Doxorubicin induces atypical NF-κB activation through c-Abl kinase activity in breast cancer cells

Abstract

Purpose: NF-κB transcription factor has been associated with cancer development and chemoresistance. We studied the signaling pathway activated by doxorubicin (DOX) leading to NF-κB activation in breast cancer cells.

Methods: NF-κB activity was evaluated by electrophoretic mobility shift in T47D, ZR75.30 and primary culture (MBCDF) from a ductal infiltrating carcinoma. Cell viability was measured by crystal violet. Western blotting was performed to check the expression and phosphorylation of IκBα Ser-32/36. c-Abl was inhibited with Imatinib or by overexpressing a dominant negative form of c-Abl (K290R).

Results: We found a correlation between sensitivity to DOX and amplitude of NF-κB activation. In cells least sensitive to DOX, NF-κB remained activated for longer time (T47D and MBCDF). The opposite effect was observed in cells sensitive to DOX (ZR75.30). DOX did not induce IκBα degradation or Ser-32/36 phosphorylation. Instead, there were modifications in the levels of IκBα tyrosine phosphorylation, suggesting an atypical NF-κB activation. In DOX-resistant cells, Imatinib treatment reduced IκBα tyrosine phosphorylation and NF-κB activity. The Imatinib-DOX combination significantly enhanced cell death of T47D and MBCDF breast cancer cells. Overexpression of c-Abl K290R in T47D and MBCDF cells reduced basal and DOX-induced NF-κB activation as well as IκBα tyrosine phosphorylation. In c-Abl K290R cells, DOX treatment did not mimic the combination Imatinib-DOX-induced cell death.

Conclusions: Inhibition of c-Abl inactivated IκBα/NF-κB pathway is associated with IκBα tyrosine phosphorylation in breast cancer cells. These results also raise the potential use of a combined therapy with Imatinib and DOX for breast cancer patients.

Fig. 1

DOX induced different kinetics of NF-κB activation in various breast cancer cells. a MBCDF, T47D and ZR75.30 were treated with 1 μg/ml of DOX at the indicated time points. EMSA for NF-κB DNA-binding activity was performed using a ³²P-labeled double-stranded oligonucleotide corresponding to the NF-κB consensus site and 10 μg of nuclear protein. Numbers above the lanes represent fold increase over the control. b DOX dose-dependent curve was plotted on MBCDF, T47D and ZR75.30 breast cancer cells. Cells were seeded in 24-well plate at 10⁴ cells/cm², and DOX was added at 0, 0.1, 0.5 and 1 μg/ml. Cell viability was evaluated by crystal violet 48 h after DOX treatment. Results are expressed as mean ± SD of the percentage of OD of untreated cells. Experiments were seeded in triplicate, and graphs are representative of three independent experiments with similar results

Fig. 2

IκBα is neither Ser-32/36 phosphorylation nor degraded by DOX; instead, DOX affects its phosphorylation at tyrosine residues in breast cancer cells. a MBCDF, T47D and ZR-75-30 and breast cancer cells were treated with 20 μM of the proteasome inhibitor MG-132 2 h before the addition of DOX (1 μg/ml) or TNF (5 ng/ml). Whole-cell lysates were collected at the indicated time points. pSer-32/36 ΙκΒα was immunodetected using a phosphor-specific antibody. Membrane was stripped and reblotted for ΙκΒα. b MBCDF, T47D and ZR75.30 cells were treated with either 1 μg/ml of DOX or 5 ng/ml of TNF. Whole-cell lysates were obtained at the time points indicated. Degradation of ΙκΒα was detected by Western blot analysis using anti-ΙκΒα antibody. Membranes were stripped and reblotted with anti-tubulin antibody as loading control. c MBCDF, T47D and ZR75.30 cells were treated with DOX. Cells were harvested at 0.5 and 1 h after treatment with 1 μg/ml of DOX. Whole protein extracts were immunoprecipitated overnight with anti-ΙκΒα antibody at 4 °C. A phosphotyrosine Western blotting was performed using 4G10 antibody, and then membranes were stripped and reblotted with anti-ΙκΒα antibody as loading control

