Optimisation of MTGase production in Pichia pastoris under controlled growth rates and oxygen conditions

Although p53-inactivating mutations have been described in the majority of human cancers, their role in prostate cancer is controversial as mutations are uncommon, particularly in early lesions. p53 is activated by hypoxia and other stressors and is primarily regulated by the Mdm2 protein. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and other eicosanoids, is also induced by hypoxia. COX-2 and resultant prostaglandins increase tumor cell proliferation, resistance to apoptosis, and angiogenesis. Previous reports indicate a complex, reciprocal relationship between p53 and COX-2. To elucidate the effects of COX-2 on p53 in response to hypoxia, we transfected the COX-2 gene into the p53-positive, COX-2-negative MDA-PCa-2b human prostate cancer cell line. The expression of functional p53 and Mdm2 was compared in COX-2+ versus COX-2- cells under normoxic and hypoxic conditions. Our results demonstrated that hypoxia increases both COX-2 protein levels and p53 transcriptional activity in these cells. Forced expression of COX-2 increased tumor cell viability and decreased apoptosis in response to hypoxia. COX-2+ cells had increased Mdm2 phosphorylation in either normoxic or hypoxic conditions. Overexpression of COX-2 abrogated hypoxia-induced p53 phosphorylation and promoted the binding of p53 to Mdm2 protein in hypoxic cells. In addition, COX-2-expressing cells exhibited decreased hypoxia-induced nuclear accumulation of p53 protein. Finally, forced expression of COX-2 suppressed both basal and hypoxia-induced p53 transcriptional activity, and this effect was mimicked by the addition of PGE2 to wild-type cells. These results demonstrated a role for COX-2 in the suppression of hypoxia-induced p53 activity via both direct effects and indirect modulation of Mdm2 activity. These data imply that COX-2-positive prostate cancer cells can have impaired p53 function even in the presence of wild-type p53 and that p53 activity can be restored in these cells via inhibition of COX-2 activity.

Effect of Nitric Oxide Donor on IL-6 and PDGF-BB: Comparison of Diabetic Versus Normal Tissue Under Hypoxic and Normoxic Conditions

Decision letter: Hypoxia truncates and constitutively activates the key cholesterol synthesis enzyme squalene monooxygenase

CD133 Identifies a Human Bone Marrow Stem/Progenitor Cell Sub-population With a Repertoire of Secreted Factors That Protect Against Stroke

Hypoxia-inducible factor 1 alpha (HIF1alpha) and its related factor, HLF, activate expression of a group of genes such as erythropoietin in response to low oxygen. Transfection analysis using fusion genes of GAL4DBD with various fragments of the two factors delineated two transcription activation domains which are inducible in response to hypoxia and are localized in the C-terminal half. Their sequences are conserved between HLF and HIF1alpha. One is designated NAD (N-terminal activation domain), while the other is CAD (C-terminal activation domain). Immunoblot analysis revealed that NADs, which were rarely detectable at normoxia, became stabilized and accumulated at hypoxia, whereas CADs were constitutively expressed. In the mammalian two-hybrid system, CAD and NAD baits enhanced the luciferase expression from a reporter gene by co-transfection with CREB-binding protein (CBP) prey, whereas CAD, but not NAD, enhanced beta-galactosidase expression in yeast by CBP co-expression, suggesting that NAD and CAD interact with CBP/p300 by a different mechanism. Co-transfection experiments revealed that expression of Ref-1 and thioredoxin further enhanced the luciferase activity expressed by CAD, but not by NAD. Amino acid replacement in the sequences of CADs revealed a specific cysteine to be essential for their hypoxia-inducible interaction with CBP. Nuclear translocation of thioredoxin from cytoplasm was observed upon reducing O2 concentrations.

Bufalin induces apoptosis in myeloma cells through DNA damage at hypoxic conditions

Microcystins (MCs) are highly liver-specific and evidenced as a liver tumor promoter. Oxidative stress is one of the most important toxicity mechanisms of MCs, which is tightly related to oxygen concentration. The effects of MCs on animals and cell lines in normoxia and the mechanisms have been well studied, but such effects in different oxygen conditions were still unclear. The aim of the present study was to explore the cellular response of the human hepatocellular carcinoma cell line (HepG2) to MC-LR exposure under hypoxic (1% O2 ) and normoxic (21% O2 ) conditions. We examined cell viability, mitochondrial membrane potential (MMP), superoxide dismutase (SOD) activity and gene expression posttoxin exposure. Cell viability was increased by MC-LR in normoxia although decreased in hypoxia. MC-LR markedly induced MMP loss under hypoxic condition but only slightly MMP loss under normoxic condition. SOD activity was significantly induced by MC-LR in hypoxia, indicating prolonged oxidative stress. Inhibitory apoptosis protein (c-IAP2) was significantly up-regulated by MC-LR under normoxic condition, suggesting that c-IAP2 played an important role in the promotion of cell proliferation by MC-LR. These results indicate that MC-LR promotes cell proliferation under normoxic condition whereas induces cell apoptosis under hypoxic condition.

