Role of hypoxia-inducible factors in the dexrazoxane-mediated protection of cardiomyocytes from doxorubicin-induced toxicity

Abstract

Background and purpose: Iron aggravates the cardiotoxicity of doxorubicin, a widely used anticancer anthracycline, and the iron chelator dexrazoxane is the only agent protecting against doxorubicin cardiotoxicity; however, the mechanisms underlying the role of iron in doxorubicin-mediated cardiotoxicity and the protective role of dexrazoxane remain to be established. As iron is required for the degradation of hypoxia-inducible factors (HIF), which control the expression of antiapoptotic and protective genes, we tested the hypothesis that dexrazoxane-dependent HIF activation may mediate the cardioprotective effect of dexrazoxane.

Experimental approach: Cell death, protein levels (by immunoblotting) and HIF-mediated transcription (using reporter constructs) were evaluated in the rat H9c2 cardiomyocyte cell line exposed to low doses of doxorubicin with or without dexrazoxane pretreatment. HIF levels were genetically manipulated by transfecting dominant-negative mutants or short hairpin RNA.

Key results: Treatment with dexrazoxane induced HIF-1α and HIF-2α protein levels and transactivation capacity in H9c2 cells. It also prevented the induction of cell death and apoptosis by exposure of H9c2 cells to clinically relevant concentrations of doxorubicin. Suppression of HIF activity strongly reduced the protective effect of dexrazoxane. Conversely, HIF-1α overexpression protected against doxorubicin-mediated cell death and apoptosis also in cells not exposed to the chelator. Exposure to dexrazoxane increased the expression of the HIF-regulated, antiapoptotic proteins survivin, Mcl1 and haem oxygenase.

Conclusions and implications: Our results showing HIF-dependent prevention of doxorubicin toxicity in dexrazoxane-treated H9c2 cardiomyocytes suggest that HIF activation may be a mechanism contributing to the protective effect of dexrazoxane against anthracycline cardiotoxicity.

© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Figures

Figure 1

Dexrazoxane (DRZ) induces HIF-α expression and transactivation capacity. (A) Immunoblot analysis of the nuclear extracts of untreated H9c2 cells (C) and cells treated with desferrioxamine (DFO) for 24 h, or different concentrations of DRZ for 3 h, using anti-HIF-1α and anti-HIF-2α antibodies. The blots were reprobed using the antibody against TFIID as a loading control. The panel shows one representative blot and the densitometric quantification relative to C-values. (B) Relative luciferase activity (RLA) in untreated H9c2 cells (C) and cells exposed to DFO or dexrazoxane, as described above. The cells were transiently transfected with the empty pGL3 vector (ev) or a construct in which luciferase was controlled by an HRE multimer and cotransfected using a control vector containing the Renilla luciferase gene. When appropriate, the cells were also cotransfected with an expression vector coding for a dominant-negative mutant of the constitutive HIF-1β subunit (ΔARNT). Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity and normalized to the activity recorded in untreated cells (arbitrarily set to 1). Mean values ± SD. *P < 0.001; ***P < 0.01; #P < 0.05, n = 3. HIF, hypoxia-inducible factor; HRE, hypoxia response element; TFIID, transcription factor II D.

Figure 2

Dexrazoxane (DRZ) protects H9c2 cells from apoptotic cell death. (A) H9c2 cells were left untreated (C), or exposed for 24 h to doxorubicin (DOX), for 3 h to dexrazoxane alone or pretreated with DRZ for 3 h and then exposed to doxorubicin. Viability was evaluated by the MTT assay and DOX toxicity was calculated as the percentage of viable cells after drug exposure. (B) H9c2 cells were treated as described for panel A, and apoptosis was determined by measuring caspase-3 activity. (C) H9c2 cells were treated as described for panel A, and apoptosis was determined by measuring Annexin V-FITC as described in Methods. (D) H9c2 cells were treated as described for panel A, and apoptosis was determined by measuring cytochrome c (Cyt c) release. α-Tubulin was used as a loading control. The figure shows one representative immunoblot and the densitometric quantification relative to C-values. Mean values ± SD. *P < 0.001; **P < 0.005 ***P < 0.01; #P < 0.05, n = 5 for experiments reported in panels A and B, and 3 for experiments reported in panel C and D. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Figure 3

Doxorubicin (DOX) does not inhibit HIF expression and transactivation capacity. (A) Immunoblot analysis of nuclear extracts of untreated H9c2 cells (C) and cells exposed for 24 h to doxorubicin, for 3 h to dexrazoxane (DRZ) alone or pretreated with DRZ for 3 h and then exposed to DOX, using the anti-HIF-1α antibody. The blots were reprobed using the antibody against TFIID as a loading control. The panel shows one representative blot and the densitometric quantification relative to C-values. (B) Relative luciferase activity (RLA) in H9c2 cells transiently transfected with a construct in which luciferase was controlled by an HRE multimer and treated as described for panel A. The cells were cotransfected using a control vector containing the Renilla luciferase gene. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity and normalized to the activity recorded in untreated cells (arbitrarily set to 1). Mean values ± SD. *P < 0.001; ***P < 0.01; #P < 0.05, n = 3. HIF, hypoxia-inducible factor; TFIID, transcription factor II D.

