Eg5蛋白靶向抑制剂S—Trityl—L—Cysteine对神经母细胞瘤增殖和分裂的影响

Effects of S-trityl-L-cysteine, an Eg5 inhibitor, on proliferation and cell division in neuroblastoma

目的研究驱动蛋白Eg5在神经母细胞瘤中表达及其靶向抑制剂S-三苯甲基-L-半胱氨酸（S-Trityl—L-Cysteine，STLC）对神经母细胞瘤的诱导作用。方法通过免疫荧光及Westernblot检测Eg5蛋白在神经母细胞瘤中的表达水平，通过相差显微镜观察STLC对细胞形态学的影响，采用CCK-8法、流式细胞术检测STLC对细胞增值、凋亡及周期的影响，采用FISH法检测STLC对原癌基因MYCN扩增的影响，并经DAPI细胞核染色实验观察STLC对细胞核形态变化的影响。结果免疫荧光及Western blot结果显示Eg5蛋白在神经母细胞瘤中稳定表达。细胞形态学观察结果表明，细胞经不同浓度的STLC诱导，当浓度≥5μmol／L，诱导时间为72h时，细胞形态变圆，出现大量凋亡样小体。CCK-8细胞增殖能力检测及流式细胞术分析结果显示，STLC具有促进细胞凋亡，抑制细胞分裂与增殖的作用，将细胞周期阻滞在G2／M期，且其诱导作用呈时间剂量依赖性。FISH实验证实STLC对MYCN基困扩增无影响。结论神经母细胞瘤组织及细胞株中均表达Eg5蛋白，STLC通过抑制Eg5，影响神经母细胞瘤细胞的分裂及增值，从而促进细胞凋亡。结果表明Eg5有望作为神经母细胞瘤分子治疗的靶点。

Objective To determine the expression levels of Eg5 in neuroblastoma and elucidate the effect of Eg5 specific inhibitor S-trityl-L-cysteine （STLC） on cell proliferation and cell division in neuroblastoma. Methods Immunofluorescence and Western blot was performed for detecting the expression of Eg5 in neuroblastoma tissues and cells. After an induction of STLC, cell morphology was observed under phase contrast microscope. And cell proliferation, cell cycle and cell apoptosis were examined with CCK8 assay and flow cytometry. MYCN expression was assayed by fluorescence in situ hybridization （FISH） after STLC induction. Nuclear staining by 4＂, 6-diamidino-2-phenylindole （DAPI） was performed for observing the morphological changes. Results Eg5 was expressed in neuroblastoma tissues and cells. There were changes of cell morphology and apoptotic bodies were observed. Eg5 inhibitor effectively prevented the proliferation and arrests cells at G2/M phase and promoted cell apoptosis in dose and time-dependent ways. STLC had no effect on the expression of MYCN. Conclusions Eg5 is expressed both in neuroblastoma tissues and cells. And STLC suppresses the proliferation and cell division, arrests cells at mitosis and induces cell apoptosis. Eg5 may become a therapeutic target for neuroblastoma.