Monoclonal antibody to total Src and alpha tubulin were obtained from Upstate Biotech nology and Sigma Aldrich, respectively. Rab bit polyclonal antibodies against casein kinase 2alpha. NF kappaB p65, phospho NF selleck chem kappaB p65 and caspase 3 were obtained from Cell Signaling Technology. HeLa cell lines, untreated and treated with TNFalpha were used as a positive con trol for casein kinase 2alpha and NF Inhibitors,Modulators,Libraries kappaB p65 phos pho NF kappaB p65, respectively, according to the manufacturers protocol. In vitro viability assays Measurement of metabolic activity by a WST 1 colori metric assay was used as a read out system for cell viability in response to kinase inhibitors. Dasatinib was used to inhibit Src pathway. TBB was used to inhibit casein kinase 2, which is an important kinase in atypical NF kappaB signalling.
After harvesting, 2000 cells well of every cell line and primary culture were seeded into 96 well flat bottom plates. After 24 hours, increasing concentrations of the drugs were added or 0,1% DMSO as vehicle control, each condition in quadrupli cate. Ten percent serum supplementation was used for all experiments. After 3 days of treatment, absorbance was measured on Inhibitors,Modulators,Libraries a Victor Multilabel Inhibitors,Modulators,Libraries Counter 1420 Inhibitors,Modulators,Libraries 042 at 450 nm, and was corrected for background and averaged. GIST882 and Jurkat cell lines were used as positive con trols for dasatinib and TBB experiments, respectively y. In combination experiments, 2000 cells were pla ted overnight followed by treatment with dasatinib which was added 30 minutes after TBB administration. In these experiments, increasing concentrations of dasa tinib at IC50 concentrations of TBB were used.
Background Cisplatin is a chemotherapeutic agent widely used in the treatment of several solid tumors, among them testicular cancer, lung cancer, breast cancer, and bladder cancer. Resistance to cisplatin is a serious obstacle to effective cancer therapy. Clinically relevant levels of resistance can emerge quickly after treatment. Beside Inhibitors,Modulators,Libraries intrinsic resistance, acquired or gradually developing resistance has been observed in tumors under therapy. Several mechanisms underlying resistance have been described, like decreased exposure to the drug, U0126 chemical structure e. g. via reduced drug accumulation drug target interaction or increased detox ification response, diminished cell cycle effects, reduced apoptotic responses, or increased DNA repair. A number of these biological processes are controlled on a post transcriptional level by noncoding micro RNA spe cies. We created an in vitro model of acquired cisplatin resistance by long term exposure of three well estab lished germ cell tumor cell lines to cisplatin, resulting in sublines with significantly increased resistance to cispla tin.