resulting in cell proliferation, and apoptosis resistance in models of human cancer [14,15]. While our previous studies have shown that the resistance of HNSCC to CDDP-based therapy can be overcome with inhibition of TrkB pathways, the mechanisms for this phenomenon has yet to be elucidated [13]. In previous studies, we reported that both TrkB and BDNF were overexpressed in HNSCC, and that modulation of TrkB expression altered the invasive phenotype in this disease [9]. Moreover, we identified TrkB as a potential target for therapy in CDDP-resistant HNSCC [13]. These findings led us to investigate the mechanisms underlying CDDP-resistance induced through BDNF\TrkB in HNSCC. In the current study, we demonstrate a novel molecular mechanism of action for TrkB/BDNF in modulating the response of HNSCC to cisplatin-based treatment, primarily through differential apoptotic response. These results provide a potential strategic approach for reversing cisplatin resistance in aggressive HNSCC. Materials and Methods Cell lines and Reagents The head and neck squamous carcinoma cell (HNSCC) lines OSC-19 [9,13,16] , HN-5 [9,13], and UMSCC1 [13] cells were maintained with DMEM supplemented with 2 mM L-glutamine, 0.1 mM MEM non-essential amino acids, 1 mM sodium pyruvate, MEM vitamin solution 100 U/ml streptomycin, penicillin, vitamin and 10% FBS as described previously. OSC-19 and HN-5 cell lines were treated with low dose of cisplatin for six months to generate a cisplatin resistant strains of both cell lines. For end-point PCR analysis, PCR primer set for GAPDH (VHPS-3541) was purchased from Real Time Primers (Elkins Park, PA, USA). Three AKT siRNA oligo pairs (59 ‘CUCACAGCCCUGAAGUACU 39, 59 AGUACUUCAGGGCUGUGAG 39; 59 GAGACUGACAC- CAGGUAUU 39, 59 AAUACCUGGUGUCAGUCUC 39; 59 GUGCCAUGAUCUGUAUUUA 39, 59 UAAAUACAGAU- CAUGGCAC 39) and a non-target universal negative control (59 GCGCGAUAGCGCGAAUAUATT 39, 59 UAUAUUCGCG- CUAUCGCGCTT 39) were purchased from Sigma-Aldrich (Saint Louis, MO) to knock down AKT. A pool of the three AKT siRNA oligos was used to knock down AKT. The following antibodies were used for western blot analysis: goat anti-rabbit antibody (Santa Cruz, sc-2004), goat ant-mouse antibody (sc- 2005); p-Akt(4058), Akt1(2938), p-Src(2101), Src(2108), p-MAPK(9101), MAPK(9102), Bim(2819), GST-p(3369), PARP(9532), Cleaved PARP(9541), and BAX were from Cell Signaling (Danvers, MA); TrkB(sc-8316), GSK-3b(sc-9166), and MDR1(sc-55510) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and GAPDH (AM4300, Ambion, Austin, TX). Cisplatin and MTT purchased from Sigma (St. Louis, Mo). An Akt inhibitor, triciribine (TCN) was obtained from Berry Associates (Dexter, MI). Western blotting (WB) All western blotting analysis was performed as described previously [17]. Briefly, five to fifty microgram of total protein was loaded to 8 to 12% SDS-PAGE, and transferred onto PDVF membrane. The membrane was blocked with 5% milk in TBST and incubated with the appropriate primary and secondary antibodies and developed with Lumi-Light Western Blotting detection kit (Roche Applied Sciences, Indianapolis, IN). Densi- tometry data were analyzed by using Un-Scan-It gel Software (Silk Scientific Corp., Orem, UT) and statistical analysis was performed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon modified Newman- Keuls-Student procedure, where appropriate. Results are reported BDNF Contributes to Cisplatin Resistance as mean +/2 SEM. A p value of ,0.05 was considered significant and all are paired and two-tailed. Plasmid Transfections Short-hairpin RNA (shRNA) constructs targeting TrkB (Cat. TR320436, Origene, Rockville, MD) were introduced into cells via retroviral infection or with lipofectamine2000 reagent according to the manufacturer’s protocol, as previously described [16]. For TrkB over- expression, pcDNA3.1 plasmids containing the full length cDNA sequences for human TrkB was stably transfected into UMSCC1 cells and selected with G418. TrkB expression was confirmed by SDS- PAGE gel analysis and end-point RT-PCR analysis. RT-PCR Extraction of total RNAs was conducted by using the Qiagen RNeasy Mini kit (Valencia, CA). The quality and quantity of extracted RNA was analyzed by using Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) at the absorbance ratio of 260 and 280 nm. About 700 ng RNA was used as the template for semi-quantitative RT-PCR analysis to determine expression of TrkB at mRNA level. To determine expression level of TrkB at HNSCC, we conducted one-step RT-PCR reaction with SuperScriptH III One-Step RT- PCR System with PlatinumHTaq (12574-026, Invitrogen, Carls- bad, CA) with the Gene Amp PCR System 9700 (AB Applied Biosystems, Carlsbad, CA), according to manufacturer’s instruc- tion. For RT-PCR, the primers used were human TrkB 59 CCC ACT CAC ATG AAC AAT GG 39 (forward) and 59 TCA GTG ACG TCT GTG GAA GG 39 (reverse); human BDNF 59 AAA CAT CCG AGG ACA AGG TG 30 (forward) and 59 AGA AGA GGA GGC TCC AAA GG 39 (reverse); GAPDH (Real Time Primers, Elkins Park, PA), 59 GAG TCA ACG GAT TTG GTC GT 39 (forward) and 59 TTG ATT TTG GAG GGA TCT CG 39 (reverse). After a reverse transcription reactions at 55uC for 30 minutes, there was an initial 10-minute denaturation at 95uC followed by the 40 cycles run consists of a 15-second denaturation step at 94uC and an annealing step at 60uC for 30 seconds, and extension step at 68uC for 1 minute. The density of bands was determined by using Un-Scan-It gel Software (Silk Scientific Corp., Orem, UT) and statistical analysis was performed by utilizing either conventional Student’s t test. The PCR results were averaged with three independent experiments, which were used in the statistical analysis. Results were normalized to GAPDH. Cell Proliferation Assay with MTT To calculate IC50 of CDDP in various HNSCCs, MTT assay was conducted. Fifteen hundred cells were seeded into each well of 96-well plates and cultured overnight. Cells were treated with defined concentrations of CDDP for three days, followed by the MTT assay. Briefly, 25 ul of 5 mg/ml MTT was added to each well. After three hour incubation with MTT, the media was removed carefully. The formed dye was dissolved with 100 ul DMSO and the absorbance of each well was read using a micro- plate reader. Six or eight samples were treated with same concentration of CDDP. Experiments were repeated at least three times. CDDP IC50 was calculated using the GraphPad Prism Software (GraphPad Software, Inc.). ELISA Assay To determine expression level of BDNF, we performed ELISA assay with BDNF EmaxH ImmunoAssay System (G7610, Promega, Madison, WI) based on the manufacturer’s instructions. Briefly, cells were cultured to 80% confluence followed by replacement of PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30246