BDNF Contributes to Cisplatin Resistance Figure 3. Differential response of OSC-19P and OSC-19CR to CDDP. Cell lysate was collected following 5 uM of CDDP and separated with SDS-PAGE gel. Membrane was then incubated with indicated antibodies. A. Expression of TrkB was determined in both OSC-19P and OSC-19CR following 5 uM of CDDP treatment. B. Phosphorylation of Akt, Src, MAPK, and STAT-3 was determined following 5 uM CDDP treatment. Levels of phosphorylated kinases were normalized with expression of total kinases. C. 5 uM of CDDP modulates expression of Bim, XiAP, and MDR1. Expression levels of Bim, XiAP, and MDR1 were determined by Western blot analysis. doi:10.1371/journal.pone.0030246.g003 complete picture of the expression of the BCL2 family of proteins in regulation of Cisplatin resistance, we analyzed the expression of anti-apoptotic molecules of the BCL2 family and showed that BCL- XL and Survivin were upregulated in the CDDP-resistant cell lines compared to their parental counterparts (Figure S1A). Among the enzymes associated with drug resistance, increased expression of multidrug resistance 1 (MDR1), and glutathion S tranferase-p (GST-p) was noted (Fig. 2B), which confirmed our previous findings vis-a`-vis the role of KiSS1 in mediating CR behavior, and is consistent with previous findings of GSK3b- mediated TrkB-induced cell protection from chemotherapy [16,18]. To determine whether downstream signaling in known pathways of apoptosis resistance was associated with these findings, we next compared the activation of intracellular cascades in the parental and CR cells. While MAPK phosphorylation was consistently elevated among the resistant cell lines, increased Src and Akt phosphorylation was also observed, suggesting that these selected pathways coordinated CDDP metabolism and the resulting inhibition of cell death in the presence of CDDP (Fig. 2C). CDDP induces differential signaling in CDDP resistance As our previous data supported the concept that activation of anti-apoptotic proteins was partially driven by BDNF in drug- sensitive cells, we explored whether this phenomenon was differentially altered in the drug-resistant cell lines. First, to examine the impact of CDDP treatment on TrkB expression, we compared the parental cells to the resistant cells for receptor expression. Western blot analysis revealed that after 24 hours of exposure to CDDP, a substantial increase in receptor expression was seen in the sensitive cells (125% increase), but a blunted response was noted in the CR cells due to elevated basal levels (25% increase) (Figure 3A). However, we observed an induction of pro-survival pathways in the CR cells, as evidenced by phosphorylation of MAPK (50% increase) and Akt (30% increase), while baseline Src activation remained elevated (Figure 3B). The results were consistent with our previous findings when comparing the basal activity of anti-apoptotic pathways in the parental and resistant lines. Additionally, CDDP induced genes favoring cell death in the parental cells, particularly the pro-apoptotic molecule, Bim. Treatment of CDDP resistant cell line with a relatively high dose of CDDP for 24 hrs did not alter the basal expression of XIAP and MDR1 suggesting a stable phenotypic change of these molecules in response to drug treatment (Figure 3C). It is interesting to note that, overall, CDDP-resistant cell lines have an elevated level of STAT3 phosphorylation, but no change is observed when cells are treated with CDDP suggesting that STAT3 may not be playing a significant role in the development of resistance in Head and Neck cell lines (Fig. 3B). These results demonstrate that CDDP resistant cell lines harbor a differential molecular response profile that may be due in part to enhanced pro-survival signaling and constitutive apoptotic resistance. BDNF regulates signaling and apoptosis in drug-sensitive HNSCC We next sought to determine whether exogenous BDNF could induce the molecular alterations that were consistent with those PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30246