BDNF Contributes to Cisplatin Resistance Figure 4. BDNF induces signals and modulates MDR1, Bim, and XiAP in CDDP sensitive cells. A. Dose-dependent up-regulation of intracellular signals, Akt, Src, and MAPK in OSC-19P. The culture reaches ,80% confluent, cells were starved for overnight with medium without serum. Lysate was collect after 24 hour treatment with 100 ng/ml BDNF and separated with SDS-PAGE gel and membrane was incubated with indicated antibodies. B. Western blot analysis of XiAP, Bim, and MDR1 following treatment with various concentration of BDNF for 24 hours (0.1, 1, 10, and 100 ng/ml, respectively). doi:10.1371/journal.pone.0030246.g004 seen in the CDDP-resistant cells. To determine whether TrkB/ BDNF signaling impacted the induction of apoptotic pathways in CDDP-sensitive cell lines, we analyzed the alterations in the phosphorylation status of various signaling molecules. After exposure to BDNF for 24 hours, stimulation with BDNF resulted in increased phosphorylation of various kinases, Akt, Src, and MAPK in parental cells, consistent with previous studies regarding its role in tumor proliferation and apoptosis (Figure 4A) [13]. The activation of these kinases was noted in a dose-dependent manner, even at low BDNF concentrations. We also measured expression levels of XiAP, Bim, and MDR1, which were previously found to be dysregulated in our system. In these experiments, we found that level of XiAP, an inhibitor of apoptosis, was induced by a 5.3-fold change under the influence of BDNF exposure (Figure 4B). In contrast, the expression of the pro-apoptotic protein Bim was reduced by 70% in a dose-dependent manner after exposure to BDNF (Figure 4B). In line with these findings, the expression of MDR1, the cisplatin-efflux complex was enhanced by 60% when cells were exposed to BDNF (Figure 4B). These results suggests that exposure to exogenous BDNF could contribute to a molecular profile that mimicked the CDDP-resistant one. Alterations in CDDP-response is mediated by TrkB Although the previous results highlighted an association between CDDP resistance and enhanced TrkB signaling, we next sought to test whether this relationship was directly mediated by receptor activity. Initially, we over-expressed TrkB in the low- expressing UMSCC1 cell line and compared the response of these genetically-altered cells to CDDP. A moderate induction of CDDP resistance was noted (Figure 5A & 5B) in cells with genetically upregulated TrkB, compared to those transfected with an empty vector, confirming our prior results [13]. As shown in Figure 5A, over-expression of TrkB increased in the IC50 from 1.2 uM (empty vector) to 2.3 uM significantly (p,0.05). Corresponding to this altered phenotype, we observed that Bim was down-regulated, suggesting that the CDDP resistance may have been due in part to suppressed apoptosis through a TrkB-directed mechanism (Figure 5C). Additionally, we found that the expression of MDR1 and XiAP were induced by genetic upregulation of TrkB, a finding that was in line with our results from exogenous BDNF exposure. To further establish this direct link, we genetically suppressed the expression of TrkB HNSCC cell lines with high levels of endogenous TrkB, HN-5 and OSC-19. In contrast to the previous experiments, the relative loss of TrkB expression initiated a more cisplatin sensitive response in HNSCC cells, with a nearly 66% reduction in IC50 value in the TrkB-knockdowns (Figures 5D and S1B). Additionally, when shRNA-based knockdown of TrkB was induced, an inverse expression change between Bim and MDR1 was seen, which was complementary to the TrkB overexpression results. Corresponding analysis by Western blotting of MDR1 expression revealed attenuated expression, and a reciprocal enhancement in Bim expression (Figure 5E). These findings correlated not only with the previous TrkB inhibition experiments providing greater evidence for a direct role of BDNF- TrkB in modulating therapy resistance in HNSCC. An interesting observation was that knock down of TrkB led to reduced BDNF expression, suggesting a possible autocrine pathway for BDNF/ TrkB in in the development of CDDP-resistance in HNSCC. In addition inhibition of TRKB in CDDP-resistant cell line increased PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e30246