Cobalt Chloride Treatment Research Articles

Hypoxia Inducible Factor 1α Inhibits the Expression of Immunosuppressive Tryptophan-2,3-Dioxygenase in Glioblastoma.

Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.

Hypoxia promotes colorectal cancer cell migration and invasion in a SIRT1-dependent manner

BackgroundHypoxic microenvironments play a significant role in the progression of colorectal cancer (CRC). Silencing information regulator 1 (SIRT1), a class III histone deacetylase, modulates the multiple biological behaviors of cancer. However, its role in CRC remains unclear. This study aims to explore the role of SIRT1 in CRC migration and invasion under hypoxia.MethodsSIRT1 protein and mRNA levels were detected by Western blotting and real-time PCR in CRC cells exposed to hypoxia (1% O2). The migration and invasion abilities of SW480 and HCT116 cells with SIRT1 overexpression or knockdown were studied with transwell assays, and the results were confirmed by those of treatment with specific SIRT1 activator (SRT1720) and inhibitor (EX527). The dual-luciferase reporter systems with a series of SIRT1 promoter truncations were used to analyze their transcriptional activities, respectively. After a bioinformatic analysis of potential transcription factors, the direct interaction between the transcription factor and SIRT1 promoter was determined by chromatin immunoprecipitation (ChIP) assays. Western blot and real-time PCR assays were used to detect the activation and acetylation levels of the NF-κB pathway.ResultsThe protein and mRNA levels of SIRT1 were significantly decreased under hypoxia, and these effects were replicated by cobalt chloride treatment. Hypoxia promoted cell migration and invasion, which were impeded by the overexpression or activation of SIRT1 and promoted by the knockdown or inhibition of SIRT1. The dual-luciferase reporter gene and ChIP analyses revealed that the core regulatory elements located 100 bp upstream of the SIRT1 promoter and early growth response factor 1 (EGR1) could interact with this DNA sequence. Subsequent rescue experiments suggested that EGR1 was essential for hypoxia-mediated SIRT1 transcriptional suppression. Western blot analyses demonstrated that SIRT1 overexpression eliminated the p65 acetylation induced by hypoxia along with the decreased MMP-2/-9, suggesting that NF-κB was a direct downstream target of SIRT1 and might regulate cell migration and invasion through MMP-2/-9.ConclusionsOur results establish for the first time that EGR1 plays an important role in regulating SIRT1 expression under hypoxia. Hypoxia promotes CRC cell migration and invasion in a SIRT1-dependent manner. And a potential SIRT1/NF-κB/MMP-2/-9 axis modulates this process.

NLRP2 is highly expressed and promotes apoptosis in a mouse model of kidney ischemia/reperfusion injury

The aim of this study was to investigate the role of nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 2 (NLRP2) in kidney ischemia/reperfusion injury. A mouse model of acute kidney ischemia/reperfusion injury was established to conduct in vivo experiments. Oxygen–glucose deprivation (OGD) and cobalt chloride treatment of the HK-2 and glomerular endothelial cell (GENC) kidney cell lines were performed for the in vitro study. Reverse transcription–quantitative polymerase chain reaction, western blotting, and immunohistochemical staining were used to analyze NLRP2 expression levels. Knockdown of NLRP2 in cells was also performed, and cell apoptosis was detected using flow cytometry. NLRP2 was expressed in normal kidney tissues; however, its expression was significantly increased in the acute kidney injury model and in OGD-treated cells. Conversely, knockdown of NLRP2 reduced apoptosis of cells. These results suggested that NLRP2 was involved in kidney damage and may be an important target for treatment of acute kidney injury.

Moringa oleifera extract attenuates the CoCl2 induced hypoxia of rat's brain: Expression pattern of HIF-1α, NF-kB, MAO and EPO

