TrkB—BDNF信号传导通路对神经母细胞瘤细胞耐药和转移的影响

Effects of TrKB-BDNF signaling pathway on chemoresistance and metastasis of neuroblastoma cells

目的TrKPrBDNF信号传导通路与神经母细胞瘤（neuroblastoma，NB）细胞的化疗耐药和转移密切相关。本研究探讨阻断TrkBBDNF信号传导通路的三条下游信号传导通路后，NB细胞SHSY5Y对化疗药物顺铂的敏感性及分泌基质金属蛋白酶-9（matrixmetalloproteinas-9，MMP-9）的影响。方法常规培养SH—SY5YNB细胞，用纳摩尔浓度全反式维甲酸（albtransRetinoicAcid，ATRA）诱导酪氨酸激酶受体B（tyrosinekinasereceptorB，TrkB）高表达，加入脑源性神经营养因子（brain-derivedneurotrophicfactor，BDNF），从而激活Trk昏BDNF信号传导通路。用磷脂酰肌醇一3激酶（P13K）抑制剂LY294002、磷脂酶C（PLC）抑制剂U73122、丝裂原活化蛋白激酶（MAPK）抑制剂U0126阻断该信号通路的相应下游信号传导通路后，加入化疗药顺铂（CDDP）。四甲基偶氮蓝比色（MTT）实验方法检测应用i种抑制剂前后细胞的存活率的变化；流式细胞仪（FCM）检测应用三种抑制剂前后细胞凋亡率的变化。酶联免疫吸附测定（ELISA）技术检测SH—SY5Y细胞培养上清中MMP-9含量。结果ATRA＋BDNF＋CDDP组NB细胞的存活率及凋亡率与对照组相比，差异无统计学意义（P〉0．05）。ATRA＋BDNF＋LY294002＋CDDP组细胞对顺铂的敏感性增加，细胞的存活率明显降低，凋亡率明显升高，与ATRA＋BDNF＋CDDP组相比，差异有统计学意义（P〈0．05）；而ATRA＋BDNF＋U73122＋CDI）P组、ATRA＋BDNF＋U0126＋CDDP组细胞对顺铂的敏感性降低，细胞的存活率明显升高，凋亡率明显降低，与ATRA＋BDNF＋CDDP组相比，差异无统计学意义（P〉0．05）。ELISA结果显示，ATRA＋BDNF组MMP-9含量明显高于对照组及ATRA组（P〈0．05）；ATRA＋BDNF＋LY294002组MMP9含量明显低于ATRA＋BDNF组（P〈O．05），与对照组及ATRA组比较差异无统计学意义（P〉0．05）；ATRA＋BDNF＋U73122组MMP-9含量明显高于对照组及ATRA组（P〈0．05），与ATRA＋BDNF组比较差异无统计学意义（P〉0．05）。AT—RA＋BDNF＋U0126组MMP-9含量明显高于对照组及ATRA组（P〈0．05），与ATRA＋BDNF组比较差异无统计学意义（P〉0．05）。结论激活TrkB-BDNF信号传导通路可增强NB细胞耐药及促进其合成、分泌MMP-9。TrkB-BDNF信号传导通路可能通过进一步激活其下游PBK／Akt通路增强耐药及促进NB细胞合成、分泌MMP-9，用LY294002阻断P13K／Akt通路后可逆转耐药及抑制NB细胞合成、分泌MMP9，从而抑制NB的侵袭、转移。

Objective To present the changes in the sensitivity of SH-SYSY cells to cisplatin （CDDP） and the synthesis and secretion of matrix metalloproteinases-9（MMP-9） before and after blockage of TrkB-BDNF downstream signaling pathways. Methods Human neuroblastoma （NB） cell line SH-SY5Y （SYSY） was cultured. High expression of tyrosine kinase receptor B （TrkB） was in- duced by nM all-trans retinoid acid （ATRA）. Ectogenid brain-derived neurotrophic factor （BDNF） was then added to activate the TrkB-BDNF signaling pathway. The three downstream signaling path- ways were respectively inhibited by LY294002 （PI3K pathway inhibitor）, U73122（PLC pathway inhibitor） and U0126（MAPK pathway inhibitor）. Cell viability was assessed by methyl thiazolyl tetrazo- lium （MTT） assay. Cell apoptosis rate was detected by flow cytometry （FCM）. MMP-9 concentra- tions in the SY5Y cell culture supernatants were measured by enzyme-linked immunosorbent assay （ELISA）. Results The viability and apoptosis rate of SY5Y cells treated with ATRA, BDNF and CDDP were not different from the control group （P〈0. 05）. However, the sensitivity of SY5Y cells to CDDP increased in the ATRA ＋ BDNF ＋ LY294002 ＋ CDDP group, the viability rate decreased and the apoptosis rate increased in SY5Y cells when compared to the ATRA ＋ BDNF ＋ CDDP group （P〈 0. 05）. However, sensitivity of SYSY cells to CDDP decreased in the ATRA＋ BDNF＋ U73122 ＋ CD- DP group and in the ATRA ＋ BDNF ＋ U0126 ＋ CDDP group, the viability and the apoptosis rate of the two groups had no significant difference when compared to the ATRA ＋ BDNF ＋ CDDP group （P〈 0. 05）. The results of ELISA implied that MMP-9 protein levels in neuroblastoma ceils cultured in ser- um-free media in the ATRA ＋ BDNF group were significantly higher than those of the control group and ATRA alone group （P〈0. 05）. MMP-9 protein levels in the ATRA ＋ BDNF ＋ LY294002 group were significantly lower than those of the ATRA ＋ BDNF group and showed no significant difference when compared to the control group and the ATRA alone group. MMP-9 protein levels in neuroblas- toma cells cultured in serum-free media in the ATRA ＋ BDNF ＋ U73122 group were significantly high- er than those of the control group and ATRA alone group and had no significant difference when com- pared with the ATRA ＋ BDNF group. MMP-9 protein levels in neuroblastoma cells cultured in serum- free media in the ATRA ＋ BDNF ＋ U0126 group were significantly higher than those of the control group and ATRA alone group and had no significant difference when compared to the ATRA ＋ BDNF group. Conclusions Activation of TrkB-BDNF signaling pathway can increase the ehemoresistenee and the synthesis and secretion of MMP-9 in human neuroblastoma cells. TrkB-BDNF signaling path- way may act through activating its downstream PI3K/Akt pathway to increase the sensitivity of NB cells to CDDP and promote the synthesis and secretion of MMP-9. Blocking the TrKB-BDNF down- stream signaling pathway PI3K/Akt with LY294002 can reverse chemoresistance and inhibit the syn- thesis and secretion of MMP-9, thus limiting neuroblastoma tumor invasion and metastasis.