Nanocomposite liposomes for pH-controlled porphyrin release into human prostate cancer cells.

It is both challenging and desirable to have drug sensitizers released at acidic tumor pH for photodynamic therapy in cancer treatment. A pH-responsive carrier was prepared, in which fumed silica-attached 5,10,15,20-tetrakis(4-trimethylammoniophenyl)porphyrin (TTMAPP) was encapsulated into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) nanocomposite liposomes. The sizes of agglomerates were determined by dynamic light scattering to be 115 nm for silica and 295 nm for silica-TTMAPP-DOPC liposomes. Morphological changes were also found in TEM images, showing liposome formation at pH 8.5 but collapse upon silanol protonation. TTMAPP release is enhanced from 13% at pH 7.5 to 80% at pH 2.3, as determined spectrophotometrically through dialysis membranes. Fluorescence emission of TTMAPP encapsulated in the dry film of liposomes was reduced to half at pH 8.6 when compared to that at pH 5.4, while the production of singlet oxygen was quintupled at pH 5.0 compared to pH 7.6. Upon treatment of human prostate cancer cells with liposomes containing 6.7 µM, 13 µM, 17 µM and 20 µM TTMAPP, the cell viabilities were determined to be 60%, 18%, 20% and 5% at pH 5.4; 58%, 30%, 25% and 10% at pH 6.3; and 90%, 82%, 68% and 35% at pH 7.4, respectively. Light-induced apoptosis in cancerous cells was only observed in the presence of liposomes at pH 6.3 and pH 5.4 but not at pH 7.4, as indicated by chromatin condensation.

Rapidly progressive course of Trypanosoma cruzi infection in mice heterozygous for hexamethylene bis-acetamide inducible 1 (Hexim1) gene.

Infection with Trypanosoma cruzi causes Chagas disease and results in myocardial inflammation and cardiomyopathy. Downregulated Hexim1 expression, as in Hexim1+/- mice, reduces cardiac inflammation and fibrosis following ischemic stress. We asked whether reduced expression of Hexim1 would also afford protection against T. cruzi-induced cardiomyopathy. C57BL/6J (wild type - WT) and Hexim1+/- mice were infected with sub-lethal doses of T. cruzi (Brazil strain), and cardiac function, serologic markers of inflammation and tissue pathology were examined. Infected Hexim1+/- mice had compromised cardiac function, altered expression of both pro- and anti-inflammatory cytokines, and increased inflammation and fibrosis. Cardiac failure was evidenced by severely diminished heart rate, compensatory increase in respiratory rate, and abnormally high left ventricular mass with severe transmural inflammation. Lungs displayed intense peribronchial inflammation and fibrosis extending into the parenchyma. We also observed Smad3-serine208 phosphorylation in hearts and lungs of infected mice, suggesting increased TGF-ß signaling pathway activity. This was more pronounced in Hexim1+/- mice and correlated with increased fibrosis in these tissues. Conspicuous splenomegaly in the Hexim1+/- mice most likely resulted from the observed extensive white pulp expansion. T. cruzi infection induced colonic dilatation and marked villous atrophy in both the WT and Hexim1+/- mice but more so in the latter. The profound exacerbation of pathologic findings suggests a protective role for Hexim1 in T. cruzi infection.

Hexim1 heterozygosity stabilizes atherosclerotic plaque and decreased steatosis in ApoE null mice fed atherogenic diet.

Hexim-1 is an inhibitor of RNA polymerase II transcription elongation. Decreased Hexim-1 expression in animal models of chronic diseases such as left ventricular hypertrophy, obesity and cancer triggered significant changes in adaptation and remodeling. The main aim of this study was to evaluate the role of Hexim1 in lipid metabolism focused in the progression of atherosclerosis and steatosis. We used the C57BL6 apolipoprotein E (ApoE null) crossed bred to C57BL6Hexim1 heterozygous mice to obtain ApoE null - Hexim1 heterozygous mice (ApoE-HT). Both ApoE null backgrounds were fed high fat diet for twelve weeks. Then, we evaluated lipid metabolism, atherosclerotic plaque formation and liver steatosis. In order to understand changes in the transcriptome of both backgrounds during the progression of steatosis, we performed Affymetrix mouse 430 2.0 microarray. After 12 weeks of HFD, ApoE null and ApoE-HT showed similar increase of cholesterol and triglycerides in plasma. Plaque composition was altered in ApoE-HT. Additionally, liver triglycerides and steatosis were decreased in ApoE-HT mice. Affymetrix analysis revealed that decreased steatosis might be due to impaired inducible SOCS3 expression in ApoE-HT mice. In conclusion, decreased Hexim-1 expression does not alter cholesterol metabolism in ApoE null background after HFD. However, it promotes stable atherosclerotic plaque and decreased steatosis by promoting the anti-inflammatory TGFß pathway and blocking the expression of the inducible and pro-inflammatory expression of SOCS3 respectively.