Hypoxia-induced oxidative stress and apoptosis are the major hallmark explanations underlying brain dysfunction. Hypoxia in the current study was induced by Cobalt chloride (CoCl2) treatment in rats. The aim of this experiment was to explore the potential ameliorative potency of Moringa oleifera ethanolic extract (MO) against experimentally induced hypoxia on the structure and function of the rat’s brain. Fifty male rats were allocated to five groups (10 rats each): a control group, a MO-treated group (400 mg/kg bw, orally), a CoCl2-treated group (40 mg/kg bw/day, orally), a prophylaxis group, and a therapeutic co-treated group. Oxidative stress biomarkers and monoamine neurotransmitter were evaluated in brain tissue. In addition, qRT-PCR for expression pattern of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. Glial fibrillary acidic protein (GFAP), apoptotic markers (BCL-2 and caspase 3) were detected immunohistochemically in brain cells. The results revealed a significantly lower concentration of GABA, monoamine neurotransmitter in hypoxic rat’s brain. Moreover, an evident up-regulation of the mRNA expression of HIF-1α, EPO, CYTO, NF-kB, and MAO-A. There was marked encephalopathy manifested by pyknotic neurons with eosinophilic cytoplasm, vacuolations and cerebral congestions in the hypoxic rat brains. Additionally, the score of neuronal expression occupied by GFAP- positive astroglia, Caspase-3 and microglial CD68 were elevated but Bcl-2 expression was found decreased in the hypoxic group than control. The endpoints of this study clearly stated that MO ethanolic extract suggestively counteracted neurotoxic impacts caused by hypoxia, particularly when it administered prior to and concurrently with CoCl2 administration.

Indoxyl glucuronide, a protein-bound uremic toxin, inhibits hypoxia-inducible factor‒dependent erythropoietin expression through activation of aryl hydrocarbon receptor

Renal anemia is common among chronic kidney disease (CKD) patients, and is mainly caused by inadequate erythropoietin (EPO) production from kidneys due to dysfunction of intracellular hypoxia-inducible factor (HIF) signaling in renal EPO-producing cells. We have previously shown that indoxyl sulfate (IS), a representative protein-bound uremic toxin accumulated in the blood of CKD patients, inhibits hypoxia-induced HIF activation and subsequent EPO production through activation of aryl hydrocarbon receptor (AHR). In this study, we further investigated the effects of other protein-bound uremic toxins on HIF-dependent EPO expression using EPO-producing HepG2 cells. We found that indoxyl glucuronide (IG) and IS, but not p-cresyl sulfate, phenyl sulfate, 3-indoleacetic acid or hippuric acid, inhibited hypoxia mimetic cobalt chloride-induced EPO mRNA expression. Furthermore, IG at concentrations similar to the blood levels in CKD patients inhibited the transcriptional activation of HIF induced by both cobalt chloride treatment and hypoxic culture. IG also induced CYP1A1 mRNA expression and nuclear translocation of AHR protein, indicating that IG activates AHR signaling. Blockade of AHR by a pharmacological antagonist CH-223191 abolished the IG-induced inhibition of HIF activation. Collectively, this study is the first to elucidate the biological effects of IG to inhibit HIF-dependent EPO production through activation of AHR. Our data suggests that not only IS but also IG contributes to the impairment of HIF signaling in renal anemia.

Cellular adaptive response and regulation of HIF after low dose gamma-radiation exposure

Purpose: Cellular damage due to low dose of γ-radiation (≤0.1 Gy) is generally extrapolated from observing the effects at higher doses. These estimations are not accurate. This has led to uncertainties while assessing the radiation risk factors at low doses. Although there are reports on the radiation induced adaptive response, the mechanism of action is not fully elucidated, leading to the uncertainties. One of the outcomes of low dose radiation exposure is believed to be an adaptive response. The mechanism of adaptive response is not fully understood. Therefore, the study was undertaken to understand the role of hypoxia inducible factor (HIF) on radiation induced adaptive response.Materials and methods: Human breast cancer cell line MCF-7 cells pre-exposed to low dose γ-radiation (0.1 Gy; priming dose) were exposed to 2 Gy (challenging dose) 8 h after the priming dose and studied for the adaptive response. Cell death was measured by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was measured by fluorescence-activated cell sorting analysis. DNA damage was measured by alkaline comet assay. HIF transcription activity was assayed using transiently transfected plasmid having HIF consensus sequence and luciferase as the reporter gene.Results: Cells when exposed to 0.1 Gy priming dose 8 h prior to the higher dose (2 Gy; challenging dose) results in lower amount of radiation induced damages compared to the cells exposed to 2 Gy alone. Cobalt chloride treatment in place of priming dose also results in the protection to cells when exposed to challenging dose. There was up-regulation of HIF activity when cells were exposed to priming dose, indicating the role of HIF in radiation induced response.Conclusion: Results indicate the γ-radiation induced adaptive response. One of the mechanism proposed is up-regulation of HIF after low dose exposure, which protects the cells from damages when they are exposed to challenging dose of 2 Gy radiation.

