Stabilization Of Hypoxia-inducible Factor-1α Research Articles

Isoliquiritigenin Inhibits Proliferation and Induces Apoptosis via Alleviating Hypoxia and Reducing Glycolysis in Mouse Melanoma B16F10 Cells.

Isoliquiritigenin (ISL) is a licorice chalcone. According to CN104758274, CN101658513 and US009089546, it is claimed that ISL has anti-inflammatory, anti-oxidative, and anti-tumoral effects. This study aimed to investigate the potential therapeutic effect of ISL in mouse melanoma B16F10 cells. Sulforhodamine B (SRB) colorimetric assay was used to test the effects of ISL on proliferation. Commercial assay kits were applied to assess glucose uptake, lactate production and ATP levels. Measurement of apoptosis was involved with Hoechst 33258, JC-1 and annexin V-FITC/PI staining. H2DCFDA probe was employed to detect ROS generation. Quantitative RT-PCR and western blot were utilized to measure the mRNA and protein levels. ISL abated hypoxia-inducible factor 1α (HIF-1α) stability and reduced a series of glycolysis-relevant enzymes expression, including glucose transporters 1/4 (GLUT 1/4), hexokinase 2 (HK2), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Exposure to ISL induced the mitochondrial membrane potential depolarization and increased intracellular reactive oxygen species (ROS) level. ISL could effectively inhibit proliferation and alleviate hypoxia in mouse melanoma B16F10 cells via inducing apoptosis and reducing the expression of significant enzymes in the glycolysis. ISL significantly inhibited B16F10 cell proliferation via inducing apoptosis, and alleviated hypoxia by recovering mitochondrial function and reversing high glycolysis. Our findings propose that ISL can be a promising therapeutic agent for the melanoma via reliving hypoxia of microenvironment and targeting energy metabolism system of cancer cells. Consistent with WO2015079213 and WO2014084494, targeting glycolysis can be an effective means to anti-cancer.

Role of prolyl hydroxylase domain proteins in the regulation of insulin secretion.

Type 2 diabetes is associated with impaired nutrient‐regulated anaplerosis and insulin secretion in pancreatic β‐cells. One key anaplerotic substrate that may be involved in regulating insulin release is α‐ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of β‐cell function. The oxygen‐sensing PHDs regulate the stability of hypoxia‐inducible factor 1α (HIF1α) as well as other proline‐containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on β‐cell function. We demonstrate that ethyl‐3,4‐dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose‐stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA‐mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA‐mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion.

Hif1a Deletion Reveals Pro-Neoplastic Function of B Cells in Pancreatic Neoplasia.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths worldwide, with an exceedingly low 5-year survival rate. PDAC tumors are characterized by an extensive desmoplastic stromal response and hypovascularity, suggesting that tumor hypoxia could regulate PDAC initiation and/or progression. Using a well-defined, autochthonous Kras(G12D)-driven murine model, as well as human tumors, we demonstrate that hypoxia and stabilization of hypoxia-inducible factor 1α (HIF1α), a principal mediator of hypoxic adaptation, emerge early during preinvasive stages of PDAC. Surprisingly, pancreas-specific Hif1a deletion drastically accelerated Kras(G12D)-driven pancreatic neoplasia and was accompanied by significant increases in intrapancreatic B lymphocytes, featuring prominent influx of a rare "B1b" B-cell subtype. Finally, treatment of HIF1α-deficient mice with B cell-depleting αCD20 monoclonal antibodies inhibited progression of pancreatic intraepithelial neoplasia (PanIN). Our data reveal a previously unrecognized role for B cells in promoting pancreatic tumorigenesis and implicate HIF1α as a critical regulator of PDAC development. We show here that pancreas-specific Hif1a deletion promotes PDAC initiation, coincident with increased intrapancreatic accumulation of B cells, and that B-cell depletion suppresses pancreatic tumorigenesis. We therefore demonstrate a protective role for HIF1α in pancreatic cancer initiation and uncover a previously unrecognized function of B cells.

Volatile anesthetics suppress glucose-stimulated insulin secretion in MIN6 cells by inhibiting glucose-induced activation of hypoxia-inducible factor 1.

