异硫氰酸苄酯抑制人胶质瘤U87MG细胞体外侵袭和诱导凋亡的作用和机制

Effect and Mechanism of Benzyl Isothiocyanate Inhibiting Invasion and Inducing Apoptosis of Human Glioma U87MG Cell Lines

关键词:  异硫氰酸苄酯  脑胶质瘤  U87MG细胞  侵袭

Key words:  benzyl isothiocyanate  gliomas  U87MG cell lines  invasion

摘要:

目的 研究异硫氰酸苄酯对人胶质瘤U87MG细胞体外侵袭和凋亡的影响，并初步探讨其作用机制。方法 采用MTS实验考察异硫氰酸苄酯对肿瘤细胞体外增殖的抑制作用；2，5 μmol·L^-1异硫氰酸苄酯作用24 h后，采用Transwell实验、黏附实验和划痕实验观察异硫氰酸苄酯对人胶质瘤细胞侵袭、黏附和迁移能力的影响；应用Real-time-PCR和Western blot法检测相应浓度异硫氰酸酯处理后人胶质瘤U87MG细胞中MMP-2、MMP-9、CD44、Survivin、Bcl-2、Net1、RohA、caspase-3、caspase-8和p-AKT的表达变化；应用报告基因技术检测NF-κB转录活性的变化；采用ELISA法测定胞内8-OH-dG含量；10，20 μmol·L^-1异硫氰酸酯作用24 h后，采用流式细胞术观察其对细胞凋亡的作用。结果 异硫氰酸苄酯可显著抑制人胶质瘤U87MG细胞体外增殖；与0 μmol·L^-1组相比，2，5 μmol·L^-1异硫氰酸苄酯对人胶质瘤细胞U87MG侵袭、黏附和迁移能力有明显的抑制作用；不同浓度异硫氰酸苄酯处理后，肿瘤细胞MMP-2、MMP-9、CD44、Survivin、Bcl-2、NET1和RhoA的mRNA和蛋白表达、AKT磷酸化水平和NF-κB转录活性明显下调，caspase-3和caspase-8表达以及8-OH-dG含量显著上调；10，20 μmol·L^-1异硫氰酸苄酯可显著诱导细胞凋亡。结论 异硫氰酸苄酯抑制人胶质瘤细胞U87MG的侵袭能力，诱导细胞凋亡，其机制可能与抑制AKT/NF-κB信号转导途径，进而调节侵袭和凋亡相关基因表达有关。

Abstract:

OBJECTIVE To explore the effect and mechanism of benzyl isothiocyanate on the invasion inhibition and apoptosis induce of human glioma cancer U87MG cell lines. METHODS MTS assay was employed to determine the effect of benzyl isothiocyanate on the proliferation of tumor cells. After treatment with 2 and 5 μmol·L^-1 benzyl isothiocyanate, Transwell assay, adhesion assay and scratch assay were employed to evaluate the potential of invasive, adhesion and migration in U87MG human glioma cells. Real-time-PCR and western blot assay was employed to evaluate the mRNA and protein expression levels of MMP-2, MMP-9, CD44, Survivin, Bcl-2, caspase-3, caspase-8, Net1, RohA and p-AKT. Report gene assay was employed to evaluate the transcriptional activity of NF-κB, ELISA assay was employed to detect the level of 8-OH-dG, flow cytometry assay was employed to evaluate the apoptosis rate with 10 and 20 μmol·L^-1 benzyl isothiocyanate treatment. RESULTS benzyl isothiocyanate significantly inhibited the proliferation of U87MG. With 2 and 5 μmol·L^-1 benzyl isothiocyanate, the invasion of U87MG cells was inhibited, the expression of MMP-2, MMP-9, CD44, Survivin, Bcl-2, NET1, RhoA, p-AKT and the transcriptional activity of NF-κB was down regulated, as well as an increase in activity of caspase-3, caspase-8 and 8-OH-dG level. Apoptosis rate was improved with 10 and 20 μmol·L^-1 benzyl isothiocyanate treatment compared with 0 μmol·L^-1 group. CONCLUSION Benzyl isothiocyanate can significantly inhibit the invasion and induce the apoptosis of human glioma U87MG cell lines via regulation of metastasis and apoptosis-related genes, which may be related to AKT/NF-κB pathways.