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

Abstract

Objective To present the changes in the sensitivity of SH-SY5Y 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 (SY5Y) was cultured.High expression of tyrosine kinase receptor B (TrkB) was induced 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 pathways were respectively inhibited by LY294002 (PI3K pathway inhibitor),U73122(PLC pathway inhibitor) and U0126(MAPK pathway inhibitor).Cell viability was assessed by methyl thiazolyl tetrazolium (MTT) assay.Cell apoptosis rate was detected by flow cytomctry (FCM).MMP-9 concentrations 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 SY5Y cells to CDDP decreased in the ATRA + BDNF + U73122 + CDDP 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 cells cultured in serum-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 neuroblastoma cells cultured in serum-free media in the ATRA + BDNF + U73122 group were significantly higher than those of the control group and ATRA alone group and had no significant difference when compared with the ATRA + BDNF group.MMP-9 protein levels in neuroblastoma cells cultured in serumfree 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 chemoresistence and the synthesis and secretion of MMP-9 in human neuroblastoma cells.TrkB-BDNF signaling pathway 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 TrKP-BDNF downstream signaling pathway PI3K/Akt with LY294002 can reverse chemoresistance and inhibit the synthesis and secretion of MMP-9,thus limiting neuroblastoma tumor invasion and metastasis. Key words: Neuroblastoma; Signal transduction; Matrix metalloproteinase-9