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Testosterone Antagonizes Doxorubicin-Induced Senescence of Cardiomyocytes.

ABSTRACT: Chronic cardiotoxicity is less common in male than in female patients receiving doxorubicin and other anthracyclines at puberty and adolescence. We hypothesized that this sex difference might be secondary to distinct activities of sex hormones on cardiomyocyte senescence, which is thought to be central to the development of long-term anthracycline cardiomyopathy.H9c2 cells and neonatal mouse cardiomyocytes were exposed to doxorubicin with or without prior incubation with testosterone or 17β-estradiol, the main androgen and estrogen, respectively. Testosterone, but not 17β-estradiol, counteracted doxorubicin-elicited senescence. Downregulation of telomere binding factor 2, which has been pinpointed previously as being pivotal to doxorubicin-induced senescence, was also prevented by testosterone, as were p53 phosphorylation and accumulation. Pretreatment with the androgen receptor antagonist flutamide, the phosphatidylinositol 3 kinase inhibitor LY294002, and the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester abrogated the reduction in senescence and the normalization of telomere binding factor 2 levels attained by testosterone. Consistently, testosterone enhanced the phosphorylation of AKT and nitric oxide synthase 3. In H9c2 cells, doxorubicin-stimulated senescence was still observed up to 21 days after treatment and increased further when cells were rechallenged with doxorubicin 14 days after the first exposure to mimic the schedule of anthracycline-containing chemotherapy. Remarkably, these effects were also inhibited by testosterone.Testosterone protects cardiomyocytes against senescence caused by doxorubicin at least in part by modulating telomere binding factor 2 via a pathway involving the androgen receptor, phosphatidylinositol 3 kinase, AKT, and nitric oxide synthase 3. This is a potential mechanism by which pubescent and adolescent boys are less prone to chronic anthracycline cardiotoxicity than girls.

SUBMITTER: Altieri P

PROVIDER: S-EPMC4859360 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other

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Project description:Background: Leydig cells secrete the steroid hormone, testosterone, which is essential for male fertility and reproductive health. Stress increases the secretion of glucocorticoid [corticosterone, (CORT) in rats] that decreases circulating testosterone levels in part through a direct action on its receptors in Leydig cells. Intratesticular CORT level is dependent on oxidative inactivation of CORT by 11?-hydroxysteroid dehydrogenase 1 (HSD11B1) in rat Leydig cells. Pain may cause the stress, thus affecting testosterone production in Leydig cells. Methods: Adult male Sprague-Dawley rats orally received vehicle control or 5 or 10 mg/kg dehydroepiandrosterone (DHEA) 0.5 h before being subjected to pain stimulation for 1, 3, and 6 h. In the present study, we investigated the time-course changes of steroidogenic gene expression levels after acute pain-induced stress in rats and the possible mechanism of DHEA that prevented it. Plasma CORT, luteinizing hormone (LH), and testosterone (T) levels were measured, and Leydig cell gene expression levels were determined. The direct regulation of HSD11B1 catalytic direction by DHEA was detected in purified rat Leydig, liver, and rat Hsd11b1-transfected COS1 cells. Results: Plasma CORT levels were significantly increased at hour 1, 3, and 6 during the pain stimulation, while plasma T levels were significantly decreased starting at hour 3 and 6. Pain-induced stress also decreased Star, Hsd3b1, and Cyp17a1 expression levels at hour 3. When 5 and 10 mg/kg DHEA were orally administered to rats 0.5 h before starting pain stimulation, DHEA prevented pain-mediated decrease in plasma T levels and the expression of Star, Hsd3b1, and Cyp17a1 without affecting plasma CORT levels. DHEA was found to modulate HSD11B1 activities by increasing its oxidative activity and decreasing its reductive activity, thus decreasing the intracellular CORT levels in Leydig cells. Conclusion: Stress induced by acute pain can inhibit Leydig cell T production by upregulation of corticosterone. DHEA can prevent the negative effects of excessive corticosterone by modulating HSD11B1 activity.

