Figure 3

Intracellular accumulation and cytotoxicity of doxorubicin in HT29, HT29-dx and HT29 iNOS^- cells. (A) Cells were grown in RPMI containing 5 μmol/L doxorubicin for 24 h, then the drug accumulation was measured as described in the Methods section. Measurements were done in duplicate and data are represented as mean ± SE (n = 3). Versus HT29 CTRL: * p < 0.02. (B) After an incubation of 24 h in the absence (CTRL) or in the presence of 5 μmol/L doxorubicin (doxo), LDH activity was measured in the extracellular medium, as reported under the Methods section. Measurements were done in triplicate and data are represented as mean ± SE (n = 4). Versus HT29: * p < 0.05. (C) Western blot detection of nitrated MRP3, total MRP3, nitrated Pgp, total Pgp and GAPDH. HT29, HT29-dx and HT29-iNOS^- cells were incubated for 24 h in the absence (-) or in the presence (+) of 5 μmol/L doxorubicin, then cellular lysates were immunoprecipitated with an anti-nitrotyrosine polyclonal antibody. Western blotting for MRP3 and Pgp was performed on the immunoprecipitated proteins (see Methods for details). Under the same experimental conditions, an aliquot of cells was lysed and directly probed with an anti-Pgp or an anti-MRP3 antibody to detect total MRP3 or Pgp. The expression of the housekeeping protein GAPDH was measured as equal control loading. The results shown here are representative of two similar experiments.