Fig. 3

IκBα tyrosine phosphorylation and NF-κB DNA-binding activity are modified by Imatinib. a Immunoprecipitation of IκBα from MBCDF and T47D cells treated with or without 10 μM of Imatinib 2 h before adding 1 μg/ml of DOX for 30 min. Whole-cell lysates were immunoprecipitated as in Fig. 2b. Western blotting of anti-phosphotyrosine was performed, and then the membranes were stripped and reblotted for total ΙκΒα as loading control. b MBCDF and T47D breast cancer cells were treated with 0.1, 1 and 10 μM of Imatinib for 30 min. EMSA for NF-κB was performed as in Fig. 1a. c MBCDF and T47D breast cancer cells were treated with 1 μM of Imatinib for 2 h before the addition of 1 μg/ml of DOX. EMSA for NF-κB was performed as in Fig. 1a. Numbers above the lanes represent fold increase over the control

Fig. 4

Imatinib increases the DOX-induced cell death of resistant breast cancer cells. a MBCDF and T47D cells were seeded as in Fig. 1b, and then cells were treated with increasing doses of Imatinib. Cell viability was evaluated by crystal violet assay 48 h after Imatinib addition. b T47D and MBCDF cells were seeded as in a, and then cells were stimulated with 1, 5 and 10 μM of Imatinib before adding 0.1 μg/ml of DOX. Cell viability was evaluated by crystal violet assay 48 h after the addition of drugs. Results in a and b are mean ± SD of a plate seeded in triplicate, and they are representative of three independent experiments. c Pictures from cytotoxicity assays were taken under inverted microscope at the magnification of 400 × 48 h after the addition of drugs

Fig. 5

Overexpression of c-Abl K290R interferes with NF-κB DNA-binding activity and IκBα tyrosine phosphorylation. a MBCDF and T47D breast cancer cells were transfected with a plasmid containing the c-Abl K290R cDNA, and clones overexpressing c-Abl K290R were isolated using 100 μg/ml of geneticin. Western blot analysis of T47D-EV (transfected with empty vector), T47D-C6 (transfected with pcDNA3 c-Abl K290R), MBCDF-EV (transfected with empty vector) and MBCDF-C5 (transfected with pcDNA3 c-Abl K290R). Equal amounts of protein whole-cell lysates from each clone were analyzed by Western blotting using a polyclonal antibody against c-Abl. Membranes were stripped and reblotted for tubulin as loading control. b T47D-EV, T47D-C6, MBCDF-EV and MBCDF-C6 cells were treated with 1 μg/ml of DOX for 30 min, and then nuclear protein was obtained to perform EMSA for NF-κB. Numbers above the lanes represent fold increase over the control. c T47D-EV, T47D-C6, MBCDF-EV and MBCDF-C6 cells were treated as in b, and then whole protein lysates were immunoprecipitated with anti-IκBα antibody as in Fig. 3b. Western blotting of anti-phosphotyrosine and anti-IκBα was performed

Fig. 6

Overexpression of c-Abl K290R partially increases DOX-induced cell death. a T47D-EV, T47D-C6, MBCDF-EV and MBCDF-C6 cells were seeded as in Fig. 1b. DOX was added at 0.1 and 0.5 μg/ml. Cell viability was evaluated by crystal violet assay after 48 h. b T47D-EV, T47D-C6, MBCDF-EV and MBCDF-C6 cells were seeded as in a, and then cells were stimulated with 1, 5 and 10 μM of Imatinib before adding 0.1 μg/ml of DOX. Cell viability was evaluated 48 h after drug addition by crystal violet assays. Results in a and b are mean ± SD of a plate seeded in triplicate, and it is representative of three independent experiments