Introduction: The purpose of this study is to quantify the effects of combination trastuzumab and radiation therapy in HER2+ breast cancer. Breast tumors can become hypoxic which increases radioresistance partly due to the upregulation of hypoxia inducible factors (HIFs). HIF-1α promotes cancer cell survival and has been found to be upregulated by phosphatidylinositol-3 kinase (P13K) signaling. Trastuzumab, a targeted therapy, has been used in combination with radiation in the adjuvant setting to treat HER2+ patients. Trastuzumab causes cell cycle arrest by inhibiting P13K pathways and been shown to increase tumor oxygenation in vivo. There is evidence that trastuzumab sensitizes HER2+ breast cancer to radiation therapy; however, longitudinal studies are limited and fail to quantify sensitization over time, reducing clinical relevance. There are three main goals to this study: 1) quantify the effects of combination trastuzumab and radiation therapy in normoxic and hypoxic conditions in vitro, 2) quantify the regulation of P13K when treated with trastuzumab in hypoxic conditions in vitro, and 3) test the hypothesis that trastuzumab sensitizes HER2+ breast cancer to radiation therapy in an in vivo model of HER2+ breast cancer. Experimental Design: For in vitro studies, BT474-GFP expressing HER2+ breast cancer cells were plated in a 96 well plate. 24 hours later, cell media was changed to simulate hypoxic (50 or 100 μM CoCl2) or normoxic (0 μM CoCl2) conditions and then treated with trastuzumab and/or radiation (various dosing and timing) and imaged using the IncuCyte ZOOM System every 6 hours for 7 days. Cell number was automatically quantified and surviving fractions (SF) were calculated for each time point. An enzyme-linked immunosorbent assay (ELISA) was used to quantify HIF-1α and P13K protein levels in cells. For in vivo studies, 107 BT474 HER2+ breast cancer cells were implanted subcutaneously into athymic nude mice. After reaching 250 mm3, tumors were treated with trastuzumab (10 mg/kg, 2 doses), and/or radiation (5 or 10 Gy, single dose), or saline and were measured with calipers for six weeks. A non-parametric Wilcoxon rank sum test was used to determine statistical differences in tumor size between treatment groups. Degree of synergy over time in combination treatment groups both in vitro and in vivo was determined using a Bliss independence model. Results and Discussion: The SF of cells 5 days after 10 Gy irradiation under hypoxic conditions was significantly higher at 51.2% (50 μM CoCl2, P < 0.05) and 65.5% (100 μM CoCl2, P < 0.01) than normoxic conditions with a SF of 44.8%. Cells treated with 3 μg/ml of trastuzumab 24 hours prior to 10 Gy irradiation had a significant decrease in SF 5 days post irradiation under both normoxic (SF 40.8%) and hypoxic conditions (100 μM CoCl2 - SF 58.8%) compared to either monotherapy (P < 0.05). In vivo, treatment with trastuzumab and radiation results in a faster rate of tumor regression. Mice (n = 4) had a significant (P < 0.05) 45% decrease in tumor size 7 days after 10 Gy irradiation when treated in combination with trastuzumab compared to control (n = 5) or radiation alone (n = 5). Mice treated with radiation alone did not have a significant decrease in tumor size until 14 days after radiation therapy. Conclusion: Quantifying the synergy between trastuzumab and radiation over time will elucidate optimal combination regimens and has the potential to decrease the amount of therapy needed to achieve tumor regression. Systematically evaluating the radiosensitizing effect of trastuzumab in vitro under normoxic and hypoxic conditions may provide mechanistic insight on how to overcome radioresistance in HER2+ breast cancer. We acknowledge the support of CPRIT RR160005, NCI R01CA186193, and ACS RSG-18-006-01-CCE. Citation Format: Meghan Bloom, Jack Virostko, Anna Sorace, Thomas Yankeelov. Quantifying the effects of combination trastuzumab and radiation therapy in HER2 positive breast cancer under normoxic and hypoxic conditions [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-25.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

In this study we investigated the influence of oxygen availability on a phenotypic microtiter screen to identify new, natural product inhibitors of growth for the bovine mastitis-causing microorganisms; Streptococcus uberis, Staphylococcus aureus, and Escherichia coli. Mastitis is a common disease in dairy cattle worldwide and is a major cause of reduced milk yield and antibiotic usage in dairy herds. Prevention of bovine mastitis commonly relies on the application of teat disinfectants that contain either iodine or chlorhexidine. These compounds are used extensively in human clinical settings and increased tolerance to chlorhexidine has been reported in both Gram-positive and Gram-negative microorganisms. As such new, non-human use alternatives are required for the agricultural industry. Our screening was conducted under normoxic (20% oxygen) and hypoxic (<1% oxygen) conditions to mimic the conditions on teat skin and within the mammary gland respectively, against two natural compound libraries. No compounds inhibited E. coli under either oxygen condition. Against the Gram-positive microorganisms, 12 inhibitory compounds were identified under normoxic conditions, and 10 under hypoxic conditions. Data revealed a clear oxygen-dependency amongst compounds inhibiting growth, with only partial overlap between oxygen conditions. The oxygen-dependent inhibitory activity of a naturally occurring quinone, β-lapachone, against S. uberis was subsequently investigated and we demonstrated that this compound is only active under normoxic conditions with a minimum inhibitory concentration and minimum bactericidal concentration of 32 μM and kills via a reactive oxygen species-dependent mechanism as has been demonstrated in other microorganisms. These results demonstrate the importance of considering oxygen-availability in high-throughput inhibitor discovery.

