Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.

Targeting aquaporin function: potent inhibition of aquaglyceroporin-3 by a gold-based compound.

Aquaporins (AQPs) are membrane channels that conduct water and small solutes such as glycerol and are involved in many physiological functions. Aquaporin-based modulator drugs are predicted to be of broad potential utility in the treatment of several diseases. Until today few AQP inhibitors have been described as suitable candidates for clinical development. Here we report on the potent inhibition of AQP3 channels by gold(III) complexes screened on human red blood cells (hRBC) and AQP3-transfected PC12 cells by a stopped-flow method. Among the various metal compounds tested, Auphen is the most active on AQP3 (IC(50) = 0.8±0.08 µM in hRBC). Interestingly, the compound poorly affects the water permeability of AQP1. The mechanism of gold inhibition is related to the ability of Au(III) to interact with sulphydryls groups of proteins such as the thiolates of cysteine residues. Additional DFT and modeling studies on possible gold compound/AQP adducts provide a tentative description of the system at a molecular level. The mapping of the periplasmic surface of an homology model of human AQP3 evidenced the thiol group of Cys40 as a likely candidate for binding to gold(III) complexes. Moreover, the investigation of non-covalent binding of Au complexes by docking approaches revealed their preferential binding to AQP3 with respect to AQP1. The high selectivity and low concentration dependent inhibitory effect of Auphen (in the nanomolar range) together with its high water solubility makes the compound a suitable drug lead for future in vivo studies. These results may present novel metal-based scaffolds for AQP drug development.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

PARP-1 regulates metastatic melanoma through modulation of vimentin-induced malignant transformation.

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.

EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer.

Circulating tumor cells (CTCs) are frequently associated with epithelial-mesenchymal transition (EMT).The objective of this study was to detect EMT phenotype through Vimentin (VIM) and Slug expression in cytokeratin (CK)-negative CTCs in non-metastatic breast cancer patients and to determine the importance of EGFR in the EMT phenomenon. In CK-negative CTCs samples, both VIM and Slug markers were co-expressed in the most of patients. Among patients EGFR+, half of them were positive for these EMT markers. Furthermore, after a systemic treatment 68% of patients switched from CK- to CK+ CTCs. In our experimental model we found that activation of EGFR signaling by its ligand on MCF-7 cells is sufficient to increase EMT phenotypes, to inhibit apoptotic events and to induce the loss of CK expression. The simultaneous detection of both EGFR and EMT markers in CTCs may improve prognostic or predictive information in patients with operable breast cancer.

Leptin is an anti-apoptotic effector in placental cells involving p53 downregulation.

Leptin, a peripheral signal synthetized by the adipocyte to regulate energy metabolism, can also be produced by placenta, where it may work as an autocrine hormone. We have previously demonstrated that leptin promotes proliferation and survival of trophoblastic cells. In the present work, we aimed to study the molecular mechanisms that mediate the survival effect of leptin in placenta. We used the human placenta choriocarcinoma BeWo and first trimester Swan-71 cell lines, as well as human placental explants. We tested the late phase of apoptosis, triggered by serum deprivation, by studying the activation of Caspase-3 and DNA fragmentation. Recombinant human leptin added to BeWo cell line and human placental explants, showed a decrease on Caspase-3 activation. These effects were dose dependent. Maximal effect was achieved at 250 ng leptin/ml. Moreover, inhibition of endogenous leptin expression with 2 µM of an antisense oligonucleotide, reversed Caspase-3 diminution. We also found that the cleavage of Poly [ADP-ribose] polymerase-1 (PARP-1) was diminished in the presence of leptin. We analyzed the presence of low DNA fragments, products from apoptotic DNA cleavage. Placental explants cultivated in the absence of serum in the culture media increased the apoptotic cleavage of DNA and this effect was prevented by the addition of 100 ng leptin/ml. Taken together these results reinforce the survival effect exerted by leptin on placental cells. To improve the understanding of leptin mechanism in regulating the process of apoptosis we determined the expression of different intermediaries in the apoptosis cascade. We found that under serum deprivation conditions, leptin increased the anti-apoptotic BCL-2 protein expression, while downregulated the pro-apoptotic BAX and BID proteins expression in Swan-71 cells and placental explants. In both models leptin augmented BCL-2/BAX ratio. Moreover we have demonstrated that p53, one of the key cell cycle-signaling proteins, is downregulated in the presence of leptin under serum deprivation. On the other hand, we determined that leptin reduced the phosphorylation of Ser-46 p53 that plays a pivotal role for apoptotic signaling by p53. Our data suggest that the observed anti-apoptotic effect of leptin in placenta is in part mediated by the p53 pathway. In conclusion, we provide evidence that demonstrates that leptin is a trophic factor for trophoblastic cells.

