Inhibitory effects and mechanisms of tetrandrine on proliferation and stemness marker expression of neuroblastoma stem cells

doi:10.3969/j.issn.2095-4344.1573

Abstract

Abstract:

BACKGROUND: Studies have shown that tetrandrine can inhibit the characteristics of glioma stem cells by down-regulating the expression of β-catenin. However, its effect on neuroblastoma stem cells has not been reported.
OBJECTIVE: To explore the function and mechanism of tetrandrine on proliferation and stemness marker expression of neuroblastoma stem cells.
METHODS: Serum-free suspension method was used to obtain SK-N-SH stem cells. SK-N-SH stem cells were treated with tetrandrine, and the inhibitory effect of tetrandrine on the proliferation of neuroblastoma stem cells was detected by MTT method. Immunofluorescence staining was used to detect Nestin expression in the cells after intervention with tetrandrine. The tumorsphere-forming ability of SK-N-SH stem cells was determined by tumorsphere-forming assay. The mRNA levels of CD133, Oct-4 and Nestin were tested by qRT-PCR. The protein level of CD133, Oct-4, Nestin, p-GSK3β and β-catenin were tested by western blot.
RESULTS AND CONCLUSION: Tetrandrine inhibited the proliferation of SK-N-SH stem cells (P < 0.05). After treated with tetrandrine, the mRNA levels of CD133, Oct-4 and Nestin in SK-N-SH stem cells were significantly decreased (P < 0.05). Fluorescence intensity of Nestin protien was weakened and the tumorsphere-forming ability of SK-N-SH stem cells was abated (P < 0.05). Tetrandrine also inhibited the protein levels of CD133, Oct-4, Nestin, p-GSK3β and β-catenin (P < 0.05). In conclusion, tetrandrine inhibits the proliferation and stemness marker expression of neuroblastoma stem cells, and the mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neuroblastoma, Neoplastic Stem Cells, Benzylisoquinolines, Tissue Engineering

CLC Number:

Xie Dongke, He Xia, Liao Kainan. Inhibitory effects and mechanisms of tetrandrine on proliferation and stemness marker expression of neuroblastoma stem cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(5): 729-733.

Figures/Tables 1

2.1 神经母细胞瘤SK-N-SH干细胞鉴定结果普通培养基培养神经母细胞瘤细胞，见图1A，无血清培养基培养2周可见悬浮生长的细胞球，离心吹打后传代培养获得新细胞球，见图1B。提取SK-N-SH细胞和SK-N-SH干细胞的蛋白和mRNA，通过Western blot和qRT-PCR检测CD133、Oct-4和Nestin表达，发现SK-N-SH干细胞中CD133、Oct-4、Nestin的蛋白和mRNA表达明显升高，见图1C和图1D，证实成功获取SK-N-SH干细胞。 2.2 粉防己碱抑制SK-N-SH干细胞增殖使用0，0.625，1.25，2.5，5.0，7.5，10.0 µmol/L粉防己碱处理SK-N-SH干细胞24 h，MTT实验检测SK-N-SH干细胞增殖能力的变化。结果显示，粉防己碱抑制SK-N-SH干细胞增殖能力，呈浓度依赖性，粉防己碱对细胞的半数抑制浓度(IC50)为3.749 µmol/L，差异有显著性意义(P < 0.05)，见图2。 2.3 免疫荧光检测粉防己碱处理后SK-N-SH干细胞Nestin蛋白表达使用5 µmol/L粉防己碱处理SK-N-SH干细胞，应用细胞免疫荧光检测SK-N-SH干细胞Nestin蛋白表达，结果显示对照组Nestin表达阳性，而粉防己碱处理组Nestin表达减弱，见图3。 2.4 粉防己碱抑制SK-N-SH干细胞的干细胞标志物表达使用2.5 µmol/L和5 µmol/L粉防己碱处理SK-N-SH干细胞，蛋白提取后使用Western blot检测CD133、Oct-4和Nestin蛋白表达，结果发现粉防己碱抑制SK-N-SH干细胞CD133、Oct-4和Nestin蛋白表达，见图4A。使用qRT-PCR检测CD133、Oct-4和Nestin mRNA表达，结果发现粉防己碱抑制SK-N-SH干细胞CD133、Oct-4和Nestin mRNA表达，差异均有显著性意义(P < 0.05)，见图4B。 2.5 粉防己碱抑制SK-N-SH干细胞克隆球形成能力使用2.5，5 µmol/L粉防己碱处理SK-N-SH干细胞，通过肿瘤干细胞克隆球形成实验检测粉防己碱对SK-N-SH干细胞干性的影响。结果显示，粉防己碱处理后干细胞克隆球体积减小，见图5A。与对照组相比，粉防己碱处理组克隆球数量减少，见图5B，并呈浓度依赖性，差异有显著性意义(P=0.019和P=0.002)。 2.6 粉防己碱抑制SK-N-SH干细胞Wnt/β-catenin信号通路使用2.5，5 µmol/L粉防己碱处理SK-N-SH干细胞并提取蛋白，通过Western blot检测p-GSK3β和β-catenin蛋白表达，见图6。结果显示，2.5，5 µmol/L粉防己碱明显抑制p-GSK3β和β-catenin蛋白水平，提示粉防己碱抑制SK-N-SH干细胞Wnt/β-catenin信号通路。 "

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