Proper glycemic control is one of the most important goals in perioperative patient management. Insulin secretion from pancreatic β-cells in response to an increased blood glucose concentration plays the most critical role in glycemic control. Several animal and human studies have indicated that volatile anesthetics impair glucose-stimulated insulin secretion (GSIS). A convincing GSIS model has been established, in which the activity of ATP-dependent potassium channels (KATP) under the control of intracellular ATP plays a critical role. We previously reported that pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected in response to glucose stimulation and that MIN6 cells overexpressing HIF-1α were resistant to glucose-induced hypoxia. Genetic ablation of HIF-1α or HIF-1β significantly inhibited GSIS in mice. Moreover, we previously reported that volatile anesthetics suppressed hypoxia-induced HIF activation in vitro and in vivo.To examine the direct effect of volatile anesthetics on GSIS, we used the MIN6 cell line, derived from mouse pancreatic β-cells. We performed a series of experiments to examine the effects of volatile anesthetics (sevoflurane and isoflurane) on GSIS and demonstrated that these compounds inhibited the glucose-induced ATP increase, which is dependent on intracellular hypoxia-induced HIF-1 activity, and suppressed GSIS at a clinically relevant dose in these cells.

Inhibitory effect of withaferin A on Helicobacter pylori‑induced IL‑8 production and NF‑κB activation in gastric epithelial cells.

Withaferin A (WA), a withanolide purified from Withania somnifera, has been known to exert anti-inflammatory effects. The present study sought to determine the effects of WA on Helicobacter (H.) pylori-mediated inflammation in the AGS gastric epithelial cell line. Cellular production of interleukin (IL)-8 and vascular endothelial growth factor (VEGF) was measured by ELISA. Western blot analysis was performed to determine the activation of nuclear factor (NF)-κB and mitogen-activated protein kinases (MAPKs) as well as hypoxia-inducible factor 1α stabilization. Bacterial growth was also examined by measuring the optical density. Pre-treatment or co-treatment with WA efficiently reduced IL-8 production by AGS cells in response to H. pylori infection. H. pylori-induced activation of NF-κB, but not MAPKs, was also inhibited by pre-treatment of WA in the cells. However, WA did not affect VEGF production and HIF-1α stabilization induced by H. pylori in AGS cells. In addition, WA did not influence the growth of H. pylori, suggesting that the anti-inflammatory effect of WA was not due to any bactericidal effect. These findings indicate that WA is a potential preventive or therapeutic agent for H. pylori-mediated gastric inflammation.

Hypoxic induction of T-type Ca(2+) channels in rat cardiac myocytes: role of HIF-1α and RhoA/ROCK signalling.

T-type Ca(2+) channels are expressed in the ventricular myocytes of the fetal and perinatal heart, but are normally downregulated as development progresses. Interestingly, however, these channels are re-expressed in adult cardiomyocytes under pathological conditions. We investigated low voltage-activated T-type Ca(2+) channel regulation in hypoxia in rat cardiomyocytes. Molecular studies revealed that hypoxia induces the upregulation of Cav 3.2 mRNA, whereas Cav 3.1 mRNA is not significantly altered. The effect of hypoxia on Cav 3.2 mRNA was time- and dose-dependent, and required hypoxia inducible factor-1α (HIF-1α) stabilization. Patch-clamp recordings confirmed that T-type Ca(2+) channel currents were upregulated in hypoxic conditions, and the addition of 50 μm NiCl2 (a T-type channel blocker) demonstrated that the Cav 3.2 channel is responsible for this upregulation. This increase in current density was not accompanied by significant changes in the Cav 3.2 channel electrophysiological properties. The small monomeric G-protein RhoA and its effector Rho-associated kinase I (ROCKI), which are known to play important roles in cardiovascular physiology, were also upregulated in neonatal rat ventricular myocytes subjected to hypoxia. Pharmacological experiments indicated that both proteins were involved in the observed upregulation of the Cav 3.2 channel and the stabilization of HIF-1α that occurred in response to hypoxia. These results suggest a possible role for Cav 3.2 channels in the increased probability of developing arrhythmias observed in ischaemic situations, and in the pathogenesis of diseases associated with hypoxic Ca(2+) overload.