Figure 4

Suppression of HIF activity blocks dexrazoxane (DRZ)-mediated cardioprotection. (A) H9c2 cells were transfected with the expression vector ΔARNT or an empty vector (ev) and treated as indicated in Figure 3A. Viability was evaluated by means of the MTT assay. (B) H9c2 cells were transfected and treated as described for panel A, and apoptosis was evaluated by measuring caspase-3 activity. Mean values ± SD. *P < 0.001; ***P < 0.01; n = 5. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Figure 5

Knockdown of HIF-1α blocks dexrazoxane (DRZ)-mediated cardioprotection. (A) Transfection efficiency in H9c2 cells. The figure shows a merged image with DAPI-stained nuclei (blue) and punctuate fluorescence of rhodamine-labelled siRNA (red). (B) Relative luciferase activity (RLA) and HIF-1α protein levels in untreated H9c2 cells (C) or cells exposed to dexrazoxane for 3 h. The cells were transiently transfected with a HRE multimer and also cotransfected with vectors containing the negative control shRNA (NC) or shRNA targeting HIF-1α (HIF-1α shRNA). Luciferase activity was determined after 48 h, corrected for transfection efficiency on the basis of Renilla luciferase activity and normalized to the activity recorded in untreated cells (arbitrarily set to 1). HIF-1α protein levels were determined by immunoblot analysis of the nuclear extracts, as described in the legend to Figure 1. (C) H9c2 cells were transfected with vectors containing the negative control shRNA (NC), shRNA targeting GFP (GFP shRNA) or HIF-1α (HIF-1α shRNA) and treated as indicated in Figure 3A. Cell viability was evaluated by means of the MTT assay. Mean values ± SD. *P < 0.001; **P < 0.005 ***P < 0.01; n = 3. siRNA, small interfering RNA; shRNA, short hairpin RNA; HIF, hypoxia-inducible factor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Figure 6

HIF-1α overexpression is cardioprotective in the absence of dexrazoxane (DRZ). (A) Relative luciferase activity (RLA) and HIF-1α protein levels in untreated H9c2 cells (C), exposed to dexrazoxane for 3 h or transfected with a construct that induced the overexpression of HIF-1α (pcMV4 HIF-1α). The cells were transiently transfected with a construct in which luciferase was controlled by an HRE multimer. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity and normalized to the activity recorded in untreated cells (arbitrarily set to 1). HIF-1α protein levels were determined by immunoblot analysis of the nuclear extracts, as described in the legend to Figure 1. (B) Untreated H9c2 cells (C) or cells exposed to doxorubicin (DOX) for 24 h were transiently transfected with the empty pGL3 vector (ev) or the pcMV4 HIF-1α vector. Cell viability was evaluated by means of the MTT assay. (C) H9c2 cells were treated and transfected as described for panel B and apoptosis was determined as described in Figure 4B. Mean values ± SD. *P < 0.001; ***P < 0.01; #P < 0.05, n = 3. HIF, hypoxia-inducible factor; HRE, hypoxia response element; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium.

Figure 7

Dexrazoxane (DRZ) induces the expression of antiapoptotic genes. Immunoblot analysis of cytosolic extracts of untreated H9c2 cells (C), and cells exposed for 24 h to doxorubicin (DOX), for 3 h to dexrazoxane alone or pretreated with DRZ for 3 h and then exposed to DOX. When appropriate, the cells were also transfected with the expression vector ΔARNT. In some cases, the cells were exposed to DMOG for 24 h or to BSO and H2O2 for 3 h, and then washed and re-incubated for 2 h. Antibodies against the indicated proteins were used, and the blots were reprobed using the antibody against α-tubulin as a loading control. The panels show one representative blot and the densitometric quantification relative to C-values. Mean values ± SD. *P < 0.001; **P < 0.005, n = 3. DMOG, dimethyloxalyl glycine; BSO, buthionine sulphoximine.