Low Oxygen Tension and Synthetic Nanogratings Improve the Uniformity and Stemness of Human Mesenchymal Stem Cell Layer

ObjectiveIschemic brain injury has been shown to be increased in endothelial nitric oxide synthase (eNOS) knockout mice but reduced in neuronal nitric oxide synthase (nNOS) knockout mice compared to the wild‐type mice. It has been proposed that NO derived from nNOS in neurons is detrimental whereas NO derived from eNOS in endothelial cells is protective after stroke. However, the functional mechanism underlying this protection or damage has never been studied. Mitochondria play an important role in a wide range of physiological and pathophysiological processes. Besides the generation of cellular energy, mitochondria are also involved in reactive oxygen species (ROS) production and the activation of cell death pathways. Therefore, the effect of NOS inhibitors on mitochondrial respiration is important to investigate as mitochondria determine the fate of cell survival after stroke or ischemic injury. Our objective is to determine the effects of NOS inhibition on mitochondrial bioenergetics in primary rat cortical neurons under normoxic and oxygen‐glucose deprivation‐reoxygenation (OGD‐R) conditions.MethodsCortical neurons were isolated from brain and exposed to normoxic and OGD‐R conditions with and without non‐specific NOS inhibitor N‐ω‐propyl‐L‐arginine (L‐NAME). Oxygen consumption rates (OCR) and respiratory parameters were measured using Seahorse XFe24 cell mito‐stress test (Agilent technologies). Cell viability was measured using CCK‐8 kit to determine the effect of NOS inhibition under hypoxic and normoxic conditions. Electron spin resonance (ESR) spectroscopy (Bruker Bio‐Spin spectrometer, Germany) was used to measure ROS using spin probe CMH.ResultsIn neurons, OCR showed a decrease in basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition compared to normoxic control. Interestingly, NOS inhibition under normoxic conditions increased basal respiration, maximal respiration, spare respiratory capacity, and non‐mitochondrial respiration but not proton leak whereas, NOS inhibition decreased basal respiration, maximal respiration, spare respiratory capacity, proton leak and non‐mitochondrial respiration under hypoxic condition. Thus, NOS inhibition has opposite effects on mitochondrial respiratory parameters under normoxic and hypoxic conditions. Viability studies showed NOS inhibition has improved cell viability under hypoxic conditions. In contrast, NOS inhibition under normoxic conditions showed a decrease in cell viability. ROS measurements by ESR have shown that L‐NAME treatment increased ROS levels under normoxic conditions, however, NOS inhibition decreased ROS levels under hypoxic conditions.ConclusionsNOS inhibition has opposite effects on mitochondrial bioenergetics, viability and superoxide levels under hypoxia compared to normoxia. nNOS appears to inhibit ROS generation under hypoxia but promotes the ROS generation following hypoxia possibly due to uncoupling of nNOS. Thus, the change in the generation of free radicals may underlie the diverse effects of nNOS in neurons following exposure to normoxia and hypoxiaSupport or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

Differential phenotypic behaviors of human degenerative nucleus pulposus cells under normoxic and hypoxic conditions: influence of oxygen concentration during isolation, expansion, and cultivation

We investigated inter-individual variation in metabolism, swimming performance and the relationship between metabolism and swimming performance under normoxic and hypoxic oxygen conditions ([O2]) in juvenile black carp (Mylopharyngodon piceus). We measured the standard metabolic rate (SMR), critical swimming speed (Ucrit), active metabolic rate (AMR) and aerobic scope (AS) of 40 fish (body weight, 9.7–11.0 g; body length, 8.4–8.8 cm) at 20 °C under normoxic (100% air saturation) and hypoxic (30% air saturation) conditions. Hypoxia resulted in a significant decrease in all the investigated parameters (p < 0.05). The SMR, Ucrit and AMR exhibited consistent individual differences (repeatability) (p ≤ 0.022), whereas the AS had no consistency across different water [O2] conditions (p = 0.088). The SMR was positively correlated with the Ucrit, AMR and AS (p ≤ 0.002) under normoxic conditions. The SMR was also positively correlated with the Ucrit and AMR under hypoxic conditions (p ≤ 0.003) but there was no correlation between the SMR and AS (p = 0.141). The slope of the correlation between the SMR and Ucrit was shallower under hypoxic conditions than under normoxic conditions (F1,76 = 13.844, p < 0.001), which indicated that the swimming performance decreased more profoundly under hypoxic conditions in individuals with a high SMR.