Abstract 1384: CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of triple-negative breast cancer

Abstract Antiangiogenic agents are designed to impede the development of new tumor blood vessels, thereby starving tumors of oxygen and nutrients to ultimately block tumor growth. Despite advances made in developing antiangiogenic therapies, clinical and preclinical data suggest that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenic agents and patients frequently fail to demonstrate significantly better overall survival. Acquired resistance to antiangiogenic agents has been attributed in part to the induction of intra-tumoral hypoxia and the concomitant stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. In this preclinical model, we demonstrate that CRLX101, an investigational nanoparticle-drug conjugate (NDC) containing the payload camptothecin (CPT), can reverse the stimulatory effects of hypoxia on the CSC population in an orthotopic triple-negative xenograft model. The payload, camptothecin, a topoisomerase 1 inhibitor, inhibits HIF-1α protein accumulation at concentrations below those that were cytotoxic to tumor cells, and inhibits hypoxia-mediated CSC induction at these low concentrations in vitro. In an orthotopic triple-negative breast cancer model, CRLX101 is synergistic with bevacizumab. Tumor reimplantation experiments confirm that the combination therapy effectively targets both the bulk tumor cell and CSC populations. Results generated from these preclinical studies support the clinical research of combining antiangiogenic agents with CRLX101 to increase patient survival. CRLX101 is currently in phase 2 clinical trials in combination with bevacizumab in both recurrent ovarian cancer and metastatic renal cell carcinoma. Citation Format: Sarah J. Conley, Trenton L. Baker, Joseph P. Burnet, Rebecca L. Thiesen, Douglas Lazarus, Christian G. Peters, Shawn G. Clouthier, Scott Eliasof, Max S. Wicha. CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, reverses the HIF-1α-mediated increase in cancer stem cells caused by bevacizumab in a preclinical model of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1384. doi:10.1158/1538-7445.AM2015-1384

The role of iNOS in cholesterol-induced liver fibrosis

Accumulation of cholesterol in the liver is associated with the development of non-alcoholic steatohepatitis-related fibrosis. However, underlying mechanisms are not well understood. The present study investigated the role of inducible nitric oxide synthase (iNOS) in cholesterol-induced liver fibrosis by feeding wild-type (WT) and iNOS-deficient mice with control or high-cholesterol diet (HCD) for 6 weeks. WT mice fed with HCD developed greater liver fibrosis, compared with iNOS-deficient mice, as evident by Sirius red staining and higher expression levels of profibrotic genes. Enhanced liver fibrosis in the presence of iNOS was associated with hypoxia-inducible factor-1α stabilization, matrix metalloproteinase-9 expression, and enhanced hepatic DNA damage. The profibrotic role of iNOS was also demonstrated in vivo using a selective inhibitor of iNOS as well as in vitro in a rat liver stellate cell line (HSC-T6). In conclusion, these findings suggest that iNOS is an important mediator in HCD-induced liver fibrosis.

Nitric Oxide And Oxygen: Actions And Interactions In Health And Disease

Nitric oxide (NO) inhibits cell respiration reversibly and in competition with O2 through the inhibition of the mitochondrial cytochrome c oxidase (Complex IV). At concentrations lower than those required to inhibit respiration, endogenous NO enhances the reduction of the electron transport chain, thus enabling cells to maintain their O2 consumption. This however facilitates the release of superoxide anion, which initiates the transcriptional activation of NF-kB as an early signal of a stress response. Through free radical formation, long-term inhibition of mitochondrial respiration by NO leads to persistent inhibition of Complex I. This is dependent on the S-nitrosylation of a specific thiol in the active form of this protein. S-nitrosylation of Complex I might indicate the early stages of a pathological process. Inhibition of mitochondrial respiration by low concentrations of NO at critical O2 concentrations also leads to prevention of the stabilization of hypoxia-inducible factor-1α (HIF-1α) due to the redistribution of O2 towards non-respiratory O2-dependent targets. This prevents the cell from registering a state of hypoxia at low O2 concentrations. On the other hand, at higher concentrations such as those generated in certain forms of cancer, NO increases the expression of HIF-1α by an action most probably involving a free radical mechanism. It is likely that the interactions between oxygen and NO, either at the mitochondria or in the cell in general, play a role in the initiation and development of neoplastic transformation and spreading. The ways in which these interactions operate remain unclear and are likely to vary from cancer to cancer.

