Division of Cancer Glycosylation Research, Tohoku Medical and Pharmaceutical University, Sendai, Japan; Division of Cell Recognition Study, Tohoku Medical and Pharmaceutical University, Sendai, Japan.
St Petersburg State Pediatric Medical University, Litovskaya ul., 2, St Petersburg, 194100, Russia.
Biochim Biophys Acta Gen Subj. 2017 Nov;1861(11 Pt A):2778-2788. doi: 10.1016/j.bbagen.2017.07.023. Epub 2017 Jul 29.
Glioblastoma multiforme is one of the most malignant tumors of the human central nervous system characterized by high degree of invasiveness. Focusing on this invasive nature, we investigated whether ganglioside-specific sialidase NEU3 might be involved, because gangliosides are major components of brain tissues, and cell surface sialic acids, as target residues of sialidase catalysis, are thought to be closely related to cell invasion.
NEU3 mRNA levels of human glioblastoma specimens were evaluated by quantitative RT-PCR. Human glioblastoma cell lines, U251, A172, and T98G were used for cell invasion and migration by transwell and cell scratching assay. The molecules involved in the signaling cascade were investigated by western blot and immunofluorescent microscopy.
NEU3 expression was down-regulated in human glioblastoma specimens. In the human glioblastoma cell lines, NEU3 overexpression reduced invasion and migration by promoting the assembly of focal adhesions through reduced calpain-dependent proteolysis, but NEU3 silencing resulted in accelerating cell invasion via disassembly of focal adhesions. In NEU3-silenced cells, elevation of calpain activity and GM3 accumulation were observed, as results of reduced sialidase hydrolysis, localization of calpain and GM3 at the cell lamellipodium being demonstrated by immunofluorescence microscopy.
Sialidase NEU3 was found to exert a great influence on cell invasion in regulation of calpain activity and focal adhesion disassembly and consequent invasive potential of glioblastoma cells.
This first demonstration of sialidase involvement in invasive potential of gliolastoma cells may point to NEU3 as an attractive treatment target of human gliomas.
多形性胶质母细胞瘤是人类中枢神经系统中最恶性的肿瘤之一，其特征是高度侵袭性。针对这种侵袭特性，我们研究了神经节苷脂特异性唾液酸酶 NEU3 是否参与其中，因为神经节苷脂是脑组织的主要成分，细胞表面的唾液酸作为唾液酸酶催化的靶残基，被认为与细胞侵袭密切相关。
通过定量 RT-PCR 评估人胶质母细胞瘤标本中的 NEU3 mRNA 水平。使用人胶质母细胞瘤细胞系 U251、A172 和 T98G 通过 Transwell 和细胞划痕实验研究细胞侵袭和迁移。通过 Western blot 和免疫荧光显微镜研究参与信号级联的分子。
NEU3 在人胶质母细胞瘤标本中表达下调。在人胶质母细胞瘤细胞系中，NEU3 的过表达通过减少钙蛋白酶依赖性蛋白水解促进粘着斑的组装，从而减少侵袭和迁移，但 NEU3 沉默通过破坏粘着斑导致细胞侵袭加速。在 NEU3 沉默的细胞中，观察到钙蛋白酶活性和 GM3 积累升高，这是由于唾液酸酶水解减少、钙蛋白酶和 GM3 在细胞片状伪足的定位导致的，这通过免疫荧光显微镜得到证明。
神经节苷脂酶 NEU3 通过调节钙蛋白酶活性和粘着斑解体以及随后的神经胶质瘤细胞侵袭潜力，对细胞侵袭产生了巨大影响。
这首次证明唾液酸酶参与神经胶质瘤细胞的侵袭潜力，可能表明 NEU3 是人类神经胶质瘤有吸引力的治疗靶点。
