Hypoxic Activation Research Articles

Adrenal Medulla Chemo Sensitivity Does Not Compensate the Lack of Hypoxia Driven Carotid Body Chemo Reflex in Guinea Pigs.

Guinea pigs (GP), originally from the Andes, have absence of hypoxia-driven carotid body (CB) reflex. Neonatal mammals have an immature CB chemo reflex and respond to hypoxia with metabolic changes arising from direct effects of hypoxia on adrenal medulla (AM). Our working hypothesis is that adult GP would mimic neonatal mammals. Plasma epinephrine (E) has an AM origin, while norepinephrine (NE) is mainly originated in sympathetic endings, implying that specific GP changes in plasma E/NE ratio, and in blood glucose and lactate levels during hypoxia would be observed. Experiments were performed on young adult GP and rats. Hypoxic ventilation (10% O2) increased E and NE plasma levels similarly in both species but PaO2 was lower in GP than in rats. Plasma E/NE ratio in GP was higher (≈1.0) than in rats (≈0.5). The hypoxia-evoked increases in blood glucose and lactate were smaller in GP than in the rat. The AM of both species contain comparable E content, but NE was four times lower in GP than in rats. GP superior cervical ganglion also had lower NE content than rats and an unusual high level of dopamine, a negative modulator of sympathetic transmission. Isolated AM from GP released half of E and one tenth of NE than the rat AM, and hypoxia did not alter the time course of CA outflow. These data indicate the absence of direct effects of hypoxia on AM in the GP, and a lower noradrenergic tone in this species. Pathways for hypoxic sympatho-adrenal system activation in GP are discussed.

A bioreducible N-oxide-based probe for photoacoustic imaging of hypoxia

Hypoxia occurs when limited oxygen supply impairs physiological functions and is a pathological hallmark of many diseases including cancer and ischemia. Thus, detection of hypoxia can guide treatment planning and serve as a predictor of patient prognosis. Unfortunately, current methods suffer from invasiveness, poor resolution and low specificity. To address these limitations, we present Hypoxia Probe 1 (HyP-1), a hypoxia-responsive agent for photoacoustic imaging. This emerging modality converts safe, non-ionizing light to ultrasound waves, enabling acquisition of high-resolution 3D images in deep tissue. HyP-1 features an N-oxide trigger that is reduced in the absence of oxygen by heme proteins such as CYP450 enzymes. Reduction of HyP-1 produces a spectrally distinct product, facilitating identification via photoacoustic imaging. HyP-1 exhibits selectivity for hypoxic activation in vitro, in living cells, and in multiple disease models in vivo. HyP-1 is also compatible with NIR fluorescence imaging, establishing its versatility as a multimodal imaging agent.

Bevacizumab Induces Acute Hypoxia and Cancer Progression in Patients with Refractory Breast Cancer: Multimodal Functional Imaging and Multiplex Cytokine Analysis

Purpose: Bevacizumab, an antibody against endothelial growth factor, is a key but controversial drug in the treatment of metastatic breast cancer. We, therefore, aimed to determine the intrinsic resistance to bevacizumab at the physiologic and molecular levels in advanced breast cancer using PET, dynamic contrast-enhanced MRI, diffuse optical spectroscopic imaging (DOSI), and multiplex cytokine assays.Experimental Design: In total, 28 patients diagnosed with advanced stage III/IV breast cancer receiving single-agent bevacizumab for 1 week followed by paclitaxel combined with bevacizumab underwent 18F-fluorodeoxyglucose (FDG)-PET, 18F-fluoromisonidazole (FMISO)-PET, and MRI at both baseline and two courses after treatment initiation. Hemodynamic measurement using DOSI and blood sample collection were performed at baseline and multiple times during the first week after the initiation of single-agent bevacizumab. We distinguished nonresponders from responders by serial FDG-PET based on their glycolytic changes to chemotherapy.Results: Nonresponders showed significantly higher hypoxic activity on FMISO-PET and less tumor shrinkage than responders. Hemodynamic parameters showed higher tumor blood volume and a remarkable decrease in the tissue oxygen level in nonresponders compared with responders after the infusion of single-agent bevacizumab. Multiplex cytokine assays revealed increased plasma levels of both proangiogenic and hypoxia-related inflammatory cytokines in nonresponders and decreased levels in responders.Conclusions: Nonresponders exhibited a higher degree of angiogenesis with more severe hypoxia than responders during bevacizumab treatment. These findings demonstrated that the addition of bevacizumab to paclitaxel treatment under hypoxic conditions could be ineffective and may result in acute hypoxia and increased cytokine secretion associated with cancer progression. Clin Cancer Res; 23(19); 5769-78. ©2017 AACR.

PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

The role of prolyl hydroxylase (PHD)-3 as a hypoxia inducible factor (HIF)-1α cofactor is controversial and remains unknown in skeletal tissues. We investigated whether PHD3 controls HIF-1 transcriptional activity in nucleus pulposus (NP) cells through the pyruvate kinase muscle (PKM)-2-Jumonji domain--containing protein (JMJD5) axis. PHD3-/- mice (12.5 mo old) showed increased incidence of intervertebral disc degeneration with a concomitant decrease in expression of the HIF-1α targets VEGF-A, glucose transporter-1, and lactate dehydrogenase A. PHD3 silencing decreased hypoxic activation of HIF-1α C-terminal transactivation domain (C-TAD), but not HIF-1α-N-terminal-(N)-TAD or HIF-2α-TAD. Moreover, PHD3 suppression in NP cells resulted in decreased HIF-1α enrichment on target promoters and lower expression of select HIF-1 targets. Contrary to other cell types, manipulation of PKM2 and JMJD5 levels had no effect on HIF-1 activity in NP cells. Likewise, stabilization of tetrameric PKM2 by a chemical approach had no effect on PHD3-dependent HIF-1 activity. Coimmunoprecipitation assays showed lack of association between HIF-1α and PKM2 in NP cells. Results support the role of the PHD3 as a cofactor for HIF-1, independent of PKM2-JMJD5.-Schoepflin, Z. R., Silagi, E. S., Shapiro, I. M., Risbud, M. V. PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII

Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3′5′-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

TRPC6 channels modulate the response of pancreatic stellate cells to hypoxia.

Pancreatic cancer is characterized by a massive fibrosis (desmoplasia), which is primarily caused by activated pancreatic stellate cells (PSCs). This leads to a hypoxic tumor microenvironment further reinforcing the activation of PSCs by stimulating their secretion of growth factors and chemokines. Since many of them elicit their effects via G-protein-coupled receptors (GPCRs), we tested whether TRPC6 channels, effector proteins of many G-protein-coupled receptor pathways, are required for the hypoxic activation of PSCs. Thus far, the function of ion channels in PSCs is virtually unexplored. qPCR revealed TRPC6 channels to be one of the most abundant TRPC channels in primary cultures of murine PSCs. TRPC6 channel function was assessed by comparing PSCs from TRPC6-/- mice and wildtype (wt) littermates. Cell migration, Ca2+ signaling, and cytokine secretion were analyzed as readout for PSC activation. Hypoxia was induced by incubating PSCs for 24h in 1% O2 or chemically with dimethyloxalylglycine (DMOG). PSCs migrate faster in response to hypoxia. Due to reduced autocrine stimulation, TRPC6-/- PSCs fail to increase their rate of migration to the same level as wt PSCs under hypoxic conditions. This defect could not be overcome by the stimulation with platelet-derived growth factor. In line with these results, calcium influx is increased in wt but not TRPC6-/- PSCs under hypoxia. We conclude that TRPC6 channels of PSCs are major effector proteins in an autocrine stimulation pathway triggered by hypoxia.

