粉防己碱对神经母细胞瘤干细胞增殖和干性基因表达的影响及作用机制
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摘要
背景:研究发现粉防己碱能通过下调β-catenin表达抑制神经胶质瘤干细胞特性,然而其对神经母细胞瘤干细胞的影响未见报道。目的:探讨粉防己碱对神经母细胞瘤干细胞增殖及干性标志物表达的影响,并探究其分子机制。方法:使用无血清悬浮法从神经母细胞瘤SK-N-SH细胞中获取肿瘤干细胞,给予粉防己碱干预,通过MTT法检测粉防己碱对神经母细胞瘤干细胞的增殖抑制作用,细胞免疫荧光染色检测粉防己碱干预后Nestin蛋白水平变化,克隆球形成实验检测粉防己碱干预后肿瘤干细胞成球能力变化,qRT-PCR法检测粉防己碱干预后CD133、Oct-4、Nestin mRNA表达,Western blot检测粉防己碱干预后CD133、Oct-4、Nestin、p-GSK3β和β-catenin蛋白表达。结果与结论:与未干预组比较,粉防己碱干预后SK-N-SH干细胞增殖能力降低(P <0.05),CD133、Oct-4和Nestin mRNA表达降低(P <0.05),Nestin蛋白荧光强度减弱,克隆球形成能力减弱(P <0.05),CD133、Oct-4、Nestin、p-GSK3β和β-catenin蛋白减少(P <0.05)。结果表明,粉防己碱抑制神经母细胞瘤干细胞增殖并降低干性标志物表达,可能与其抑制Wnt/β-catenin信号通路有关。
BACKGROUND:Studies have shown that tetrandrine can inhibit the characteristics of glioma stem cells by down-regulating the expression ofβ-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 m RNA levels of CD133, Oct-4 and Nestin were tested by qR T-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 m RNA 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.
BACKGROUND:Studies have shown that tetrandrine can inhibit the characteristics of glioma stem cells by down-regulating the expression ofβ-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 m RNA levels of CD133, Oct-4 and Nestin were tested by qR T-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 m RNA 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.
引文
[1]Ahmed AA,Zhang L,Reddivalla N,et al.Neuroblastoma in children:Update on clinicopathologic and genetic prognostic factors.Pediatr Hematol Oncol.2017;34(3):165-185.
[2]Tolbert VP,Matthay KK.Neuroblastoma:clinical and biological approach to risk stratification and treatment.Cell Tissue Res.2018;372(2):195-209.
[3]李雪.神经母细胞瘤多药耐药机制的研究[J].国际儿科学杂志,2016,43(10):748-753,759.
[4]Whittle SB,Smith V,Doherty E,et al.Overview and recent advances in the treatment of neuroblastoma.Expert Rev Anticancer Ther.2017;17(4):369-386.
[5]Kiyonari S,Kadomatsu K.Neuroblastoma models for insights into tumorigenesis and new therapies.Expert Opin Drug Discov.2015;10(1):53-62.
[6]Vlashi E,Pajonk F.Cancer stem cells,cancer cell plasticity and radiation therapy.Semin Cancer Biol.2015;31:28-35.
[7]Liu T,Liu X,Li W.Tetrandrine,a Chinese plant-derived alkaloid,is a potential candidate for cancer chemotherapy.Oncotarget.2016;7(26):40800-40815.
[8]Bhagya N,Chandrashekar KR.Tetrandrine--A molecule of wide bioactivity.Phytochemistry.2016;125:5-13.
[9]Zhang Y,Wen YL,Ma JW,et al.Tetrandrine inhibits glioma stem-like cells by repressingβ-catenin expression.Int J Oncol.2017;50(1):101-110.
[10]谢艳,宿玉玺,覃佳强,等.SK-N-SH神经母细胞瘤干细胞培养及鉴定[J].中国组织工程研究,2014,18(45):7260-7265.
[11]Wang T,Shigdar S,Gantier MP,et al.Cancer stem cell targeted therapy:progress amid controversies.Oncotarget.2015;6(42):44191-44206.
[12]Pützer BM,Solanki M,Herchenr?der O.Advances in cancer stem cell targeting:How to strike the evil at its root.Adv Drug Deliv Rev.2017;120:89-107.
