摘要:Sirtuins (SIRTs)為一群高度保守的菸醯胺腺嘌呤二核苷酸(NAD+)-依賴賴氨酸修飾酶的家族, 具有去乙醯酶、核糖轉移酶及其他脫醯酶的活性。哺乳類動物目前已知有七種sirtuins,名為 SIRT1-7,它們調控著許多細胞生理生化反應,像是細胞存活、代謝、老化和癌症等等。過去 許多研究指出sirtuins在不同的器官及標的中,在抗老化及癌症形成中扮演著重要的角色。 Sirtuins與血管新生的關係也被探討著,尤其SIRT1和SIRT6。軟骨肉瘤(chondrosarcoma)是 常見的惡性骨腫瘤之一,具有近端入侵和遠端轉移的能力,因此傳統的放射線療法以及化療 方法對於軟骨肉瘤效果有限。臨床上,外科手術切除仍是軟骨肉瘤主要的治療方法。由於目 前軟骨肉瘤並沒有一個很好的治療方式,若能找到一個治療的標的是很重要的。目前有關 sirtuins在軟骨肉瘤中所扮演的角色實在不清楚。因此,我們計晝探討sirtuins在軟骨肉瘤細胞 生長及腫瘤生成上之角色,並與正常人類軟骨細胞生長比較。根據本計晝預實驗結果顯示, SIRT1-7在人類正常軟骨細胞及軟骨肉瘤細胞的表現上,SIRT1和SIRT6的蛋白表現在軟骨 肉瘤細胞上的表現有顯著較正常軟骨細胞為多的現象;另外,為了探討SIRT1在軟骨肉瘤中 所扮演的角色,給予SIRT1的活化劑-白藜蘆醇(resveratrol),結果顯示白藜蘆醇在不影響正常 軟骨細胞生長的劑量下,會造成軟骨肉瘤細胞凋亡,並且會造成細胞内SIRT1、乙醯化NF-kB 和半胱氨酸蛋白酶3(caspase 3)的表現增加,給予SIRT1-siRNA處理會有效逆轉白藜蘆醇的作 用;這些結果顯示SIRT1雖然在軟骨肉瘤細胞表現較正常細胞高許多,但當其受到SIRT1活 化劑刺激時,SIRT1增加,促進NF-kB乙醯化及引起軟骨肉瘤細胞凋亡。因此,根據文獻檢 索及預試驗結果,我們擬進行下列研究:(1).以人類軟骨肉瘤細胞培養模式深入分析SIRT1-7 的蛋白與基因表現和在細胞中分布情形,以及利用小干擾核醣核酸轉殖(siRNAs)或基因過度 表現(gene overexpression)來了解SIRT1-7對於軟骨肉瘤細胞生長/死亡(凋亡、壞死性凋亡、 或自噬)調節及腫瘤生成因子之影響情形和釐清相關之分子機制,並與人類正常軟骨細胞比 較;也計晝以購買的人類正常軟骨及軟骨肉瘤組織切片測試SIRT1-7表現情形。(2).為釐清 SIRT1對於軟骨肉瘤的影響,以人類軟骨肉瘤細胞培養液引發細胞和動物血管新生模式,以 及利用軟骨肉瘤細胞異種移植動物模式,探討SIRT1在腫瘤生長及血管新生上的角色,配合 給予SIRT1活化劑-白藜蘆醇或SIRT1-siRNA,並與人類正常軟骨細胞比較。(3).為釐清SIRT6 對於軟骨肉瘤的影響,以人類軟骨肉瘤細胞培養液引發細胞和動物血管新生模式,以及利用 軟骨肉瘤細胞異種移植動物模式,探討SIRT6的角色,配合給予SIRT6活化劑-核纖層A (lamin A)或SIRT6-siRNA,並與人類正常軟骨細胞比較。此計晝之執行除了能釐清sirtuins在軟骨肉 瘤細胞/腫瘤中所扮演的角色,並且也提供了將來針對軟骨肉瘤一個可能的治療標的。期望將 來能夠提供用於臨床對於軟骨肉瘤治療上另一個不同的選擇。
Abstract: Sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein lysine modifying enzymes with deacetylase, adenosine diphosphate ribosyltransferase and other deacylase activities. Mammals have seven sirtuins, namely sirtuin 1-7 (SIRT1-7). They are key regulators for a wide variety of cellular and physiological processes such as cell survival, metabolism, aging, cancer, and so on. Several studies have suggested that sirtuins could be of great important to both antiaging and tumorigenesis, depending on its targets in specific signaling pathway or in specific cancers. The relationship between sirtuins and angiogenesis has been studied, especially SIRT1 and SIRT6. Chondrosarcoma is a primary malignant bone cancer with a potent capacity to invade locally, and cause distant metastasis and responding poorly to both chemotherapy and radiation therapy, making the management of chondrosarcomas a complicated challenge. Clinically, surgical resection remains the primary mode of therapy for chondrosarcoma. Due to the absence of an effective adjuvant therapy, this mesenchymal malignancy has a poor prognosis and therefore, it is important to explore a novel and adequate remedy target. The role of sirtuins in chondrosarcoma remains largely unknown. Therefore, in this project, we would like to investigate the role of sirtuins in cell growth and tumorigenesis of chondrosarcoma cells. In our preliminary study, we screened the protein expressions of SIRT1-7 in human normal chondrocytes and human chondrosarcoma cells. Results showed that protein levels of SIRT1 and SIRT6 expressed higher in human chondrosarcoma cells than in human chondrocytes. Moreover, we also examined the effect of SIRT1 activator resveratrol in human chondrosarcoma cells. Resveratrol upregulated SIRT1 expression and induced cell death, apoptosis, and deacetylation of NF-kB in human chondrosarcoma cells, which could be reversed by SIRT1-siRNA transfection. There are three parts in this project: (1). To investigate the protein and gene expressions of SIRT1-7 and their cellular distribution in human chondrosarcoma cells, comparing with normal human chondrocytes; the role of SIRT1-7 in cell growth/cell death (apoptosis, necroptosis, or autophagy) and tumorigenesis factors using the transfection of siRNAs or gene overexpression ; we also plan to use commercial human normal cartilage and chondrosarcoma tissue slides to test the expressions of SIRT1-7. (2). To investigate the role of SIRT1 in in vitro and in angiogenesis induced by the culture medium of human chondrosarcoma cells, and in tumorigenesis in a xenograft animal model coupled with SIRT1 activator (resveratrol) or siRNA treatments. (3). To investigate the role of SIRT6 in in vitro and in vivo angiogenesis induced by the culture medium of human chondrosarcoma cells, and in tumorigenesis in a xenograft animal model coupled with SIRT6 activator (lamin A) or siRNA treatments. Taken together, this project tries to reveal the role of sirtuins in chondrosarcoma and provide possible remedy target of chondrosarcoma in the future.