Abstract 4499: The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway

Abstract Cancer cells co-opt mechanisms of adaptation to hypoxia to minimize energy expenditure and halt proliferation thereby increasing hypoxic tolerance, a major factor contributing to chemo- and radioresistance in patients. The endoplasmic reticulum (ER) kinase PERK is one of the three signaling transducers of the unfolded protein response (UPR) that inhibits mRNA translation via eIF2α phosphorylation to alleviate hypoxia-induced ER stress. We have previously shown that accumulation of fumarate, resulting from loss-of-function (LOF) of the mitochondrial enzyme fumarate hydratase (FH), augments the antioxidant capacity of hypoxic cancer cells through upregulation of the pentose phosphate pathway (PPP). Here, we report for the first time that the fumarate-dependent increase of antioxidants generated by the PPP prevents hypoxic activation of the PERK→eIF2α axis enabling protein synthesis and proliferation leading to an impairment of hypoxic tolerance. HeLa (cervix), HCT-116 and LS174T (colon) adenocarcinoma cells were transfected with lentiviral vectors encoding for shRNAs targeting FH. Immunoblot assays showed that FH LOF impaired PERK activity under hypoxia thus inactivating eIF2α. Moreover, the ability of fumarate to prevent PERK activation was specific to hypoxia since fumarate did not affect the pharmacological activation of the UPR by thapsigargin, DTT or tunicamycin. A comprehensive analysis utilizing a RT-qPCR array to profile the mRNA expression of 84 UPR genes, showed that FH LOF inhibited the UPR in HCT-116 cells. A similar response was observed in patient-derived UOK262 kidney cancer cells whereby bi-allelic FH mutations resulted in downregulation of 72% of the UPR genes whereas FH re-introduction restored UPR signaling. We employed LS174T mucinous adenocarcinoma cells as a model to assess protein synthesis. Mucin synthesis was not affected in hypoxic FH deficient cells whereas it was abrogated in wild-type cells. Consistent with these results, FH LOF increased 3D spheroid growth and decreased clonogenic potential in HCT-116 and HeLa cells. Pharmacological inhibition of the PPP by 6-aminonicotinamide activated the PERK→eIF2α axis in FH silenced cells thereby suggesting that PPP activation by fumarate is implicated in PERK inhibition. RNAseq data analysis in melanoma, prostate, colon, breast, lung and renal cancers from TCGA cohorts shows a negative correlation between PERK and the PPP enzyme PGLS suggesting that the mechanism hereby delineated occurs in cancer patients. In conclusion, we show that fumarate impairs PERK→eIF2α UPR signaling in hypoxic cancer cells. Clinical and molecular data show that UPR activation is one of the mechanisms responsible for chemo- and radioresistance in hypoxic tumors. This study suggests that fumarate accumulation (either through extrinsic or FH LOF) represents a novel approach to target hypoxic cancer cells and improve patient prognosis. Citation Format: Luana Schito, Sergio Rey, Bradly G. Wouters, Marianne Koritzinsky. The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2017-4499

Abstract 3203: Targeting tumor hypoxia with prodrug conjugates of potent small-molecule inhibitors of tubulin polymerization

