School of Radiation Medicine and Protection, Medical College of Soochow University, Suzhou, Jiangsu Province, China.
Cancer Biother Radiopharm. 2011 Dec;26(6):705-15. doi: 10.1089/cbr.2011.0987.
Secreted protein acidic and rich in cysteine (SPARC) has been shown to play an important role in the promotion of glioma. In this study, we investigated the effects of downregulated SPARC expression on the radiosensitivity of human glioma U-87MG cells and its possible mechanism. With a small-interfering RNA (siRNA) expression plasmid vector targeting SPARC, we obtained the stably transfected cells in which the expression of SPARC was successfully downregulated. Then, the cells were irradiated with 60Co-γ-rays and analyzed by several methods, such as clonogenic assay, flow cytometry, comet assay, and western blotting. Clonogenic assay showed that downregulation of SPARC expression enhanced cell survival after radiation. Flow cytometry analysis indicated that SPARCsiRNA decreased cell apoptosis responding to irradiation. Analysis of signaling molecules with western blotting showed that the level of Akt phosphorylation was increased in irradiated U-87MG/SPARCsiRNA cells. Further, cell-cycle analysis by flow cytometry showed enhanced G2 accumulation in U-87MG/SPARCsiRNA cells after irradiation. Comet assay revealed that SPARCsiRNA promoted the repair of radiation-induced DNA damage. Our results suggest that inhibition of SPARC expression may diminish the radiosensitivity of human glioma U-87MG cells. One of the mechanisms for this effect may be associated with the reduced cell apoptosis responding to radiation, which may be contributed by the phosphoinositide 3-kinase/AKT pathway activation. Moreover, enhanced G2 accumulation and increased DNA repair may also account for the decreased radiosensitivity.
富含半胱氨酸的酸性分泌蛋白（SPARC）已被证明在促进神经胶质瘤中发挥重要作用。在这项研究中，我们研究了下调 SPARC 表达对人神经胶质瘤 U-87MG 细胞放射敏感性的影响及其可能的机制。我们使用靶向 SPARC 的小干扰 RNA（siRNA）表达质粒载体，获得了 SPARC 表达成功下调的稳定转染细胞。然后，用 60Co-γ射线照射这些细胞，并通过克隆形成实验、流式细胞术、彗星实验和 Western blot 等方法进行分析。克隆形成实验表明，下调 SPARC 表达增强了细胞对辐射后的存活能力。流式细胞术分析表明，SPARCsiRNA 减少了细胞对辐射的凋亡反应。Western blot 分析信号分子的结果表明，在照射的 U-87MG/SPARCsiRNA 细胞中，Akt 磷酸化水平增加。进一步通过流式细胞术进行细胞周期分析表明，照射后 U-87MG/SPARCsiRNA 细胞中 G2 期积累增加。彗星实验显示，SPARCsiRNA 促进了辐射诱导的 DNA 损伤的修复。我们的结果表明，抑制 SPARC 表达可能会降低人神经胶质瘤 U-87MG 细胞的放射敏感性。这种作用的机制之一可能与减少细胞对辐射的凋亡反应有关，这可能是由磷酸肌醇 3-激酶/AKT 途径的激活引起的。此外，G2 期积累增加和 DNA 修复增强也可能导致放射敏感性降低。
