Geranylgeranylacetone blocks doxorubicin-induced cardiac toxicity and reduces cancer cell growth and invasion through RHO pathway inhibition.

Doxorubicin is a widely used chemotherapy for solid tumors and hematologic malignancies, but its use is limited due to cardiotoxicity. Geranylgeranylacetone (GGA), an antiulcer agent used in Japan for 30 years, has no significant adverse effects, and unexpectedly reduces ovarian cancer progression in mice. Because GGA reduces oxidative stress in brain and heart, we hypothesized that GGA would prevent oxidative stress of doxorubicin cardiac toxicity and improve doxorubicin's chemotherapeutic effects. Nude mice implanted with MDA-MB-231 breast cancer cells were studied after chronic treatment with doxorubicin, doxorubicin/GGA, GGA, or saline. Transthoracic echocardiography was used to monitor systolic heart function and xenografts evaluated. Mice were euthanized and cardiac tissue evaluated for reactive oxygen species generation, TUNEL assay, and RHO/ROCK pathway analysis. Tumor metastases were evaluated in lung sections. In vitro studies using Boyden chambers were performed to evaluate GGA effects on RHO pathway activator lysophosphatidic acid (LPA)-induced motility and invasion. We found that GGA reduced doxorubicin cardiac toxicity, preserved cardiac function, prevented TUNEL-positive cardiac cell death, and reduced doxorubicin-induced oxidant production in a nitric oxide synthase-dependent and independent manner. GGA also reduced heart doxorubicin-induced ROCK1 cleavage. Remarkably, in xenograft-implanted mice, combined GGA/doxorubicin treatment decreased tumor growth more effectively than doxorubicin treatment alone. As evidence of antitumor effect, GGA inhibited LPA-induced motility and invasion by MDA-MB-231 cells. These anti-invasive effects of GGA were suppressed by geranylgeraniol suggesting GGA inhibits RHO pathway through blocking geranylation. Thus, GGA protects the heart from doxorubicin chemotherapy-induced injury and improves anticancer efficacy of doxorubicin in breast cancer.

Cardiac-specific over-expression of epidermal growth factor receptor 2 (ErbB2) induces pro-survival pathways and hypertrophic cardiomyopathy in mice.

BACKGROUND: Emerging evidence shows that ErbB2 signaling has a critical role in cardiomyocyte physiology, based mainly on findings that blocking ErbB2 for cancer therapy is toxic to cardiac cells. However, consequences of high levels of ErbB2 activity in the heart have not been previously explored. METHODOLOGY/PRINCIPAL FINDINGS: We investigated consequences of cardiac-restricted over-expression of ErbB2 in two novel lines of transgenic mice. Both lines develop striking concentric cardiac hypertrophy, without heart failure or decreased life span. ErbB2 transgenic mice display electrocardiographic characteristics similar to those found in patients with Hypertrophic Cardiomyopathy, with susceptibility to adrenergic-induced arrhythmias. The hypertrophic hearts, which are 2-3 times larger than those of control littermates, express increased atrial natriuretic peptide and ß-myosin heavy chain mRNA, consistent with a hypertrophic phenotype. Cardiomyocytes in these hearts are significantly larger than wild type cardiomyocytes, with enlarged nuclei and distinctive myocardial disarray. Interestingly, the over-expression of ErbB2 induces a concurrent up-regulation of multiple proteins associated with this signaling pathway, including EGFR, ErbB3, ErbB4, PI3K subunits p110 and p85, bcl-2 and multiple protective heat shock proteins. Additionally, ErbB2 up-regulation leads to an anti-apoptotic shift in the ratio of bcl-xS/xL in the heart. Finally, ErbB2 over-expression results in increased activation of the translation machinery involving S6, 4E-BP1 and eIF4E. The dependence of this hypertrophic phenotype on ErbB family signaling is confirmed by reduction in heart mass and cardiomyocyte size, and inactivation of pro-hypertrophic signaling in transgenic animals treated with the ErbB1/2 inhibitor, lapatinib. CONCLUSIONS/SIGNIFICANCE: These studies are the first to demonstrate that increased ErbB2 over-expression in the heart can activate protective signaling pathways and induce a phenotype consistent with Hypertrophic Cardiomyopathy. Furthermore, our work suggests that in the situation where ErbB2 signaling contributes to cardiac hypertrophy, inhibition of this pathway may reverse this process.

Detection of dose response in chronic doxorubicin-mediated cell death with cardiac technetium 99m annexin V single-photon emission computed tomography.

The aim of this study was to evaluate whether technetium 99m hydrazinonicotinamide (99mTc-HYNIC)-annexin V single-photon emission computed tomography (SPECT) would detect dose-dependent doxorubicin (DOX)-mediated cell death in the heart compared with functional echocardiography. Adult female Sprague-Dawley rats were treated with DOX (cumulative dose of 15 or 7.5 mg/kg) or saline (n = 7) and monitored by echocardiography. Rats were injected with 7 to 8 mCi 99mTc-HYNIC-annexin V and imaged 1 hour postinjection using a small animal dual-head SPECT/computed tomography (CT) system with multipinhole technology. Two regions of interest were drawn in the myocardium and soft tissue regions to calculate the cardiac uptake ratio (CUR) of reconstructed images. Myocardium and blood were harvested for radioactivity measurements or TUNEL assay. Biodistribution of 99mTc-HYNIC-annexin V uptake, CUR from SPECT/CT fused cardiac images, and TUNEL of myocardium demonstrated a dose-dependent toxicity response, with the cumulative 15 mg/kg DOX treatment showing the greatest degree of cell death. In contrast, echocardiography detected functional deficits only at the highest DOX dose. In vivo molecular imaging of DOX-induced cardiac toxicity with 99mTc-HYNIC-annexin V detects dose-dependent cell death before ventricular deficits are observed with echocardiography. 99mTc-HYNIC-annexin V SPECT-based molecular imaging may provide an attractive new technique for assessing early changes in myocardial function in patients undergoing DOX therapy.