Cell-permeable mitochondrial ubiquinol–cytochrome c reductase binding protein induces angiogenesis in vitro and in vivo

Ubiquinol–cytochrome c reductase binding protein (UQCRB), a component of the mitochondrial complex III, has been recently implicated in angiogenesis. Targeting mitochondria to balance vascular homeostasis has been widely recognized. However, the effect of UQCRB replenishment by direct delivery remains unknown. To explore the biological function of UQCRB in angiogenesis, a novel protein transduction domain (PTD)-conjugated UQCRB fusion protein was generated. PTD-UQCRB localized to mitochondria as does endogenous UQCRB. Treatment with PTD-UQCRB generated mitochondrial reactive oxygen species (mROS) without cytotoxicity, following hypoxia inducible factor-1α (HIF-1α) stabilization and downstream vascular endothelial growth factor (VEGF) expression. Accordingly, PTD-UQCRB induced angiogenesis in vitro and PTD-UQCRB pro-angiogenic activity was further validated in matrigel plug assay and in cutaneous wound-healing mouse models in vivo. Together, these results demonstrate that UQCRB plays a role in angiogenesis and the developed cell-permeable PTD-UQCRB can be utilized as a pro-angiogenic agent.

Leukemia stem cells in T-ALL require active Hif1α and Wnt signaling

AbstractThe Wnt signaling pathway has been shown to play important roles in normal hematopoietic stem cell biology and in the development of both acute and chronic myelogenous leukemia. Its role in maintaining established leukemia stem cells, which are more directly relevant to patients with disease, however, is less clear. To address what role Wnt signaling may play in T-cell acute lymphoblastic leukemia (T-ALL), we used a stably integrated fluorescent Wnt reporter construct to interrogate endogenous Wnt signaling activity in vivo. In this study, we report that active Wnt signaling is restricted to minor subpopulations within bulk tumors, that these Wnt-active subsets are highly enriched for leukemia-initiating cells (LICs), and that genetic inactivation of β-catenin severely reduces LIC frequency. We show further that β-catenin transcription is upregulated by hypoxia through hypoxia-inducible factor 1α (Hif1α) stabilization, and that deletion of Hif1α also severely reduces LIC frequency. Of note, the deletion of β-catenin or Hif1α did not impair the growth or viability of bulk tumor cells, suggesting that elements of the Wnt and Hif pathways specifically support leukemia stem cells. We also confirm the relevance of these findings to human disease using cell lines and patient-derived xenografts, suggesting that targeting these pathways could benefit patients with T-ALL.

Drug-induced regeneration in adult mice.

Whereas amphibians regenerate lost appendages spontaneously, mammals generally form scars over the injury site through the process of wound repair. The MRL mouse strain is an exception among mammals because it shows a spontaneous regenerative healing trait and so can be used to investigate proregenerative interventions in mammals. We report that hypoxia-inducible factor 1α (HIF-1α) is a central molecule in the process of regeneration in adult MRL mice. The degradation of HIF-1α protein, which occurs under normoxic conditions, is mediated by prolyl hydroxylases (PHDs). We used the drug 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA), a PHD inhibitor, to stabilize constitutive expression of HIF-1α protein. A locally injectable hydrogel containing 1,4-DPCA was designed to achieve controlled delivery of the drug over 4 to 10 days. Subcutaneous injection of the 1,4-DPCA/hydrogel into Swiss Webster mice that do not show a regenerative phenotype increased stable expression of HIF-1α protein over 5 days, providing a functional measure of drug release in vivo. Multiple peripheral subcutaneous injections of the 1,4-DPCA/hydrogel over a 10-day period led to regenerative wound healing in Swiss Webster mice after ear hole punch injury. Increased expression of the HIF-1α protein may provide a starting point for future studies on regeneration in mammals.