[13]Agarwal S,Lakoma A,Chen Z,et al.G-CSF Promotes Neuroblastoma Tumorigenicity and Metastasis via STAT3-Dependent Cancer Stem Cell Activation.Cancer Res.2015;75(12):2566-2579.
[14]Guan B,Mu L,Zhang L,et al.MicroRNA-218 inhibits the migration,epithelial-mesenchymal transition and cancer stem cell properties of prostate cancer cells.Oncol Lett.2018;16(2):1821-1826.
[15]Sun JH,Luo Q,Liu LL,et al.Liver cancer stem cell markers:Progression and therapeutic implications.World J Gastroenterol.2016;22(13):3547-3557.
[16]Hayakawa Y,Fox JG,Wang TC.The Origins of Gastric Cancer From Gastric Stem Cells:Lessons From Mouse Models.Cell Mol Gastroenterol Hepatol.2017;3(3):331-338.
[17]Czarnecka AM,Solarek W,Kornakiewicz A,et al.Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.Oncol Rep.2016;35(3):1433-1442.
[18]Luo M,Clouthier SG,Deol Y,et al.Breast cancer stem cells:current advances and clinical implications.Methods Mol Biol.2015;1293:1-49.
[19]Zage PE,Whittle SB,Shohet JM.CD114:A New Member of the Neural Crest-Derived Cancer Stem Cell Marker Family.J Cell Biochem.2017;118(2):221-231.
[20]Khalil MA,Hraběta J,Groh T,et al.Valproic Acid Increases CD133Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma.PLoS One.2016;11(9):e0162916.
[21]樊珈榕,李万福,梁挺.干细胞标志物CD133在神经母细胞瘤中的表达[J].中国组织工程研究,2015,19(50):8090-8094.
[22]Lin Y,Wang Y,Liu X,et al.A novel derivative of tetrandrine(H1)induces endoplasmic reticulum stress-mediated apoptosis and prosurvival autophagy in human non-small cell lung cancer cells.Tumour Biol.2016;37(8):10403-10413.
[23]N B,K R C.Tetrandrine and cancer-An overview on the molecular approach.Biomed Pharmacother.2018;97:624-632.
[24]杨敬,朱安祥,胡军,等.粉防己碱的作用机制研究进展[J].国际中医中药杂志,2018,40(3):286-289.
[25]Kim M,Jho EH.Cross-talk between Wnt/β-catenin and Hippo signaling pathways:a brief review.BMB Rep.2014;47(10):540-545.
[26]姚彬,张庆华.Wnt/β-catenin信号通路在卵巢癌干细胞增殖、迁移和侵袭中的作用[J].中国组织工程研究,2018,22(25):4001-4006.
[27]Takebe N,Miele L,Harris PJ,et al.Targeting Notch,Hedgehog,and Wnt pathways in cancer stem cells:clinical update.Nat Rev Clin Oncol.2015;12(8):445-464.
[28]Wang R,Sun Q,Wang P,et al.Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.Oncotarget.2016;7(5):5754-5768.
[29]Zhu JY,Yang X,Chen Y,et al.Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin and Sonic Hedgehog Pathways.Phytother Res.2017;31(4):680-688.
[30]Chen Z,Zhou L,Wang L,et al.HBO1 promotes cell proliferation in bladder cancer via activation of Wnt/β-catenin signaling.Mol Carcinog.2018;57(1):12-21.
[2]Tolbert VP,Matthay KK.Neuroblastoma:clinical and biological approach to risk stratification and treatment.Cell Tissue Res.2018;372(2):195-209.
[3]李雪.神经母细胞瘤多药耐药机制的研究[J].国际儿科学杂志,2016,43(10):748-753,759.
[4]Whittle SB,Smith V,Doherty E,et al.Overview and recent advances in the treatment of neuroblastoma.Expert Rev Anticancer Ther.2017;17(4):369-386.
[5]Kiyonari S,Kadomatsu K.Neuroblastoma models for insights into tumorigenesis and new therapies.Expert Opin Drug Discov.2015;10(1):53-62.
[6]Vlashi E,Pajonk F.Cancer stem cells,cancer cell plasticity and radiation therapy.Semin Cancer Biol.2015;31:28-35.
[7]Liu T,Liu X,Li W.Tetrandrine,a Chinese plant-derived alkaloid,is a potential candidate for cancer chemotherapy.Oncotarget.2016;7(26):40800-40815.
[8]Bhagya N,Chandrashekar KR.Tetrandrine--A molecule of wide bioactivity.Phytochemistry.2016;125:5-13.
