Doxorubicin enhances oxysterol levels resulting in a LXR-mediated upregulation of cardiac cholesterol transporters.

The anthracycline-mediated cardiotoxicity is still not completely understood. To examine the impact of cholesterol metabolism and transport in this context, cholesterol and oxysterol levels as well as the expression of the cholesterol transporters ABCA1 and ABCG1 were analyzed in doxorubicin-treated HL-1 murine cardiomyocytes as well as in mouse model for acute doxorubicin-induced cardiotoxicity. Doxorubicin-treated HL-1 cells exhibited enhanced cholesterol (153±20% of control), oxysterol (24S-hydroxycholesterol: 206±29% of control) and cholesterol precursor levels (lathosterol: 122±12% of control; desmosterol: 188±10% of control) indicating enhanced cholesterol synthesis. Moreover, abca1 and abcg1 were upregulated on mRNA, protein and functional level caused by a doxorubicin-mediated activation of the nuclear receptor LXR. In addition, the oxysterols not only induced the abca1 and abcg1 in HL-1 cells but also enhanced the expression of endothelin-1 and transforming growth factor-ß, which have already been identified as important factors in doxorubicin-induced cardiotoxicity. These in vitro findings were verified in a murine model for acute doxorubicin-induced cardiotoxicity, demonstrating elevated cardiac (2.1±0.2vs. 3.6±1.0ng/mg) and systemic cholesterol levels (105.0±8.4vs. 130.0±4.3mg/dl), respectively, as well as enhanced oxysterol levels such as cardiac 24S-hydroxycholesterol (2.1±0.2vs. 3.6±1.0ng/mg). In line with these findings cardiac mRNA expression of abca1 (303% of control) and abcg1 (161% of control) was induced. Taken together, our data demonstrate enhanced cholesterol and oxysterol levels by doxorubicin, resulting in a LXR-dependent upregulation of abca1 and abcg1. In this context, the cytotoxic effects of oxysterols and their impact on cardiac gene expression should be considered as an important factor in doxorubicin-induced cardiotoxicity.

Protective effects of endothelin receptor A and B inhibitors against doxorubicin-induced cardiomyopathy.

The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways.

Acute exposure to doxorubicin results in increased cardiac P-glycoprotein expression.

Doxorubicin is a frequently used anticancer drug, but its use is restricted due to the occurrence of severe side effects, namely strong cardiotoxicity. It is known from cancer cells that doxorubicin enhanced the expression of its efflux pump P-glycoprotein (P-gp), which may modulate local drug concentrations. We therefore studied the cardiac expression of P-gp in doxorubicin-treated mice. Mice were treated with doxorubicin, and P-gp expression was studied after 1, 3, and 5 days. Thereby, we could show a significant upregulation of abcb1a (162 ± 15% of control) and abcb1b (418 ± 110% of control) mRNA transcripts after 3 days. On protein level, western blot analysis and immunofluorescence staining revealed a similar finding 5 days after doxorubicin administration. In addition, these results could be confirmed by in vitro models using primary rat cardiomyocytes and the murine cardiomyocyte-like HL-1 cells. Besides an enhanced mRNA and protein expression, doxorubicin-treated HL-1 cells also demonstrated an enhanced P-gp function as assessed by a daunorubicin accumulation assay. Our in vivo and in vitro results demonstrate a cardiac upregulation of P-gp in doxorubicin-treated mice on expression and functional level. This finding may be relevant for cardiac tissue concentrations of P-gp substrates and may represent a mechanism in cardiac self-protection against xenobiotics.