Methylene blue-induced neuronal protective mechanism against hypoxia-reoxygenation stress

Brain ischemia and reperfusion (I/R) injury occurs in various pathological conditions, but there is no effective treatment currently available in clinical practice. Methylene blue (MB) is a century-old drug with a newly discovered protective function in the ischemic stroke model. In the current investigation we studied the MB-induced neuroprotective mechanism focusing on stabilization and activation of hypoxia-inducible factor-1α (HIF-1α) in an in vitro oxygen and glucose deprivation (OGD)-reoxygenation model. Methods: HT22 cells were exposed to OGD (0.1% O2, 6h) and reoxygenation (21% O2, 24h). Cell viability was determined with the calcein AM assay. The dynamic change of intracellular O2 concentration was monitored by fluorescence lifetime imaging microscopy (FLTIM). Glucose uptake was quantified using the 2-[N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino]-2-Deoxy-d-Glucose (2-NBDG) assay. ATP concentration and glycolytic enzyme activity were examined by spectrophotometry. Protein content changes were measured by immunoblot: HIF-1α, prolyl hydroxylase 2 (PHD2), erythropoietin (EPO), Akt, mTOR, and PIP5K. The contribution of HIF-1α activation in the MB-induced neuroprotective mechanism was confirmed by blocking HIF-1α activation with 2-methoxyestradiol-2 (2-MeOE2) and by transiently transfecting constitutively active HIF-1α. Results: MB increases cell viability by about 50% vs. OGD control. Compared to the corresponding control, MB increases intracellular O2 concentration and glucose uptake as well as the activities of hexokinase and G-6-PDH, and ATP concentration. MB activates the EPO signaling pathway with a corresponding increase in HIF-1α. Phosphorylation of Akt was significantly increased with MB treatment followed by activation of the mTOR pathway. Importantly, we observed, MB increased nuclear translocation of HIF-1α vs. control (about three folds), which was shown by a ratio of nuclear:cytoplasmic HIF-1α protein content. Conclusion: We conclude that MB protects the hippocampus-derived neuronal cells against OGD-reoxygenation injury by enhancing energy metabolism and increasing HIF-1α protein content accompanied by an activation of the EPO signaling pathway.

Nijmegen breakage syndrome protein 1 (NBS1) modulates hypoxia inducible factor-1α (HIF-1α) stability and promotes in vitro migration and invasion under ionizing radiation

Hypoxia-inducible factor (HIF) is a heterodimer transcription factor complex that monitors the cellular response to the oxygen levels in cells. Hypoxia-inducible factor-1α (HIF-1α) has been shown to be stabilized by ionizing radiation (IR) and its stabilization promotes tumor progression and metastasis. Nijmegen breakage syndrome protein 1 (NBS1), a component of the MRE11–RAD50–NBS1 complex, plays an important role in the cellular response to DNA damage but its overexpression contributes to transformation and has been found to correlate with metastasis. However, whether NBS1 participates in IR-induced metastasis needs to be further determined. The aim of this study is to investigate whether radiation-induced HIF-1α stabilization is regulated by NBS1 and thereby promotes tumor cell migration/invasion. Here, we show that both NBS1 and HIF-1α expression are up-regulated after exposure to IR, and NBS1 increases HIF-1α expression at the protein level. In addition, IR treatment promotes the epithelial–mesenchymal transition (EMT) and in vitro cell migration and invasion activity, which could be abolished by suppression of NBS1. Furthermore, NBS1 directly interacts with HIF-1α and reduces the ubiquitination of HIF-1α⋅ Co-expression of HIF-1α and NBS1 in primary tumors of patients with lung adenocarcinoma correlates with a worse prognosis. These results provide a new function of NBS1 in stabilizing HIF-1α under IR, which leads to enhanced cancer cell migration and invasion.

CRLX101, an investigational camptothecin-containing nanoparticle-drug conjugate, targets cancer stem cells and impedes resistance to antiangiogenic therapy in mouse models of breast cancer.

