tress, including enhanced lipid pero idation and decreased levels of reduced glutathione, it will be of interest to determine the role protein inhibitor of different antio idant effectors in retinoic acid protection of etoposide induced apoptosis. It is tempting to speculate that retinoic acid is able to regulate the sensitivity to chemotherapeutic agents induced apoptosis by increasing antio idant defense components through NF B proteins in certain cellular conte ts such as T47D breast cancer cells. Conclusions This study illustrates the multiplicity of pathways induced by retinoids in breast cancer cells that can cause markedly different responses depending on the specific cellular conte t retinoids can signal towards cell death or cell survival.
Moreover, the results of this study support an important role for the NF B pathway in retinoic acid signaling and retinoic acid mediated resistance to cancer therapy mediated apoptosis in breast cancer cells, independently of cIAP2. Our data support the use of NF B pathway activation as a mar ker for screening that will help to develop novel reti noids, or retinoid based combination therapies with improved efficacy. Additionally, this study further vali dates current efforts aimed to inhibit NF B signaling pathways to improve clinical therapies. Methods Cell culture and treatment H3396, T47D, ZR75 1 cells were cultured in RPMI or Dulbecco in the case of SK BR 3 cells, containing red phenol with 10% foetal calf serum, 100 U ml penicillin, 100 U ml streptomycin, and 2 mM glutamine. For the T47D cell line, medium was supplemented with 0,6 ug ml insulin.
9 cis RA and BMS493 were dissolved in ethanol and used at 1 10 6 M unless otherwise indi cated. TRAIL, TNFa, antiFAS antibody, Do orubicin, camptothecin and etoposide were used according to the suppliers instructions. Measurement of apoptosis Sub G1 cell population was quantified by single staining according to standard proce dures. Briefly, the cells were trypsinized and 2,5 105 cells were washed with PBS 1�� and incubated overnight at 4 C in a hypotonic buffer containing propidium iodide. DNA fragmentation assays were performed using the Cell Death Detection Elisa kit following the manufacturers recommendations. This kit measures the enrichment of histone comple ed DNA fragments in the cytoplasm of apoptotic cells.
Oil Red O staining Cells, grown in coverslips, were fi ed with cold 10% For malin Calcium Acetate for 30 min. After fi ation, cover slips were transferred to 60% isopropanol for 1 2 minutes at room temperature. Cells were stained with freshly filtered Batimastat Oil Sunitinib 341031-54-7 Red O for 20 min at RT and washed in running water to remove the e cess of the staining solution, followed by counterstaining with hemato ylin. The coverslips were then mounted in gly cerin jelly. RNase protection assays Total RNA was e tracted with Trizol. RNase protection assays were performed according to the suppliers instructions. Routinely, 4 ug total RNA and 6 8 105 cpm of uridine tripho sphate probe set
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