Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target.
Validation of SAG/RBX2/ROC2 E3 ubiquitin ligase as an anticancer and radiosensitizing target.
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DOI：
10.1158/1078-0432.ccr-09-1592
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发表时间：
2010-02-01
期刊：
影响因子：
--
通讯作者：
Sun Y
中科院分区：
文献类型：
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作者：
Jia L;Yang J;Hao X;Zheng M;He H;Xiong X;Xu L;Sun Y
SAG（对凋亡敏感的基因，也称为RBX 2或ROC 2）最初被克隆为氧化还原诱导的抗氧化蛋白，后来被表征为SCF E3泛素连接酶的RING组分。SAG过表达在体内外均抑制由多种刺激诱导的细胞凋亡。SAG mRNA在人肺肿瘤组织中过表达，与患者生存率低相关。为了研究SAG是否作为抗癌靶点，我们确定了SAG沉默对细胞增殖、存活和放射敏感性的影响。使用肿瘤组织阵列通过免疫组织化学染色评估SAG蛋白在人肿瘤中的表达。通过siRNA沉默来敲低癌细胞中的SAG表达。通过体外细胞生长和存活测定以及体内原位异种移植肿瘤模型评价SAG沉默的抗癌作用。SAG沉默在人癌细胞中的放射增敏作用通过克隆形成存活测定来确定。通过流式细胞仪分析、半胱天冬酶-3激活试验和凋亡相关蛋白的蛋白质印迹法评价细胞凋亡诱导。与正常组织相比，SAG在多种人类肿瘤组织中过表达。SAG沉默选择性地抑制癌细胞增殖，抑制体内肿瘤生长并使耐辐射癌细胞对辐射敏感。从机制上讲，SAG沉默诱导细胞凋亡与积累的Noxa，而SAG过表达降低Noxa水平和缩短Noxa蛋白半衰期。研究结果表明，SAG E3泛素连接酶在癌细胞增殖和肿瘤生长中起重要作用，可能成为一个有前途的抗癌和放射增敏靶点。
SAG (Sensitive to Apoptosis Gene, also known as RBX2 or ROC2) was originally cloned as a redox inducible antioxidant protein and was later characterized as a RING component of SCF E3 ubiquitin ligases. SAG overexpression inhibits apoptosis induced by many stimuli both in vitro and in vivo. SAG mRNA was over-expressed in human lung tumor tissues with the correlation to a poor patient survival. To investigate whether SAG serves as an anticancer target, we determined the effect of SAG silencing on cell proliferation, survival and radiosensitivity. SAG protein expression in human tumors was evaluated by immunohistochemistry staining using tumor tissue arrays. SAG expression in cancer cells was knocked down by siRNA silencing. The anticancer effects of SAG silencing were evaluated by in vitro assays for cell growth and survival, and by an in vivo orthotopic xenograft tumor model. Radiosensitization of SAG silencing in human cancer cells was determined by clonogenic survival assay. Apoptosis induction was evaluated by FACS analysis, caspase-3 activation assay, and western blotting of apoptosis-associated proteins. SAG was overexpressed in multiple human tumor tissues, compared to their normal counterparts. SAG silencing selectively inhibited cancer cell proliferation, suppressed in vivo tumor growth and sensitized radiation-resistant cancer cells to radiation. Mechanistically, SAG silencing induced apoptosis with accumulation of Noxa, while SAG over-expression reduced Noxa levels and shortened Noxa protein half-life. The findings demonstrated that SAG E3 ubiquitin ligase plays an essential role in cancer cell proliferation and tumor growth, and may serve as a promising anticancer and radiosensitizing target.