Effects of bedding substrates on microsomal enzymes in rabbit liver.

Previous studies have reported that housing rats and mice on softwood beddings induce microsomal enzymes. To date, no published studies investigate effects of softwood beddings on microsomal induction in rabbits. The purpose of this study was to determine whether microsomal enzymes, primarily cytochromes P450 3A and 2B, were induced in rabbits exposed to commonly used bedding substrates. Rabbits were placed in cages 7.6 cm above 1 sheet of 24 x 36 in. postconsumer recycled paper, approximately 16 cups (130 ounces) of pine shavings, or no substrate. Positive-control rabbits were given either rifampin (50 mg/kg) or phenobarbital (60 mg/kg) intraperitoneally once daily for 5 d. At 2, 7, and 14 d after placement in test cages, rabbits were euthanized and the livers harvested. Microsomal pellets were prepared from the livers and used in an erythromycin Ndemethylase assay (to determine CYP3A activity) and a pentoxyresorufin-O-deethylation assay (to determine CYP2B activity). Although the levels of enzyme induction varied slightly in both assays, statistical significance was not reached compared to the positive-control levels. These results indicate that neither CYP450 3A or 2B enzymes are induced by exposure of rabbits to pine shavings or paper substrate as noncontact bedding for up to 14 d.

Heat shock protein 90 and ErbB2 in the cardiac response to doxorubicin injury.

A major drawback to doxorubicin as a cancer-treating drug is cardiac toxicity. To understand the mechanism of doxorubicin cardiac toxicity and the potent synergic effect seen when doxorubicin is combined with anti-ErbB2 (trastuzumab), we developed an in vivo rat model that exhibits progressive dose-dependent cardiac damage and loss of cardiac function after doxorubicin treatment. The hearts of these animals respond to doxorubicin damage by increasing levels of ErbB2 and the ErbB family ligand, neuregulin 1beta, and by activating the downstream Akt signaling pathway. These increases in ErbB2 protein levels are not due to increased ErbB2 mRNA, however, suggesting post-transcriptional mechanisms for regulating this protein in the heart. Accordingly, levels of heat shock protein 90 (HSP90), a known ErbB2 protein stabilizer and chaperone, are increased by doxorubicin treatment, and coimmunoprecipitation reveals binding of HSP90 to ErbB2. Isolated cardiomyocytes are more susceptible to doxorubicin after treatment with HSP90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin, suggesting that the HSP90 is protective during doxorubicin treatment. Thus, our results provide one plausible mechanism for the susceptibility of the heart to anti-ErbB2 therapy post-doxorubicin therapy in subclinical and clinical conditions. Additionally, these results suggest that further testing is needed for HSP90 inhibitors under various conditions in the heart.

Validation of the use of long-term indwelling jugular catheters in a rat model of cardiotoxicity.

Doxorubicin administered to rats induces a dose-dependent cardiomyopathy. Both doxorubicin administration and the presence of indwelling catheters have been associated with thrombus formation. We sought to determine feasibility of drug delivery and degree of thrombogenesis related to long-term indwelling catheter use in a cardiotoxicity model. Rats receiving doxorubicin or saline via jugular catheters coated with end-point immobilized heparin were compared to rats receiving similar treatments via direct jugular intravenous injection (venotomy). Onset of cardiotoxicity, defined by reduction in fractional shortening to 45% or less, was determined by echocardiography. Thrombogenesis was assessed by observation of atrial thrombi and pulmonary emboli as determined by post-mortem and histologic examination. Significantly more of the doxorubicin-treated and catheterized group (87.5%) developed cardiotoxicity relative to the doxorubicin-treated-venotomized group (28.6%), as indicated by an earlier and more precipitous decline in fractional shortening in the doxorubicin-treated-catheterized rats. Despite this change, rats from catheterized groups demonstrated improved weight maintenance relative to venotomy groups. Although the number of pulmonary emboli did not differ significantly between groups, 50% of the doxorubicin-treated-catheterized animals developed vegetative endocarditis. Despite alteration of the model-induced cardiac disease, we submit that the more reliable and early induction of the desired endpoint, in addition to improved weight maintenance, represent model refinements. The ease of drug delivery with minimal restraint and no anesthesia is an additional and important benefit. The development of vegetative endocarditis represents an opportunity to study the formation and prevention of this condition.

Sex differences in cell death.

Female patients experience substantial neuroprotection after experimental stroke compared with male patients, a finding attributed to the protective effects of gonadal hormones. This study examined the response of male- and female-derived organotypic hippocampal slices to oxidative and excitotoxic injury. Both oxygen and glucose deprivation and N-methyl-D-aspartic acid exposure led to neuronal death; however, female-derived cultures sustained less injury than male-derived cultures. Cell death after oxygen and glucose deprivation was ameliorated in male cultures, but not female cultures, by the addition of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor. These studies have relevance to researchers investigating neuroprotective agents in mixed sex experiments.