Louis, MO, USA) was added for 5 min at 37?C

Louis, MO, USA) was added for 5 min at 37?C. cascade (13). Amphiregulin (AREG) is a transmembrane glycoprotein from the EGFR family, interacting with EGFR, and regulating cellular growth and proliferation (14). AREG engages adjacent cells juxtacrine signaling. After processing proteolytic membrane proteases, AREG also functions autocrine and paracrine signaling. Elevated AREG expression is associated with chronic inflammation and tumor growth (15). In contrast to monoclonal antibodies that block the human epidermal growth factor receptor (HER) tyrosine kinases directly (cetuximab for HER1, trastuzumab for HER2), selective tyrosine kinase inhibitors (TKIs) inhibit the intracellular receptor signaling cascade by inhibiting phosphorylation thereby preventing activation. This inhibition can occur through competition with the substrate, adenosine triphosphate (ATP), inhibition of the phosphorylating enzyme or by deactivating it through conformational change (16). Erlotinib and gefitinib belong to the first generation of TKI. Erlotinib is approved for the treatment of non-small cell lung cancer (NSCLC) and pancreatic cancer, with significant improvement of therapeutic overall response rates (17). Gefitinib is approved in the therapy of NSCLC and under investigation for different solid cancer types with TKI mutations such as breast cancer. An important aspect of therapy with TKI is the development of therapeutic resistance. Amplification of the proto-oncogene hepatocyte growth factor receptor (leading to activation of HER3 signaling and T790M mutations have been identified as important mechanisms of therapeutic resistance to TKI therapy Mcl1-IN-4 (18,19). Afatinib is a member of the irreversible second-generation TKIs of the HER family (20). The down-regulation of HER signaling is achieved through covalent binding to kinase domains, resulting in irreversible inhibition of autophosphorylation (21). Afatinib is approved in the therapy of metastatic NSCLC with mutations/deletions (22). Dasatinib and nilotinib are small-molecule TKIs, acting through competitive binding of ATP-binding sites, resulting in dysregulation of tyrosine kinase enzymatic activity. Dasatinib and nilotinib have been investigated in hematopoietic malignancies and inhibit platelet-derived growth factor- receptor signaling, ephrin receptor kinases and mast/stem cell growth factor receptor (23). Dasatinib has also been shown to inhibit sarcoma tyrosine kinase (SRC) family kinases, a major means of resistance to anti-HER2 therapy in patients with breast cancer Mcl1-IN-4 (24-26). As both surface proteins AREG and CD44 have a strong association with EGFR signaling pathways influencing tumor progression and therapeutic response, we aimed to investigate the effect of different selective TKIs on the expression of CD44 and AREG in HPV+ and HPV? SCC. Materials and Methods The HPV? UMSCC cell lines were kindly provided by T.E. Carey, Ph.D. University of Michigan, Ann Arbor, MI, USA. UMSCC-11A cell line originated from a primary squamous cell carcinoma of the epiglottis, whereas UMSCC-14C originated from a skin metastasis of an oral SCC after radiation, chemotherapy and surgery. The CERV-196 cell line is positive for HPV16 and was provided from poorly differentiated SCC of the uterine cervix and acquired from Cell Lines Service GmbH, Eppelheim, Germany. HPV? cells were cultured with Eagles minimum essential medium (Gibco, Life Technologies, Carlsbad, CA, USA) and supplemented with 2 mM of L-glutamine, 10% fetal calf serum and Pen-Strep (Gibco, Life Technologies). Cultured Mcl1-IN-4 HPV+ cells were supplemented with 2 mM L-glutamine, 1.0 g/l sodium bicarbonate, 1.0 g/l sodium pyruvate, 0.1 Mcl1-IN-4 mM non-essential amino acids and 10% Col4a2 of fetal bovine serum (Gibco, Life Systems). Cell cultures were cultivated under standardized conditions (37?C, 5% CO2, 95% humidity). For subcultures, 0.05% trypsin/0.02% EDTA remedy (Sigma Aldrich, St. Louis, MO, USA) was added for 5 min at 37?C. Incubation time ranged from 24 to 96 hours. Nilotinib, dasatinib, gefitinib, erlotinib and afatinib were provided by the Oncological Division, University Hospital Mannheim GmbH. The medicines were dissolved in dimethylsulfoxide at a concentration of 20 mol/l. Cell proliferation assay was performed in 96-well microtiter plates (alamarBlue?, AbD Serotec, Oxford, UK). To determine the protein concentrations of CD44 and AREG in treated and untreated cells, a sandwich ELISA technique was applied. For both proteins, DuoSet ELISA development packages (R&D Systems, Inc., Minneapolis, MN, USA; and Bio-Techne GmbH, Wiesbaden, Germany) were used (DY7045-05 for CD44, and DY989 for AREG) and performed in accordance with the manufacturers instructions. The optical denseness was measured at a wavelength of 450 nm with wavelength correction arranged to 540 nm with an MRX Microplate Reader (DYNEX Systems, Chantilly, VA, USA). Concentrations were identified in pg/ml and the detection range was 7.8-1,500 pg/ml for.