Abstract Regions of pronounced hypoxia in malignant tumors represent an opportunity for selectivity in drug activation, and hence targeting and delivery. In this study, a series of bioreductively activatable prodrug conjugates (BAPCs) of small-molecule inhibitors of tubulin polymerization were synthesized and evaluated in biochemical and biological studies. The BAPCs are designed to be biologically inert until selectively cleaved under hypoxic conditions, by reductase enzymes found in the tumor, to release potent effector anticancer agents. BAPCs were evaluated in the following assays: (1) cytotoxicity using the regular sulforhodamine B assay (normoxic conditions) to confirm their reduced cytotoxicity in cancer cells in culture compared to the effector agents; (2) cleavage under anoxic conditions by the reductase enzyme, NADPH cytochrome P450 oxidoreductase (POR), that is implicated in the bioreductive cleavage of compounds with nitrothiophene and nitroimidazole triggers; and (3) differential cytotoxicity under hypoxic versus normoxic conditions in cancer cell lines with the established bioreductive compound tirapazamine (TPZ) as the control. The POR assay was modified by the addition of protocatechuate 3,4-dioxygenase to ensure anoxic conditions, and Triton X-100 to facilitate solubilization of the BAPCs. A series of unsubstituted, methyl, and gem-dimethyl nitrothiophene- and nitroimidazole-triggered prodrug conjugates (BAPCs) of two experimental anticancer agents (KGP03 and KGP18) were synthesized. KGP03 and KGP18 were inspired by the natural product combretastatin A-4 (CA4). A unique attribute of the selected anticancer agents is that both bind tubulin at the colchicine site, inhibit tubulin polymerization into microtubules, and thus function as pronounced cytotoxic, anti-proliferative agents in human cancer cell lines and as vascular disrupting agents (VDAs). The assays were validated with a series of nitrothiophene analogues of CA4 (Thomson et al., Mol. Cancer Ther. 2006 5:2886). In our evaluation of this series, the gem-dimethyl nitrothiophene analogue of CA4 was efficiently cleaved by POR and gave an average differential hypoxia cytotoxicity ratio (HCR: GI50 values of normoxic/hypoxic conditions) of 41 in the human A549 lung carcinoma cell line. The corresponding prodrug of combretastatin A-1 had an HCR = 26. The monomethyl nitroimidazole BAPCs of KGP03 and KGP18 produced positive HCRs in our initial assays. In preliminary studies, the CA4 BAPC demonstrated antivascular activity in an orthotopic syngeneic breast tumor mouse model (4T1/BALB/c). In the POR assay, the gem-dimethyl nitrothiophene analogue of KGP03 was completely cleaved in 24 h. Biological evaluation of these BAPCs indicates the occurrence of selective, hypoxic activation resulting in release of the potent inhibitors of tubulin polymerization (CA4, CA1, KGP03, KGP18). Citation Format: Kevin G. Pinney, Mary Lynn Trawick, Ralph P. Mason, Li Liu, David J. Chaplin, Blake A. Winn, Laxman Devkota, Tracy E. Strecker, Jeni Gerberich, Alex Winters, Yifan Wang, Matthew T. MacDonough. Targeting tumor hypoxia with prodrug conjugates of potent small-molecule inhibitors of tubulin polymerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3203. doi:10.1158/1538-7445.AM2017-3203

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication

SummaryCells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.

A microfluidic oxygen gradient demonstrates differential activation of the hypoxia-regulated transcription factors HIF-1α and HIF-2α.

Gas-perfused microchannels generated a linear oxygen gradient via diffusion across a 100 μm polydimethylsiloxane (PDMS) membrane. The device enabled exposure of a single monolayer of cells sharing culture media to a heterogeneous oxygen landscape, thus reflecting the oxygen gradients found at the microscale in the physiological setting and allowing for the real-time exchange of paracrine factors and metabolites between cells exposed to varying oxygen levels. By tuning the distance between two gas supply channels, the slope of the oxygen gradient was controlled. We studied the hypoxic activation of the transcription factors HIF-1α and HIF-2α in human endothelial cells within a spatial linear gradient of oxygen. Quantification of the nuclear to cytosolic ratio of HIF immunofluorescent staining demonstrated that the threshold for HIF-1α activation was below 2.5% O2 while HIF-2α was activated throughout the entire linear gradient. We show for the first time HIF-2α is subject to hyproxya, hypoxia by proxy, wherein hypoxic cells activate HIF in close-proximity normoxic cells. These results underscore the differences between HIF-1α and HIF-2α regulation and suggest that a microfluidic oxygen gradient is a novel tool for identifying distinct hypoxic signaling activation and interactions between differentially oxygenated cells.

