BackgroundThe pathophysiology of obesity and type 2 diabetes mellitus is associated with abnormalities in endocrine signaling in adipose tissue and one of the key signaling affectors operative in these disorders is the nuclear hormone transcription factor peroxisome proliferator-activated receptor-γ (PPARγ). PPARγ has pleiotropic functions affecting a wide range of fundamental biological processes including the regulation of genes that modulate insulin sensitivity, adipocyte differentiation, inflammation and atherosclerosis. To date, only a limited number of direct targets for PPARγ have been identified through research using the well established pre-adipogenic cell line, 3T3-L1. In order to obtain a genome-wide view of PPARγ binding sites, we applied the pair end-tagging technology (ChIP-PET) to map PPARγ binding sites in 3T3-L1 preadipocyte cells.Methodology/Principal FindingsCoupling gene expression profile analysis with ChIP-PET, we identified in a genome-wide manner over 7700 DNA binding sites of the transcription factor PPARγ and its heterodimeric partner RXR during the course of adipocyte differentiation. Our validation studies prove that the identified sites are bona fide binding sites for both PPARγ and RXR and that they are functionally capable of driving PPARγ specific transcription. Our results strongly indicate that PPARγ is the predominant heterodimerization partner for RXR during late stages of adipocyte differentiation. Additionally, we find that PPARγ/RXR association is enriched within the proximity of the 5′ region of the transcription start site and this association is significantly associated with transcriptional up-regulation of genes involved in fatty acid and lipid metabolism confirming the role of PPARγ as the master transcriptional regulator of adipogenesis. Evolutionary conservation analysis of these binding sites is greater when adjacent to up-regulated genes than down-regulated genes, suggesting the primordial function of PPARγ/RXR is in the induction of genes. Our functional validations resulted in identifying novel PPARγ direct targets that have not been previously reported to promote adipogenic differentiation.Conclusions/SignificanceWe have identified in a genome-wide manner the binding sites of PPARγ and RXR during the course of adipogenic differentiation in 3T3L1 cells, and provide an important resource for the study of PPARγ function in the context of adipocyte differentiation.

No abstract

BackgroundNumerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin’s multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained.MethodsTo deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers.ResultsWe show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death.ConclusionsTaken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.

An essential oil extract, derived from the rhizome of Curcuma wenyujin (CWE), possesses antioxidative, antimicrobial, and anti-inflammatory properties. However, it remains unknown how exactly CWE inhibits tumor growth. In this study, using human cervical cancer HeLa cells, the authors postulated that CWE has the ability to inhibit tumor growth. The study shows that CWE dose-dependently suppressed colony formation and inhibited the proliferation of HeLa cells through blockade of cell cycle progression at G1 phase and apoptosis. CWE-induced G1 arrest was associated with retinoblastoma protein dephosphorylation and reduced amounts of cyclins D1 and D3, and cyclin-dependent kinase 4 and 6 proteins. CWE treatment resulted in apoptosis in HeLa cells as evidenced by morphological changes, caspase activation and PARP cleavage, which can be reversed by a pan-caspase inhibitor. It was observed that CWE treatment activated the mitochondrial apoptotic pathway indicated by a decrease in Mcl-1 and Bcl-xL levels, resulting in mitochondrial membrane potential loss and caspases 9 activation. CWE-treated cells displayed reduced PTEN, AKT, and STAT3 phosphorylation and downregulation of NFkB signaling, providing a mechanism for the G1 arrest and apoptosis observed. Furthermore, CWE inhibited tumor growth of HeLa in a xenograft mouse tumor model, suggesting that CWE inhibited tumorigenesis by inhibiting cell proliferation and inducing apoptosis. These findings are the first to reveal the molecular basis for the anticervical cancer action of CWE. The results suggest that CWE could be developed as a drug for the management of cervical cancer.