BDNF Contributes to Cisplatin Resistance Figure 2. CDDP resistant cells modulate expression of molecules related with cell survival. A. Apoptotic molecules, B. Drug resistance- related molecules, and C. intracellular signal molecules. doi:10.1371/journal.pone.0030246.g002 Bethesda, MD). Values were calculated from sections of at least 4 different tumors. STR profile nalysis of parental and CDDP-Resistant cell lines Extraction of genomic DBA (gDNA) was conducted by using Puregene Genomic DNA purification Kit (Gentra Systems, MN, Minnesota) following instructions provided by the manufacturer. The amount of extracted DNA was analyzed by using Nanodrop (ND-1000) spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). STR DNA fingerprinting analysis to determine isogenecity between parental cells and chemoresistant cells at the Characterized Cell Line Core (CCSG) of the MD Anderson Cancer Center. Cell lines were validated by STR DNA fingerprinting using the AmpF,STR Identifiler kit according to manufacturer instructions (Applied Biosystems cat 4322288). The STR profiles were compared to known ATCC fingerprints (ATCC.org), to the Cell Line Integrated Molecular Authentication database (CLIMA) version 0.1.200808 (http://bioinformatics. istge.it/clima/) (Nucleic Acids Research 37:D925-D932 PMCID: PMC2686526) and to the MD Anderson fingerprint database. Results TrkB is up-regulated in cisplatin-resistant head and neck squamous carcinoma cell lines We examined expression of TrkB and BDNF levels in cisplatin- resistant head and neck squamous cell carcinoma cell lines (OSC- 19CR and HN-5CR, respectively), as previously described [13]. These CR cells were confirmed for their isogenic lineage by short tandem repeat genotyping (STR, Table S1). The degree of CDDP resistance was determined with the MTT assay, and long-term exposure to CDDP induced a 3–4 fold increase in the IC50 of each cell line. (Figure 1A & 1B). We next established that both TrkB and BDNF expression was up-regulated in OSC-19CR and HN- 5CR cells, compared to the parental cell lines. These findings were confirmed by ELISA and RT-PCR for BDNF and by western blot and RT-PCR for the TrkB receptor (Figure 1C, 1D, 1E & F). These results suggested that the chemoresistant phenotype were associated with altered expression of BDNF-TrkB and confirmed our prior results [9,13,16]. CDDP-resistant cells up-regulate an anti-apoptotic expression profile As our previous studies supported the notion that downstream dysregulation in drug metabolism and enhanced apoptosis resis- tance were associated with TrkB/BDNF signaling, we next hypothesized that the observed CR phenotype was induced by alterations in apoptotic pathways. Substantial upregulation of anti- apoptotic proteins and suppression of pro-apoptotic proteins were seen in the CR cell lines. In CDDP-resistant cells, XiAP, an inhibitor of apoptosis, was up-regulated, while Bim, a pro-apoptotic maker, was down-regulated. However, we failed to observe any alteration of other apoptotic markers including Bax, Bid, Bok and a slight down regulation in the basal expression of cleaved PARP in the resistant cell lines (Figure 2A). In order to understand the PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e30246