Temporal changes in the structure of protist communities incubated under normoxic and hypoxic conditions: a metabarcoding analysis

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

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

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.

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

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.

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

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.

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.

Background: Urethral stricture is a narrowing of the urethral lumen caused by the formation of fibrotic tissue or spongiofibrosis within the urethra. In ADSCs, hypoxic culture with approximately 5% oxygen has been reported to enhance proliferation capacity and improve stem cell viability following transplantation. This study aims to investigate the effects of hypoxic culture conditions on the expression of IL-10, TNF-α, FGF-2, and myofibroblasts, as well as on fibrotic tissue formation, in comparison to ADSCs cultured under normoxic conditions. Methods: This study is experimental research conducted on male Wistar rats, using a randomized post-test-only control group design. The study consisted of three groups: (1) the control group, which received urethral injury without ADSC injection; (2) the treatment group that received ADSCs cultured under hypoxic conditions; and (3) the treatment group that received ADSCs cultured under normoxic conditions. The number of cells injected was 2 × 10⁵ cells periurethra. We evaluate IL-10, TNF-α, FGF-2, Myofibroblast, and Fibrotic tissue formation. Analysis data using SPSS 20.0 Results: The group that received ADSC injections cultured under hypoxic conditions exhibited significantly higher IL-10 expression, with a mean value of 240.83 pg/mL on day 3 and 238.83 pg/mL on day 8 (p < 0.05). TNF-α expression was lower in the group that received ADSC injections cultured under hypoxic conditions, with mean values of 113.33 ng/L on day 3 and 133.83 ng/L on day 8 (p < 0.05). The group that received ADSC injections cultured under hypoxic conditions exhibited significantly higher FGF-2 expression, with a mean value of 571.83 pg/mL on day 3 and 601.5 pg/mL on day 8 (p < 0.05). On day 14, myofibroblast expression in the group that received ADSC injections cultured under hypoxic conditions was significantly lower than in the control group and the group that received ADSC injections cultured under normoxic conditions (p < 0.05). Based on the results of this study, the area of fibrotic tissue formation in the group that received ADSC injections cultured under hypoxic conditions was lower than that observed in both the control group and the group that received ADSC injections cultured under normoxic conditions. Conclusion: The administration of 2 × 10⁵ ADSCs into periurethral tissue was shown to increase IL-10 expression, reduce TNF-α expression, increase FGF-2 expression, reduce myofibroblast expression on day 14, and reduce fibrotic tissue formation in Rat urethral tissue.

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.

Photoinduced Carbene for Effective Photodynamic Therapy Against Hypoxic Cancer Cells

Working Toward Better Tissue Repair Therapies: Exposure of MSC to Hypoxic Conditions In Vitro Increases Levels of the Hepatocyte Growth Factor Receptor c-met, Improves Motility, and Induces Phosphorylation of the Pro-Survival Protein AKT.

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.

Culture of Mobilized Human CD34+ Cells in Hypoxic Conditions Improves Lentiviral Transduction Efficiency in SCID-Repopulating Cells