[9]Zhang Y,Wen YL,Ma JW,et al.Tetrandrine inhibits glioma stem-like cells by repressingβ-catenin expression.Int J Oncol.2017;50(1):101-110.
[10]谢艳,宿玉玺,覃佳强,等.SK-N-SH神经母细胞瘤干细胞培养及鉴定[J].中国组织工程研究,2014,18(45):7260-7265.
[11]Wang T,Shigdar S,Gantier MP,et al.Cancer stem cell targeted therapy:progress amid controversies.Oncotarget.2015;6(42):44191-44206.
[12]Pützer BM,Solanki M,Herchenr?der O.Advances in cancer stem cell targeting:How to strike the evil at its root.Adv Drug Deliv Rev.2017;120:89-107.
[13]Agarwal S,Lakoma A,Chen Z,et al.G-CSF Promotes Neuroblastoma Tumorigenicity and Metastasis via STAT3-Dependent Cancer Stem Cell Activation.Cancer Res.2015;75(12):2566-2579.
[14]Guan B,Mu L,Zhang L,et al.MicroRNA-218 inhibits the migration,epithelial-mesenchymal transition and cancer stem cell properties of prostate cancer cells.Oncol Lett.2018;16(2):1821-1826.
[15]Sun JH,Luo Q,Liu LL,et al.Liver cancer stem cell markers:Progression and therapeutic implications.World J Gastroenterol.2016;22(13):3547-3557.
[16]Hayakawa Y,Fox JG,Wang TC.The Origins of Gastric Cancer From Gastric Stem Cells:Lessons From Mouse Models.Cell Mol Gastroenterol Hepatol.2017;3(3):331-338.
[17]Czarnecka AM,Solarek W,Kornakiewicz A,et al.Tyrosine kinase inhibitors target cancer stem cells in renal cell cancer.Oncol Rep.2016;35(3):1433-1442.
[18]Luo M,Clouthier SG,Deol Y,et al.Breast cancer stem cells:current advances and clinical implications.Methods Mol Biol.2015;1293:1-49.
[19]Zage PE,Whittle SB,Shohet JM.CD114:A New Member of the Neural Crest-Derived Cancer Stem Cell Marker Family.J Cell Biochem.2017;118(2):221-231.
[20]Khalil MA,Hraběta J,Groh T,et al.Valproic Acid Increases CD133Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma.PLoS One.2016;11(9):e0162916.
[21]樊珈榕,李万福,梁挺.干细胞标志物CD133在神经母细胞瘤中的表达[J].中国组织工程研究,2015,19(50):8090-8094.
[22]Lin Y,Wang Y,Liu X,et al.A novel derivative of tetrandrine(H1)induces endoplasmic reticulum stress-mediated apoptosis and prosurvival autophagy in human non-small cell lung cancer cells.Tumour Biol.2016;37(8):10403-10413.
[23]N B,K R C.Tetrandrine and cancer-An overview on the molecular approach.Biomed Pharmacother.2018;97:624-632.
[24]杨敬,朱安祥,胡军,等.粉防己碱的作用机制研究进展[J].国际中医中药杂志,2018,40(3):286-289.
[25]Kim M,Jho EH.Cross-talk between Wnt/β-catenin and Hippo signaling pathways:a brief review.BMB Rep.2014;47(10):540-545.
[26]姚彬,张庆华.Wnt/β-catenin信号通路在卵巢癌干细胞增殖、迁移和侵袭中的作用[J].中国组织工程研究,2018,22(25):4001-4006.
[27]Takebe N,Miele L,Harris PJ,et al.Targeting Notch,Hedgehog,and Wnt pathways in cancer stem cells:clinical update.Nat Rev Clin Oncol.2015;12(8):445-464.
[28]Wang R,Sun Q,Wang P,et al.Notch and Wnt/β-catenin signaling pathway play important roles in activating liver cancer stem cells.Oncotarget.2016;7(5):5754-5768.
[29]Zhu JY,Yang X,Chen Y,et al.Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin and Sonic Hedgehog Pathways.Phytother Res.2017;31(4):680-688.
[30]Chen Z,Zhou L,Wang L,et al.HBO1 promotes cell proliferation in bladder cancer via activation of Wnt/β-catenin signaling.Mol Carcinog.2018;57(1):12-21.