation and FAK-inhibited JNK and p38 pathways.