Hexim1, a Novel Regulator of Leptin Function, Modulates Obesity and Glucose Disposal.

Leptin triggers signaling events with significant transcriptional responses that are essential to metabolic processes affecting obesity and glucose disposal. We asked whether hexamethylene bis-acetamide inducible-1 (Hexim1), an inhibitor of RNA II polymerase-dependent transcription elongation, regulates leptin-Janus kinase 2 signaling axis in the hypothalamus. We subjected C57BL6 Hexim1 heterozygous (HT) mice to high-fat diet and when compared with wild type, HT mice were resistant to high-fat diet-induced weight gain and remain insulin sensitive. HT mice exhibited increased leptin-pY(705)Stat3 signaling in the hypothalamus, with normal adipocyte size, increased type I oxidative muscle fiber density, and enhanced glucose transporter 4 expression. We also observed that normal Hexim1 protein level is required to facilitate the expression of CCAAT/enhancer-binding proteins (C/EBPs) required for adipogenesis and inducible suppressor of cytokine signaling 3 (SOCS) expression. Further support on the role of Hexim1 regulating C/EBPs during adipocyte differentiation was shown when HT 3T3L1 fibroblasts failed to undergo adipogenesis. Hexim1 selectively modulates leptin-mediated signal transduction pathways in the hypothalamus, the expression of C/EBPs and peroxisome proliferator-activated receptor-γ (PPAR γ) in skeletal muscle and adipose tissue during the adaptation to metabolic stress. We postulate that Hexim1 might be a novel factor involved in maintaining whole-body energy balance.

Hexim-1 modulates androgen receptor and the TGF-ß signaling during the progression of prostate cancer.

BACKGROUND: Androgen and TGF-ß signaling are important components during the progression of prostate cancer. However, whether common molecular events participate in the activation of these signaling pathways are less understood. METHOD: Hexim 1 expression was detected by immunohistochemistry of human tissue microarrays and TRAMP mouse models. The in vivo significance of Hexim-1 was established by crossing the TRAMP mouse model of prostate cancer with Hexim-1 heterozygous mice. TRAMP C2 cell line was also modified to delete one copy of Hexim-1 gene to generate TRAMP-C2-Hexim-1+/- cell lines. RESULTS: In this report, we observed that Hexim-1 protein expression is absent in normal prostate but highly expressed in adenocarcinoma of the prostate and a characteristic sub-cellular distribution among normal, benign hyperplasia, and adenocarcinoma of the prostate. Heterozygosity of the Hexim-1 gene in the prostate cancer mice model and the TRAMP-C2 cell line, leads to increased Cdk9-dependent serine phosphorylation on protein targets such as the androgen receptor (AR) and the TGF-ß-dependent downstream transcription factors, such as the SMAD proteins. CONCLUSION: Our results suggest that changes in the Hexim-1 protein expression and cellular distribution significantly influences the AR activation and the TGF-ß signaling. Thus, Hexim-1 is likely to play a significant role in prostate cancer progression.

Early signaling by vascular endothelial growth factor and placental growth factor in human bone marrow-derived endothelial cells is mediated by superoxide.

AIMS: We investigated the role of superoxide O(2)(-) during the initiation of vascular endothelial growth factor (VEGF)- and placental growth factor (PlGF)-mediated signal transduction in bone marrow-derived endothelial cells. METHODS: BMhTERT cells were treated with VEGF or PlGF in the presence or absence of antioxidants. The signaling pathways downstream were analyzed by immunoprecipitation and Western blotting. Superoxide and reactive oxygen species (ROS) were measured using Superluminol or 2',7'-dichlorofluorescein fluorescence measurements. RESULTS: We show here that VEGF and PlGF generate extracellular and intracellular O(2)(-) that regulates their downstream signaling transduction pathways. Indeed, the extracellular O(2)(-) generated treatment of endothelial cells (using hypoxanthine/xanthine oxidase) was sufficient to initiate receptor phosphorylation of VEGF receptor 2. The PlGF treatment of endothelial cells increased the generation of intracellular ROS in an extracellular O(2)(-) dependent manner. Quenching of intracellular ROS by resveratrol inhibits PlGF- and VEGF-dependent induction of MAP kinase phosphorylation. Additionally, we found that the interaction of VEGF and PlGF with their specific receptors generates O(2)(-) in a cell-free system. Endothelial cells treated with VEGF stop proliferation in the presence of extracellular catalase, superoxide dismutase or peroxiredoxin IV. CONCLUSION: Our studies underscore the role of O(2)(-) as a critical regulator of VEGF and PlGF signal transduction initiation in endothelial cells.