Project description:BackgroundDoxorubicin, an anthracycline chemotherapeutic known to incur heart damage, decreases heart function in up to 11% of patients. Recent investigations have implicated the Wnt signaling cascade as a key modulator of cardiac tissue repair after myocardial infarction. Wnt upregulation in murine models resulted in stimulation of angiogenesis and suppression of fibrosis after ischemic insult. However, the molecular mechanisms of Wnt in mitigating doxorubicin-induced cardiac insult require further investigation. Identifying cardioprotective mechanisms of Wnt is imperative to reducing debilitating cardiovascular adverse events in oncologic patients undergoing treatment.MethodsExposing human cardiomyocyte AC16 cells to varying concentrations of Wnt10b and DOX, we observed key metrics of cell viability. To assess the viability and apoptotic rates, we utilized MTT and TUNEL assays. We quantified cell and mitochondrial membrane stability via LDH release and JC-1 staining. To investigate how Wnt10b mitigates doxorubicin-induced apoptosis, we introduced pharmacologic inhibitors of key enzymes involved in apoptosis: FR180204 and SB203580, ERK1/2 and p38 inhibitors. Further, we quantified apoptotic executor enzymes, caspase 3/7, via immunofluorescence.ResultsAC16 cells exposed solely to doxorubicin were shrunken with distorted morphology. Cardioprotective effects of Wnt10b were demonstrated via a reduction in apoptosis, from 70.1% to 50.1%. LDH release was also reduced between doxorubicin and combination groups from 2.27-fold to 1.56-fold relative to the healthy AC16 control group. Mitochondrial membrane stability was increased from 0.67-fold in the doxorubicin group to 5.73 in co-treated groups relative to control. Apoptotic protein expression was stifled by Wnt10b, with caspase3/7 expression reduced from 2.4- to 1.3-fold, and both a 20% decrease in p38 and 40% increase in ERK1/2 activity.ConclusionOur data with the AC16 cell model demonstrates that Wnt10b provides defense mechanisms against doxorubicin-induced cardiotoxicity and apoptosis. Further, we explain a mechanism of this beneficial effect involving the mitochondria through simultaneous suppression of pro-apoptotic p38 and anti-apoptotic ERK1/2 activities.

Project description:BACKGROUND:Doxorubicin (Dox) is a chemotherapy drug with limited application due to cardiotoxicity that may progress to heart failure. This study aims to evaluate the role of cardiomyocytes derived from mouse embryonic stem cells (CM-mESCs) in the treatment of Dox-induced cardiomyopathy (DIC) in mice. METHODS:The mouse embryonic stem cell (mESC) line E14TG2A was characterized by karyotype analysis, gene expression using RT-PCR and immunofluorescence. Cells were transduced with luciferase 2 and submitted to cardiac differentiation. Total conditioned medium (TCM) from the CM-mESCs was collected for proteomic analysis. To establish DIC in CD1 mice, Dox (7.5 mg/kg) was administered once a week for 3 weeks, resulting in a cumulative Dox dose of 22.5 mg/kg. At the fourth week, a group of animals was injected intramyocardially with CM-mESCs (8 × 105 cells). Cells were tracked by a bioluminescence assay, and the body weight, echocardiogram, electrocardiogram and number of apoptotic cardiomyocytes were evaluated. RESULTS:mESCs exhibited a normal karyotype and expressed pluripotent markers. Proteomic analysis of TCM showed proteins related to the negative regulation of cell death. CM-mESCs presented ventricular action potential characteristics. Mice that received Dox developed heart failure and showed significant differences in body weight, ejection fraction (EF), end-systolic volume (ESV), stroke volume (SV), heart rate and QT and corrected QT (QTc) intervals when compared to the control group. After cell or placebo injection, the Dox + CM-mESC group showed significant increases in EF and SV when compared to the Dox + placebo group. Reduction in ESV and QT and QTc intervals in Dox + CM-mESC-treated mice was observed at 5 or 30 days after cell treatment. Cells were detected up to 11 days after injection. The Dox + CM-mESC group showed a significant reduction in the percentage of apoptotic cardiomyocytes in the hearts of mice when compared to the Dox + placebo group. CONCLUSIONS:CM-mESC transplantation improves cardiac function in mice with DIC.

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Testosterone Antagonizes Doxorubicin-Induced Senescence of Cardiomyocytes.

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