Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity.

Cardiotoxicity, which may result from intense cardiac oxidative stress and inflammation, is the main limiting factor of the anticancer therapy using doxorubicin. Because statins might exert beneficial pleiotropic cardiovascular effects, among other things, by anti-inflammatory and antioxidative mechanisms, we investigated whether or not fluvastatin pretreatment can attenuate doxorubicin-induced cardiotoxicity. Five days after a single injection of doxorubicin (20 mg/kg; i.p.), left ventricular (LV) function was measured in fluvastatintreated (DoxStatin; 100 mg/kg/day, p.o.) and saline-treated (doxorubicin) mice (n = 8 per group) by a micro conductance catheter. Untreated mice served as controls (placebo; n = 8 per group). After measurement of cardiac function, LV tissues were analyzed by molecular biological and immunohistologic methods. Injection resulted in significantly impaired LV function (LV pressure, À29%; dp/dtmax, À45%; cardiac output, À68%; P < 0.05) when compared with placebo. This was associated with a significant increase in cardiac oxidative stress, inflammation and apoptotic mechanisms, as indicated by significant increased cardiac lipid peroxidation activity, protein expression of nitrotyrosine, tumor necrosis factor A and Bax (P < 0.05). In contrast, DoxStatin mice showed improved LV function (LV pressure, +24%; dp/dtmax, +87%; cardiac output, +87%; P < 0.05) when compared with untreated doxorubicin mice. This was associated with reduced cardiac expression of nitrotyrosine, enhanced expression of the mitochondrial located antioxidative SOD 2, attenuated mitochondrial apoptotic pathways, and reduced cardiac inflammatory response. Statin pretreatment attenuates doxorubicin-induced cardiotoxicity via antioxidative and antiinflammatory effects. [Cancer Res 2009;69(2):695-9]

S U M M A R Y ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the blood-brain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., ␤ -blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level.

Doxorubicin is a frequently used anticancer drug, but its therapeutic benefit is limited by acute and chronic cardiotoxicity, often leading to heart failure. The mechanisms underlying doxorubicin-induced cardiotoxicity remain unclear. It was previously shown in men that doxorubicin leads to increased endothelin-1 plasma levels. In addition, cardiacspecific overexpression of endothelin-1 in mice resulted in a cardiomyopathy resembling the phenotype following doxorubicin administration. We therefore hypothesized that endothelin-1 is involved in the pathogenesis of doxorubicin cardiotoxicity. In mice (C57Bl/10), we found that doxorubicin (20 mg/kg body weight, i.p.) impaired cardiac function with decreased ejection fraction, diminished cardiac output, and decreased end-systolic pressure points recorded by a microconductance catheter. This impaired function was accompanied by the up-regulation of endothelin-1 expression on mRNA and protein level. In vitro investigations confirmed the regulation of endothelin-1 by doxorubicin and indicated that the doxorubicin-mediated increase of endothelin-1 expression involves epidermal growth factor receptor signaling via the MEK1/2-ERK1/2 cascade, which was further confirmed by immunoblotting studies in the left ventricle of treated animals. Pretreatment of mice with the endothelin receptor antagonist bosentan (100 mg/kg body weight, p.o.) strikingly inhibited doxorubicin-induced cardiotoxicity with preserved indices of contractility. Moreover, bosentan pretreatment resulted in reduced tumor necrosis factor-A content, lipid peroxidation, and Bax expression, as well as increased GATA-4 expression. Thus, endothelin-1 plays a key role in mediating the cardiotoxic effects of doxorubicin and its inhibition may be of therapeutic benefit for patients receiving doxorubicin.

HepG-2 cells are widely used as a cell model to investigate hepatocellular carcinomas and the effect of anticancer drugs such as doxorubicin, an effective antineoplastic agent, which has broad antitumoral activity against many solid and hematological malignancies. To investigate the effect of doxorubicin on the protein pattern, we used complementary proteomic workflows including 2-D gel-based and gel-free methods. The analysis of crude HepG2 cell extracts by 2-D DIGE provided data on 1835 protein spots which was then complemented by MS-centered analysis of stable isotope labeling by amino acids in cell culture-labeled cells. The monitoring of more than 1300 distinct proteins, including proteins of the membrane fraction provides the most comprehensive overview on the proteome of the widely used model cell line HepG2. Of the proteins monitored in total, 155 displayed doxorubicin-induced changes in abundance. Functional analysis revealed major influences of doxorubicin on proteins involved in protein synthesis, DNA damage control, electron transport/mitochondrial function, and tumor growth. The strongest decrease in level was found for proteins involved in DNA replication and protein synthesis, whereas proteins with a function in DNA damage control and oxidative stress management displayed increased levels following treatment with doxorubicin compared with control cells. Furthermore, the doxorubicin-associated increase in levels of multiple forms of keratins 8, 18, and 19 and other structural proteins revealed an influence on the cytoskeleton network.