BDNF Contributes to Cisplatin Resistance Figure 5. TrkB altered sensitivity to CDDP in HNSCC. A. UMSCC1 cells were transfected with a vector containing wild-type TrkB (TrkB) or an empty vector (EV). Cells were then exposed to CDDP for addressing increasing resistance of TrkB over-expressed UMSCC1 to CDDP. MTT cell viability assay was conducted to determine the IC50 values. B. expression efficacy was confirmed by Western Blot analysis. BDNF expression level was also determined. C. Expression levels of XiAP, Bim, MDR1, GST-p, and N-cadherin were determined with over-expressed UMSCC1 cells. Both OSC-19 and HN-5 cells were stably transfected with a short-hairpin RNA construct targeting TrkB (TrkB shRNA) or a non-targeting, short-hairpin RNA construct (NT shRNA). D. Both HN-5P and HN-5CR cells were then treated with various concentration of CDDP and analyzed to address the effect of TrkB down- regulation on sensitivity to CDDP by suppressing TrkB expression. E. Down-regulation of TrkB expression was confirmed by Western blot analysis. Cell lysates of suppressing TrkB were separated with SDS-PAGE and membrane was incubated with BDNF, Bim, XiAP, and MDR1 antibodies. Expression levels of TrkB, BDNF, Bim, XiAP, and MDR1 were normalized with GAPDH. doi:10.1371/journal.pone.0030246.g005 sensitivity to CDDP treatment as compared to parental CDDP- resistant cell line (Figure S1C). Akt plays a crucial role in TrkB-induced CDDP-resistance development We next explored the intracellular signaling pathways that were responsible for mediating TrkB-induced CDDP resistance devel- opment in our system. We exposed CR cells to an inhibitor of Akt signaling and assayed for the expression of Bim, XiAP and MDR1 (Figure 6). Selective Akt pathway abrogation led to specific induction of Bim expression, and a significant decrease in MDR1 and XiAP protein expression, as determined by WB analysis. The changes in expression levels of MDR1, XiAP, and Bim in OSC- 19CR cells reverted to the levels noted in the parental cells (Figure 6A). We further substantiated these findings with genetic suppression of Akt prior to BDNF exposure. While a non-targeted construct did not alter the expected changes in Bim, MDR1 and XiAP expression, siRNA targeting Akt selectively blocked the BDNF-regulated alteration in apoptosis and drug resistance regulation (Figure 6B). Taken together, these results strongly support our hypothesis that up-regulation of TrkB/BDNF system contributes to CDDP-resistance development through modulating molecules, such as MDR1 and XiAP through Akt-dependent pathway facilitating cell survival against CDDP in HNSCCs. Expression of Bim and Ki-67 in animal models of HNSCC To determine whether our in vitro findings were relevant in animal models of HNSCC, we analyzed the expression level of Bim, XiAP, MDR1, and Ki-67 in vivo (Figure 7A–C). Mouse-borne tumors derived from parental or CR cell lines were assayed by immunohistochemistry [17]. We confirmed that Bim expression was down-regulated in CDDP-resistant OSC-19CR tumors (48%), compared to the parental tumors. In addition, protein expression of XiAP and Ki-67 expression was increased in CDDP-resistant OSC-19CR tumors (98% and 270%, respectively). However, we failed to show significant up-regulation of MDR1 expression. (data not shown). These data corroborated our hypothesis that chemoresistant tumors were associated with biological markers of apoptotic resistance and enhanced proliferative capacity [16]. Discussion Our previous study has shown that the BDNF-TrkB signaling axis contributes to tumor invasion and progression of HNSCC in xenograft animal models [9]. In continuation of our studies, we explored the role of this neurotrophic signaling axis in the development of chemotherapy resistance in HNSCC. Phenotypic characterization of cisplatin-resistant HNSCC cell lines demon- strated an upregulation of both TrkB and its ligand, brain derived neurotrophic factor (BDNF) with a concomitant increase in cell proliferation and apoptosis. Utilizing over-expression and knock- down experiments, we demonstrated the potential for the BDNF- TrkB as a realistic target in overcoming therapy resistance in HNSCC. Various mechanisms of drug resistance have been identified across differing cancer cell types. One major mechanism that has been identified is enhanced resistance to the induction of apoptosis Figure 6. Akt plays a critical role in TrkB-induced CDDP-resistance development. A. OSC-19CR cell lysate following treatment with 5 uM of TCN, an Akt inhibitor, was collected and separated with SDS-PAGE to determine expression levels of Bim, XiAP, and MDR1. B. Expression levels of Bim, XiAP, and MDR1 were determined with cell lysate following transfected with siRNA against role of Akt in CDDP-resistance development in HNSCC. doi:10.1371/journal.pone.0030246.g006 PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30246