Antiangiogenic therapies inhibit the development of new tumor blood vessels, thereby blocking tumor growth. Despite the advances in developing antiangiogenic agents, clinical data indicate that these drugs have limited efficacy in breast cancer patients. Tumors inevitably develop resistance to antiangiogenics, which is attributed in part to the induction of intra-tumoral hypoxia and stabilization of hypoxia-inducible factor 1α (HIF-1α), a transcription factor that promotes tumor angiogenesis, invasion, metastasis, and cancer stem cell (CSC) self-renewal. Here, we tested whether inhibiting HIF-1α can reverse the stimulatory effects of antiangiogenic-induced hypoxia on breast CSCs. Breast cancer cells grown under hypoxic conditions were treated with the dual topoisomerase-1 (TOPO-1) and HIF-1α inhibitor camptothecin and assessed for their CSC content. In a preclinical model of breast cancer, treatment with bevacizumab was compared to the combination treatment of bevacizumab with CRLX101, an investigational nanoparticle-drug conjugate with a camptothecin payload or CRLX101 monotherapy. While exposure to hypoxia increased the number of breast CSCs, treatment with CPT blocked this effect. In preclinical mouse models, concurrent administration of CRLX101 impeded the induction of both HIF-1α and CSCs in breast tumors induced by bevacizumab treatment. Greater tumor regression and delayed tumor recurrence were observed with the combination of these agents compared to bevacizumab alone. Tumor reimplantation experiments demonstrated that the combination therapy effectively targets the CSC populations. The results from these studies support the combined administration of dual TOPO-1- and HIF-1α-targeted agents like CRLX101 with antiangiogenic agents to increase the efficacy of these treatments.

A phase I, dose-escalation, multicenter study of ACT-PFK-158, 2HCl in patients with advanced solid malignancies explores a first-in-human inhibitor of glycolysis.

TPS494 Background: In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). This enzyme synthesizes fructose-2,6-bisphosphate (F2,6BP), which is an activator of 6-phosphofructo-1-kinase, a crucial step of glycolysis that is tightly controlled by multiple metabolic feedback mechanisms and dictates the rate of glycolytic flow. The vast majority of pancreatic ductal adenocarcinomas and approximately 50% of colon adenocarcinomas harbor activating mutations in Ras and these tumors have been reported to be highly glycolytic. PFK158 is a potent small molecule inhibitor of PFKFB3 that is selectively cytotoxic to Ras-transformed epithelial cells and displays broad anti-tumor activity causing ~80% growth inhibition in several mouse models of human-derived tumors and syngeneic murine models of colon cancer. Importantly, IND-enabling safety and toxicity studies have demonstrated that PFK158 is well tolerated in rats and dogs with an expected good therapeutic index, lending support for a phase 1 trial that is now underway. Methods: The primary objective of the study is to describe the dose limiting toxicity and to determine either the maximum tolerated dose or biological effective dose of PFK-158 in a “3+3” cohort-based dose escalation design that follows a modified Fibonacci scheme. Multiple secondary endpoints have been incorporated to assess the effects of PFK-158 on peripheral blood mononuclear cell F2,6BP activity and on glucose uptake using FDG-PET imaging. This trial is currently enrolling at two US sites; Cohort 1 has been completed without dose-limiting toxicity and Cohort 2 is enrolling with two subjects under treatment as of September 2014. In conclusion, PFK158 is the first-in-man and first-in-class PFKFB3 inhibitor to be examined in a phase I trial and may have significant clinical utility either as a monotherapy or when combined with other targeted agents. Clinical trial information: NCT02044861.

Pleiotropic actions of iron balance in diabetes mellitus.

As an essential element, iron plays a central role in many physiological processes, including redox balance, inflammation, energy metabolism, and environment sensing. Perturbations in iron homeostasis are associated with several conditions, including hyperglycemia and diabetes, both of which have been studied in patients and animal models. To clarify the pleiotropic role of iron homeostasis in diabetes development, the early studies on diseases with iron-overload, studies on clinical iron depletion therapies, associations between iron-related genetic polymorphisms and diabetes, and etiological mechanisms underlying iron perturbations-impaired insulin secretion and insulin sensitivity were carefully reviewed and discussed. Hereditary hemochromatosis, transfusion-dependent thalassemia, and excess heme iron intake can increase the risk of developing diabetes. Genetically modified mice and mice fed a high-iron diet present with discrepant phenotypes due to differences in tissue iron distribution. Moreover, several genetic polymorphisms related to iron homeostasis have been associated with the risk of developing diabetes. Tightly controlled iron metabolism is essential for insulin secretion and insulin sensitivity, and iron overload in pancreatic islets alters reactive oxygen species (ROS) generation, as well as hypoxia-inducible factor-1α (HIF-1α) stability and adenosine triphosphate (ATP) synthesis, thereby impairing the function and viability of β-cells. Decreased levels of adiponectin, macrophage-mediated inflammation, and ROS-mediated liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) activation can contribute to iron overload-induced insulin resistance, whereas iron deficiency could also participate in obesity-related inflammation, hypoxia, and insulin resistance. Because iron homeostasis is closely correlated with many metabolic processes, future studies are needed in order to elucidate the finely tuned network among iron homeostasis, carbohydrate and lipid metabolism, inflammation, and hypoxia.