Enhanced hypertrophy in ob/ob mice due to an impairment in expression of atrial natriuretic peptide.

RATIONALE: We investigated the molecular mechanism(s) that play a role in leptin signaling during the development of left ventricular hypertrophy (LVH) due to pressure overload. To this end, ob/ob leptin deficient and C57BL/6J control mice were subjected transverse aortic constriction (TAC). METHODS: Control sham C57BL/6J and ob/ob mice, along with C57BL/6J and ob/ob leptin deficient mice were subjected transverse aortic constriction (TAC) for 15 days and then evaluated for morphological, physiological, and molecular changes associated with pressure overload hypertrophy. RESULTS: Evaluation by echocardiography revealed a significant increase in left ventricular mass (LVmass) and wall thickness in ob/ob mice subjected to transverse aortic constriction (TAC) as compared to C57BL/6J. Analysis of the expression of molecular markers of LVH, such as atrial natriuretic peptide (ANP), revealed a blunted increase in the level of ANP in ob/ob mice as compared to C57BL/6J mice. We observed that leptin plays a role in modulating the transcriptional activity of the promoter of the ANP gene. Leptin acts by regulating NFATc4, a member of the nuclear factor activated T cell (NFAT) family of transcription factors in cardiomyocytes. Our in vivo studies revealed that ob/ob mice subjected to TAC failed to activate the NFATc4 in the heart, however, intraperitoneal injection of leptin in ob/ob mice restored the NFATc4 DNA-binding activity and induced expression of the ANP gene. CONCLUSION: This study establishes the role of leptin as an anti-hypertrophic agent during pressure overload hypertrophy, and suggests that a key molecular event is the leptin mediated activation of NFATc4 that regulates the transcriptional activation of the ANP gene promoter.

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: role of alphaGMR.

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with alphaGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the alphaGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.

Hypoxia-reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C.

Hypoxia and hypoxia-reperfusion (H-R) play important roles in human pathophysiology because they occur in clinical conditions such as circulatory shock, myocardial ischemia, stroke, and organ transplantation. Reintroduction of oxygen to hypoxic cells during reperfusion causes an increase in generation of reactive oxygen species (ROS), which can alter cell signaling, and cause damage to lipids, proteins, and DNA leading to ischemia-reperfusion injury. Since vitamin C is a potent antioxidant and quenches ROS, we investigated the role of intracellular ascorbic acid (iAA) in endothelial cells undergoing hypoxia-reperfusion. Intracellular AA protected human endothelial cells from H-R-induced apoptosis. Intracellular AA also prevents loss of mitochondrial membrane potential and the release of cytochrome C and activation of caspase-9 and caspase-3 during H-R. Additionally, inhibition of caspase-9 activation prevented H-R-induced apoptosis, suggesting a mitochondrial site of initiation of apoptosis. We found that H-R induced an increase in ROS in endothelial cells that was abrogated in the presence of iAA. Our results indicate that vitamin C prevents hypoxia and H-R-induced damage to human endothelium.

Granulocyte-macrophage colony-stimulating factor signals for increased glucose transport via phosphatidylinositol 3-kinase- and hydrogen peroxide-dependent mechanisms.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K(m) for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the alpha subunit of the GM-CSF receptor (alpha GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity alpha GMR and in human cells expressing the high affinity alpha beta GMR complex. We identified a Src homology 3 domain-binding motif in alpha GMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to alpha GMR was obtained by co-immunoprecipitation with antibodies to alpha GMR and p85, and an alpha GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of alpha GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H(2)O(2)), generated by ligand or antibody binding to alpha GMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-alpha GMR antibody-stimulated glucose uptake in alpha GMR-expressing oocytes, and H(2)O(2) activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to alpha GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H(2)O(2) generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated alpha subunit of the GM-CSF receptor.