Background: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme that catalyzes the oxidation of free thiols to generate disulfide bonds in a variety of proteins, including the cell surface and extracellular matrix. QSOX1 has been reported to be upregulated in a number of cancers, and the overexpression of QSOX1 has been correlated with aggressive cancers and poor patient prognosis. Glioblastoma (GBM) brain cancer has been practically impossible to treat effectively, with cells that rapidly invade normal brain tissue and escape surgery and other treatment. Thus, there is a crucial need to understand the multiple mechanisms that facilitate GBM cell invasion and to determine if QSOX1 is involved.Methods and Results: Here, we investigated the function of QSOX1 in human glioblastoma cells using two cell lines derived from T98G cells, whose proliferation, motility, and invasiveness has been shown by us to be dependent on disulfide bond-containing adhesion and receptor proteins, such as L1CAM and the FGFR. We lentivirally introduced shRNA to attenuate the QSOX1 protein expression in one cell line, and a Western blot analysis confirmed the decreased QSOX1 expression. A DNA content/cell cycle analysis using flow cytometry revealed 27% fewer knockdown cells in the S-phase of the cell cycle, indicating a reduced proliferation. A cell motility analysis utilizing our highly quantitativeSuperScratchtime-lapse microscopy assay revealed that knockdown cells migrated more slowly, with a 45% decrease in migration velocity. Motility was partly rescued by the co-culture of knockdown cells with control cells, indicating a paracrine effect. Surprisingly, knockdown cells exhibited increased motility when assayed using a Transwell migration assay. Our novel chick embryo orthotopic xenograft model was used to assess the in vivo invasiveness of knockdown vs. control cells, and tumors developed from both cell types. However, fewer invasive knockdown cells were observed after about a week.Conclusions: Our results indicate that an experimental reduction in QSOX1 expression in GBM cells leads to decreased cell proliferation, altered in vitro migration, and decreased in vivo invasion.
背景：巯基氧化酶1（QSOX1）是一种催化游离巯基氧化形成二硫键的酶，作用于多种蛋白质，包括细胞表面及细胞外基质蛋白。已有研究报道QSOX1在多种癌症中表达上调，其过表达与侵袭性癌症及不良预后相关。胶质母细胞瘤（GBM）因其细胞快速侵袭正常脑组织并逃逸手术及其他治疗，目前仍缺乏有效治疗手段。因此，深入探究促进GBM细胞侵袭的多重机制，并明确QSOX1是否参与其中至关重要。 方法与结果：本研究采用源自T98G细胞的两株细胞系探究QSOX1在人胶质母细胞瘤细胞中的功能。我们前期研究已证实该细胞系的增殖、运动及侵袭能力依赖于含二硫键的黏附蛋白与受体蛋白（如L1CAM与FGFR）。通过慢病毒转导shRNA技术，我们在其中一株细胞系中成功敲减QSOX1蛋白表达，Western blot分析证实QSOX1表达降低。流式细胞术DNA含量/细胞周期分析显示，敲减细胞处于S期的比例减少27%，表明其增殖能力减弱。采用高定量SuperScratch延时显微成像技术进行的细胞运动分析表明，敲减细胞迁移速度减缓45%。将敲减细胞与对照细胞共培养可部分恢复其运动能力，提示存在旁分泌效应。值得注意的是，Transwell迁移实验却显示敲减细胞运动能力增强。我们构建的新型鸡胚原位异种移植模型用于评估敲减细胞与对照细胞的体内侵袭能力，两种细胞均能形成肿瘤。但约一周后观察发现，敲减细胞的侵袭数量显著减少。 结论：实验结果表明，降低GBM细胞中QSOX1表达可导致细胞增殖减弱、体外迁移模式改变及体内侵袭能力下降。
QSOX1 Modulates Glioblastoma Cell Proliferation and Migration In Vitro and Invasion In Vivo
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