Interleukin 17 under hypoxia mimetic condition augments osteoclast mediated bone erosion and expression of HIF-1α and MMP-9

Interleukin 17 (IL-17) and hypoxia have been implicated to play a key role in rheumatoid arthritis (RA). In this study, the combined treatment of IL-17 and cobalt chloride (CoCl2), a hypoxia mimetic significantly increased the osteoclast formation and the expression of TRAP and MMP-9 in RAW 264.7 macrophage cells in the presence of RANKL and M-CSF. The unified effect of IL-17 and CoCl2 markedly increased osteoclast mediated bone erosion through the activation of RANKL/NF-κB/NFATc1 signaling pathway. The treatment of IL-17 in combination with CoCl2 further potentiated the protein and mRNA expression of HIF-1α and MMP-9 in rat synovial macrophages. Conversely, the blockage of HIF-1α expression with BAY87-2243 abrogated the IL-17 and CoCl2 mediated expression of HIF-1α and MMP-9. Further, the knockdown of IL-17RA using siRNA reversed the IL-17 and CoCl2 induced expression of HIF-1α in synovial macrophages. In conclusion, IL-17 synergizes with CoCl2 induced hypoxic condition to augment osteoclast mediated bone erosion and synovial macrophages mediated RA pathogenesis.

Effects of CoCl2‑simulated hypoxia on the expression levels of matrix metalloproteinases in renal adenocarcinoma cells and renal tubular epithelial cells

Renal cell carcinoma (RCC) and chronic kidney disease (CKD) are associated with hypoxia, but the effects of hypoxia on the process of angiogenesis in the two diseases are dramatically different. Some of matrix metalloproteinases (MMPs), such as MMP2 and MMP9, may have a role because they represent the most prominent family of proteinases associated with angiogenesis. In the present study, the differential response of human renal cell cancer cells (786-0), human renal tubular epithelial cells (HK-2) and human microvascular endothelial cells (HMEC-1) to hypoxia with regards to the expression of MMP2, MMP9, MMP14, TIMP2, RECK was investigated. Cobalt chloride (CoCl2) treatment was used to simulate the hypoxia environment in RCC and CKD. The expression levels of HIF-1α, RECK, MMP2, MMP9, MMP14 and TIMP2 in HK2, 786-0 and HMEC-1 cells were determined by western blot analysis after incubation with varying concentrations of CoCl2 for 24 h. It was indicated that the effects of hypoxia on the endogenous expression of RECK and MMP2 differed depending on the considered cell type. Notably, the RECK expression was significantly decreased in 786-0 cells under hypoxia, whereas this expression was slightly increased in HK2 and HMEC-1 cells. Furthermore, the MMP2 expression was significantly increased in HMEC-1 cells under hypoxia, whereas the expression was slightly decreased in HK2 and 786-0 cells. These results demonstrate that 786-0, HK2 and HMEC-1 cells respond differently under hypoxic conditions. Furthermore, MMP2 and RECK may serve divergent roles in HK2 and HMEC-1 cells under hypoxic conditions.

Cobalt treatment does not prevent glomerular morphological alterations in type 1 diabetic rats.

Early renal morphological alterations including glomerular hypertrophy and mesangial expansion occur in diabetic kidney disease and correlate with various clinical manifestations of diabetes. The present study was designed to investigate the influence of pharmacological modulation of HIF-1α (hypoxia inducible factor-1 alpha) protein levels, on these glomerular changes in rodent model of type 1 diabetes. Male wistar rats were made diabetic (Streptozotocin 45mg/kg; i.p.) and afterwards treated with HIF activator cobalt chloride for 4weeks. Renal function was assessed by serum creatinine, albumin, proteinuria levels, oxidative stress: reduced glutathione levels and catalase activity, and renal tissue HIF-1α protein levels were determined by ELISA assay. Histological analysis of kidney sections was done by haematoxylin and eosin (glomeruli diameter), periodic acid Schiff (mesangial expansion and glomerulosclerosis) and sirius red (fibrosis, tubular dilation) staining. Diabetes rats displayed reduced serum albumin levels, marked proteinuria, lower kidney reduced glutathione content, glomerular hypertrophy, glomerulosclerosis, mesangial expansion, tubular dilation and renal fibrosis. Cobalt chloride treatment normalised renal HIF-1α protein levels, reduced development of proteinuria and tubulo-interstitial fibrosis, but the glomerular morphological alterations such as glomerulosclerosis, mesangial expansion, increased glomerular diameter and tubular vacoulations were not abrogated in diabetic kidneys. Glomerular morphological abnormalities might precede the development of proteinuria and renal fibrosis in experimental model of type 1 diabetes. Pharmacological modulation of renal HIF-1α protein levels does not influence glomerular and tubular dilatory changes in diabetic kidney disease.