Enhanced antitumor activity of doxorubicin in breast cancer through the use of poly(butylcyanoacrylate) nanoparticles.

The use of doxorubicin (DOX), one of the most effective antitumor molecules in the treatment of metastatic breast cancer, is limited by its low tumor selectivity and its severe side effects. Colloidal carriers based on biodegradable poly(butylcyanoacrylate) nanoparticles (PBCA NPs) may enhance DOX antitumor activity against breast cancer cells, thus allowing a reduction of the effective dose required for antitumor activity and consequently the level of associated toxicity. DOX loading onto PBCA NPs was investigated in this work via both drug entrapment and surface adsorption. Cytotoxicity assays with DOX-loaded NPs were performed in vitro using breast tumor cell lines (MCF-7 human and E0771 mouse cancer cells), and in vivo evaluating antitumor activity in immunocompetent C57BL/6 mice. The entrapment method yielded greater drug loading values and a controlled drug release profile. Neither in vitro nor in vivo cytotoxicity was observed for blank NPs. The 50% inhibitory concentration (IC50) of DOX-loaded PBCA NPs was significantly lower for MCF-7 and E0771 cancer cells (4 and 15 times, respectively) compared with free DOX. Furthermore, DOX-loaded PBCA NPs produced a tumor growth inhibition that was 40% greater than that observed with free DOX, thus reducing DOX toxicity during treatment. These results suggest that DOX-loaded PBCA NPs have great potential for improving the efficacy of DOX therapy against advanced breast cancers.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after gamma-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced gamma-H2AX foci formation in response to gamma-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced gamma H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase.

Breast cancer 1 (BrCa1) may be behind decreased lipogenesis in adipose tissue from obese subjects.

CONTEXT Expression and activity of the main lipogenic enzymes is paradoxically decreased in obesity, but the mechanisms behind these findings are poorly known. Breast Cancer 1 (BrCa1) interacts with acetyl-CoA carboxylase (ACC) reducing the rate of fatty acid biosynthesis. In this study, we aimed to evaluate BrCa1 in human adipose tissue according to obesity and insulin resistance, and in vitro cultured adipocytes. RESEARCH DESIGN AND METHODS BrCa1 gene expression, total and phosphorylated (P-) BrCa1, and ACC were analyzed in adipose tissue samples obtained from a total sample of 133 subjects. BrCa1 expression was also evaluated during in vitro differentiation of human adipocytes and 3T3-L1 cells. RESULTS BrCa1 gene expression was significantly up-regulated in both omental (OM; 1.36-fold, p = 0.002) and subcutaneous (SC; 1.49-fold, p = 0.001) adipose tissue from obese subjects. In parallel with increased BrCa1 mRNA, P-ACC was also up-regulated in SC (p = 0.007) as well as in OM (p = 0.010) fat from obese subjects. Consistent with its role limiting fatty acid biosynthesis, both BrCa1 mRNA (3.5-fold, p<0.0001) and protein (1.2-fold, p = 0.001) were increased in pre-adipocytes, and decreased during in vitro adipogenesis, while P-ACC decreased during differentiation of human adipocytes (p = 0.005) allowing lipid biosynthesis. Interestingly, BrCa1 gene expression in mature adipocytes was restored by inflammatory stimuli (macrophage conditioned medium), whereas lipogenic genes significantly decreased. CONCLUSIONS The specular findings of BrCa1 and lipogenic enzymes in adipose tissue and adipocytes reported here suggest that BrCa1 might help to control fatty acid biosynthesis in adipocytes and adipose tissue from obese subjects.

The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation.

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.