Mycobacterium tuberculosis DevR/DosR Dormancy Regulator Activation Mechanism: Dispensability of Phosphorylation, Cooperativity and Essentiality of α10 Helix.

DevR/DosR is a well-characterized regulator in Mycobacterium tuberculosis which is implicated in various processes ranging from dormancy/persistence to drug tolerance. DevR induces the expression of an ~48-gene dormancy regulon in response to gaseous stresses, including hypoxia. Strains of the Beijing lineage constitutively express this regulon, which may confer upon them a significant advantage, since they would be ‘pre-adapted’ to the environmental stresses that predominate during infection. Aerobic DevR regulon expression in laboratory-manipulated overexpression strains is also reported. In both instances, the need for an inducing signal is bypassed. While a phosphorylation-mediated conformational change in DevR was proposed as the activation mechanism under hypoxia, the mechanism underlying constitutive expression is not understood. Because DevR is implicated in bacterial dormancy/persistence and is a promising drug target, it is relevant to resolve the mechanistic puzzle of hypoxic activation on one hand and constitutive expression under ‘non-inducing’ conditions on the other. Here, an overexpression strategy was employed to elucidate the DevR activation mechanism. Using a panel of kinase and transcription factor mutants, we establish that DevR, upon overexpression, circumvents DevS/DosT sensor kinase-mediated or small molecule phosphodonor-dependent activation, and also cooperativity-mediated effects, which are key aspects of hypoxic activation mechanism. However, overexpression failed to rescue the defect of C-terminal-truncated DevR lacking the α10 helix, establishing the α10 helix as an indispensable component of DevR activation mechanism. We propose that aerobic overexpression of DevR likely increases the concentration of α10 helix-mediated active dimer species to above the threshold level, as during hypoxia, and enables regulon expression. This advance in the understanding of DevR activation mechanism clarifies a long standing question as to the mechanism of DevR overexpression-mediated induction of the regulon in the absence of the normal environmental cue and establishes the α10 helix as an universal and pivotal targeting interface for DevR inhibitor development.

Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used invivo and invitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), thekey hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression inmurine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions.

Abstract 21: Effect of Hypoxic Preconditioning on Cortical Bone Stem Cells (CBSCs)

Rationale: Cortical Bone Stem Cells (CBSCs) improve cardiac performance when delivered in an infarcted heart, in part, through secretion of paracrine factors. However transplanted cells can die due to hypoxia or other hostile fators within the infarcted heart. Hypoxia is known to also influence several biological process including migration, proliferation and secretory activity. Objective: the aim of this study is to define the effects of hypoxia on biological properties of CBSCs. Methods and Results: CBSCs were isolated from C57Bl/6 mouse, expanded in vitro and characterized. CBSCs were subjected to hypoxia (1% O 2 ) or normoxia (21% O 2 ) condition and then several biological properties were analysed. Hypoxia resulted in upregulation of CAIX and GLUT1 expression, two HIF1α downstream targets (3.2 and 5.1 fold increase of GLUT1; 6.7 and 65 fold increase of CAIX after 6 and 24 hours respectively). This indicates an effective hypoxic response activation in our experimental conditions. Cell proliferation was assessed by CyQuant assay: no significant difference between normoxic and hypoxic cells were found after 1 and 2 days. Growth curves were determined to evaluate hypoxia effects at longer time points. Hypoxic cells had the same growing rate as cells in normoxia in the first days, but their number increased at longer time points, indicating different kinetics with respect to normoxic when cells were grown in a challenging condition like an exhausted media. CBSCs migration and chemotaxis decreased when cells were cultured at 1%O 2 . Hypoxia preconditioning increased the paracrine activity of CBSCs. VEGF expression was increased after 6 hours (3 fold), and this effect remain stable after 24 hours and 3 days of 1% O 2 . Hypoxia resulted in 2.2 and 1.9 fold increase of SDF1 after 24 hours and 3 days respectively and a 1.8 fold increase of IGF1 after 1 day. The expression of bFGF, Angiopoietin and SCF was not affected. Conclusion: CBSCs can survive hypoxia and their paracrine activity increases without adversely affecting their proliferative capacity. In vivo experiments are needed to investigate if hypoxic preconditioning will enhance the ability of CBSCs to improve the structure and function of the injured heart.

Creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system

Adequate concentrations of ATP are required to preserve physiological cell functions and protect tissue from hypoxic damage. Decreased oxygen concentration results in ATP synthesis relying increasingly on the presence of phosphocreatine. The lack of ATP through hypoxic insult to neurons that generate or regulate respiratory function, would lead to the cessation of breathing (apnea). It is not clear whether creatine plays a role in maintaining respiratory phrenic nerve (PN) activity during hypoxic challenge.The aim of the study was to test the effects of exogenously applied creatine or creatine pyruvate in maintaining PN induced respiratory rhythm against the deleterious effects of severe hypoxic insult using Working Heart-Brainstem (WHB) preparations of juvenile Swiss type mice. WHB's were perfused with control perfusate or perfusate containing either creatine [100μM] or creatine pyruvate [100μM] prior to hypoxic challenge and PN activity recorded throughout. Results showed that severe hypoxic challenge resulted in an initial transient increase in PN activity, followed by a reduction in that activity leading to respiratory apnea. The results demonstrated that perfusing the WHB preparation with creatine or creatine pyruvate, significantly reduced the onset of apnea compared to control conditions, with creatine pyruvate being the more effective substance. Overall, creatine and creatine pyruvate each produced time-dependent degrees of protection against severe hypoxic-induced disturbances of PN activity. The underlying protective mechanisms are unknown and need further investigations.

The role of metformin and resveratrol in the prevention of hypoxia-inducible factor 1α accumulation and fibrosis in hypoxic adipose tissue.

Hypoxic activation of hypoxia-inducible factor 1α (HIF-1α) and fibrosis in adipose tissue contribute to adipose dysfunction. This study was designed to investigate the effects of metformin and resveratrol on the regulation of HIF-1α and fibrosis in hypoxic adipose tissue. Mice were fed a high-fat diet to induce hypoxia and fibrosis in adipose tissue; adipose tissue incubated in vitro in 1% O2 showed a similar change. The effects of metformin and resveratrol on hypoxia, HIF-1α accumulation, endoplasmic reticulum stress and gene expressions of extracellular matrix components and pro-inflammatory cytokines were examined. Oral administration of metformin or resveratrol prevented hypoxia and reduced HIF-1α accumulation with dephosphorylation of inositol-requiring enzyme 1α and eukaryotic initiation factor 2α, indicative of suppression of hypoxic HIF-1α activation and endoplasmic reticulum stress. Metformin and resveratrol down-regulated gene expressions of Col3α, Col6α, elastin and lysyl oxidase and thereby reduced collagen deposition in adipose tissue. The increased gene expressions of TNF-α, IL-6, monocyte chemoattractant protein 1 and F4/80 were also down-regulated by metformin and resveratrol. Metformin and resveratrol had similar effects in adipose tissue exposed to 1% O2 . Metformin reduced ATP production and prevented the reduction in oxygen tension in 3T3-L1 cells, suggesting that it prevented hypoxia by limiting oxygen consumption, whereas resveratrol reduced HIF-1α accumulation by promoting its proteasomal degradation via the regulation of AMPK/SIRT1. Hypoxia and fibrosis are early causes of adipose dysfunction in obesity. Both metformin and resveratrol effectively inhibited HIF-1α activation-induced fibrosis and inflammation in adipose tissue, although by different mechanisms.

Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii

Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1–27], [1–19], [1–16] and [1–15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5′-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals.

TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions

Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation.

Hypoxia-Targeted Drug Q6 Induces G2-M Arrest and Apoptosis via Poisoning Topoisomerase II under Hypoxia.

In spite of the tremendous efforts dedicated to developing hypoxia-activated prodrugs, no agents yet have been approved for clinical therapy. In the present study, the hypoxic selective anti-cancer activity as well as the cellular target of a novel tirapazamine (TPZ) analogue, 7-methyl-3-(3-chlorophenyl)-quinoxaline-2-carbonitrile 1,4-dioxide (Q6) were investigated. Q6 implemented anti-cancer effects via poisoning topoisomerase II (topo II) under hypoxia. Modified trapped in agarose DNA immunostaining (TARDIS) assay showed more topo II–DNA cleavage complexes trapped by Q6 than TPZ at even lower concentration. In addition, by introducing ataxia-telangiectasia-mutated (ATM) kinase inhibitors caffeine and KU-60019, we displayed that Q6-triggered apoptosis was attributed, at least partially, to DNA double-strand breaks generated by the topo II-targeting effect. Collectively, Q6 stood out for its better hypoxia-selectivity and topo II-poisoning than the parental compound TPZ. All these data shed light on the research of Q6 as a promising hypoxia-activated prodrug candidate for human hepatocellular carcinoma therapy.

Abstract 4448: Identification of a small molecule inhibitor of aldolase A for the targeting of hypoxic cancer cells

Abstract Cancer cells are critically dependent on glycolysis. Currently, there is no effective pharmacotherapy that exploits this metabolic vulnerability. We have uncovered a feed forward cycle of anaerobic glycolysis and hypoxia-inducible factor-1 (HIF-1). Glycolysis under hypoxic conditions normally maintains high ATP levels, which is driven by transcriptional activity of HIF-1. Glycolysis inhibition results in a cellular energy crisis, i.e. an increased AMP:ATP ratio, leading to AMPK-mediated phosphorylation of the HIF-1 co-activator p300/CBP. This prevents HIF-1 activity, although HIF-1 protein levels are unchanged, abrogating the feed forward cycle. We identified fructose-1,6-bisphosphate aldolase A (ALDOA) as a top glycolytic enzyme target for inhibiting hypoxic cancer cell glycolysis and HIF-1 activity. Our aim was to identify small molecule inhibitors for ALDOA that may yield compounds for further preclinical development. To this end, a primary high-throughput screen (HTS) was performed with 65,936 compounds using a fluorescence-based NADH oxidation aldolase (cell-free) assay. 640 compounds were further tested in a cherry-pick confirmation screen, from which 112 hits underwent concentration-response curve validation in cell-free assays. From these, 4 hits were further tested in cell-based assays by using colorectal, breast and pancreatic cancer cell lines. A lead compound that showed micromolar potency in inhibiting ALDOA, induced cell death under hypoxic conditions with IC50 values ranging between 2 - 8 μM in the cancer cell lines tested. It also inhibited extracellular flux in a real-time glycolysis assay, inhibited extracellular lactate production under hypoxic conditions as a measure of glycolysis, and blocked hypoxia responsive element (HRE) HIF-1 reporter activity but not HIF-1 protein levels. No effect of the lead compound was observed on mitochondrial respiration. Thus, this compound provides a valuable chemical probe for inhibiting glycolysis in cancer cells. Furthermore, the work provides proof-of-concept that targeting glycolysis with a small molecule inhibitor exerts potent antitumor effects in vitro and is currently being tested in preclinical models as a first-in-class oncological agent. Citation Format: Petrus R. de Jong, Geoffrey V. Grandjean, Ashwini K. Devkota, Eun Jeong Cho, Kevin N. Dalby, Garth Powis. Identification of a small molecule inhibitor of aldolase A for the targeting of hypoxic cancer cells. [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 4448. doi:10.1158/1538-7445.AM2015-4448