Support for the hypoxia theory in the pathogenesis of infantile haemangioma

The pathogenesis of infantile haemangioma (IH) is unknown. Several mechanisms have been proposed, including hypoxia, which triggers upregulation and stabilization of hypoxia-inducible factor (HIF)1α. HIF1α stimulates downstream transcription of target genes that enhance angiogenesis. To identify possible involvement of hypoxia in the pathogenesis of IH, as hypoxia signalling constitutes a potential therapeutic target. IH tissue samples collected during the period 1991-2011 (preserved in paraffin wax) were immunohistochemically analysed for HIF1α and the known HIF1α targets: BCL2/adenovirus E1B kD-interacting protein family member 3 (BNIP3), carbon anhydrase (CA)-IX, glucose transporter (GLUT)-1, phosphorylated protein kinase B (pAKT), phosphorylated S6 protein (pS6) and vascular endothelial growth factor (VEGF). Four observers independently assessed the findings. Of the 10 IH samples, 2 appeared to be in the growth phase. In all samples, GLUT-1, BNIP3, pAKT and VEGF were positive, CA-IX was weakly positive, and HIF1α was negative. pS6 was positive in 9/10 cases and negative in 1/10. Several factors implicated in hypoxia-induced angiogenesis may be involved in IH development. However, the small sample size and retrospective approach of the study preclude definitive conclusions. Prospective studies are needed to conclusively determine which of the factors involved in the (hypoxia) cascade are required for an IH to grow, and could thus be a possible target of drugs for IH treatment.

The E3 Ubiquitin Ligase Pellino3 Protects against Obesity-Induced Inflammation and Insulin Resistance

Diet-induced obesity can induce low-level inflammation and insulin resistance. Interleukin-1β (IL-1β) is one of the key proinflammatory cytokines that contributes to the generation of insulin resistance and diabetes, but the mechanisms that regulate obesity-driven inflammation are ill defined. Here we found reduced expression of the E3 ubiquitin ligase Pellino3 in human abdominal adipose tissue from obese subjects and in adipose tissue of mice fed a high-fat diet and showing signs of insulin resistance. Pellino3-deficient mice demonstrated exacerbated high-fat-diet-induced inflammation, IL-1β expression, and insulin resistance. Mechanistically, Pellino3 negatively regulated TNF receptor associated 6 (TRAF6)-mediated ubiquitination and stabilization of hypoxia-inducible factor 1α (HIF1α), resulting in reduced HIF1α-induced expression of IL-1β. Our studies identify a regulatory mechanism controlling diet-induced insulin resistance by highlighting a critical role for Pellino3 in regulating IL-1β expression with implications for diseases like type 2 diabetes.

MicroRNA-126 Suppresses Mesothelioma Malignancy by Targeting IRS1 and Interfering with the Mitochondrial Function

MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia. MiR126 was up-regulated by oxidative stress in nonmalignant mesothelial (Met5A) and MM (H28) cell lines. In Met5A cells, rotenone inhibited MiR126 expression, but mtDNA depletion and hypoxia up-regulated MiR126. However, these various stimuli suppressed the levels of MiR126 in H28 cells. MiR126 affected mitochondrial energy metabolism, reduced mitochondrial respiration, and promoted glycolysis in H28 cells. This metabolic shift, associated with insulin receptor substrate-1 (IRS1)-modulated ATP-citrate lyase deregulation, resulted in higher ATP and citrate production. These changes were linked to the down-regulation of IRS1 by ectopic MiR126, reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promoted the expression of genes involved in gluconeogenesis and oxidative stress defense. These metabolic changes induced hypoxia-inducible factor-1α (HIF1α) stabilization. Consequently, MiR126 suppressed the malignancy of MM cells in vitro, a notion corroborated by the failure of H28(MiR126) cells to form tumors in nude mice. MiR126 affects mitochondrial energy metabolism, resulting in MM tumor suppression. Since MM is a fatal neoplastic disease with a few therapeutic options, this finding is of potential translational importance.