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The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work, we compared the effect of thyroid hormone and that of anacardic acid, a naturally occurring histone acetylase inhibitor, or both in combination, on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation, we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them, a rapid induction of the transcription factor Castor 1 (CASZ1), important for cardiomyocytes differentiation and maturation during embryonic development, was observed in the presence of AA. In contrast, thyroid hormone (T 3) was more effective in stimulating spontaneous firing, thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion, AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways.

This review is based on novel observations from our laboratory on the nuclear translocation and functional role of endothelial nitric oxide synthase (eNOS) in endothelial and prostate cancer (PCa) epithelial cells. Nitric oxide (NO), the product of eNOS, is a free radical involved in the physiology and pathophysiology of living organisms and in a variety of biological processes including the maintenance of vascular homeostasis. Of relevance in this context is the role that estrogens play in the apoptotic process and the migration of endothelial cells through the regulation of target genes such as eNOS itself. It has been shown that both estrogen and NO signaling, mediated respectively by the estrogen receptors (ERs) and eNOS, can strongly counteract endothelial senescence through a common effector, the catalytic subunit of human telomerase. Therefore, this protein has been identified as a key molecule in the aging process which, intriguingly, is considered the only risk factor in the development of PCa and one of the major determinants of cardiovascular diseases. Indeed, in both these contexts we have defined a molecular mechanism involving activation of eNOS and hypoxia-inducible factors in association with ERβ that characterizes the most aggressive form of PCa or influences endothelial cell differentiation. Altogether these data led us to postulate that activation of eNOS is a crucial requirement for the delaying of endothelial senescence as well as for the acquisition of androgen-independence and for tumor progression in the prostate microenvironment.

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-β and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERβ/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERβ antagonist or its natural ligand 5α-androstane-3β,17β-diol, eNOS inhibitors or ERβ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERβ/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERβ/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERβ/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERβ/eNOS function by 3β-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.

BackgroundHypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the "angiogenic switch" during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.Methodology/principal findingsHere we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.Conclusions/significanceThese findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.