Endothelial cells co-cultured with renal carcinoma cells significantly reduce RECK expression under chemical hypoxia.

Renal cell carcinoma (RCC) is characterized by excessive angiogenesis, while chronic kidney disease (CKD) suffers from the opposite problem-failure of reparative angiogenesis. It can be due to their different responses to hypoxic environment. But the specific molecular regulators are still unclear. This study is aimed to explore the influence of human renal cell cancer cells (786-0) and human renal tubular epithelial cells (HK-2) on RECK expression, proliferation, and angiogenesis of adjacent microvascular endothelial cells (HMEC-1) under chemical hypoxia. Cobalt chloride (CoCl2 ) treatment was used to simulate the hypoxia environment in RCC and CKD. Co-culture, cell proliferation assay, and tube formation assay were used to evaluate the influence of 786-0 or HK-2 cells on proliferation and angiogenesis of adjacent HMEC-1 cells. Effects of different environments on RECK expressions in 786-0, HK2, or HMEC-1 cells were determined by Western blot. We found that both 786-0 cells and HK2 cells can upregulate RECK expression of adjacent HMEC-1 cells in normoxic conditions. However, under hypoxia, the HMEC-1 cells co-cultured with 786-0 significantly reduced RECK expression and there was no significant change in HMEC-1 cells co-cultured with HK2 cells. We also found that 786-0 significantly enhanced the proliferation and angiogenesis of adjacent HMEC-1 cells. Our results suggested that some paracrine substances produced by 786-0 cells may reduce RECK expression of adjacent HMEC-1 cells and enhance their proliferation and in vitro angiogenic capacity.

DIPG-30. DIFFERENTIAL HYPOXIC RESPONSE IN HUMAN DIPG CELL LINES

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) are rare and aggressive childhood tumors, with an abysmal prognosis and limited experimental models available for study. A recent report showed that DIPG are hypoperfused compared to surrounding brain tissue, suggesting that the tumor cells are exposed to a hypoxic microenvironment. This stimulus may induce widespread transcriptional changes through activation of Hypoxia-inducible Factors (HIF), which have been associated with invasion, metastasis, angiogenesis, and resistance to radiation therapy and chemotherapy. Therefore, in vitro models using DIPG cells cultured in conditions of normal oxygen tension may not be representative of in vivo tumors because of artificial differences in HIF activation. METHODS: To test cellular responses to hypoxia, human DIPG cell lines with H3.1 K27M and H3.3 K27M mutations were treated with the hypoxia-mimetic compound, cobalt (II) chloride, for 24 hours. Simulated hypoxic responses were assayed using Western blot analysis, proliferation assays, and extracellular flux analysis. RESULTS: The H3.3 K27M cell line increased expression of HIF1a with cobalt (II) chloride treatment and correlated with an increase in glycolytic rates and increased expression of proteins involved in glucose transport and glycolysis. The H3.1 K27M cell line increased expression of HIF2a and not HIF1a after cobalt (II) chloride treatment, correlating with an increase in some glycolysis-associated proteins but no change in glycolytic rate. CONCLUSION: These data suggest that the effects of a hypoxic microenvironment should be considered in experimental models using cultured DIPG cell lines to understand differences in cell behavior, patient prognosis, and predicted treatment responses. Activation of specific HIF isoforms may underlie differences in hypoxic responses among cell lines.

The effects of Caffeic Acid Phenethyl Ester (CAPE) on oxidative stress and hypoxia‐induced cell damage

Oxidative stress has been implicated in pathogenesis of hypoxia and ischemia/reperfusion (I/R) injury. In previous studies, we have shown that the antioxidant CAPE exerted cardioprotection in an isolated rat heart I (30 min)/R (60 min) injury model. In this study, we further evaluated the effects of CAPE on oxidative stress and hypoxia‐induced cell damage. We evaluated the inhibition of absorbance in the phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide production spectrophotometrically in isolated rat neutrophils via reduction of exogenous cytochrome C. We found that CAPE (0.5 μM–40 μM; n=4–13) reduced phorbol 12‐myristate 13‐acetate induced neutrophil superoxide release dose‐dependently from 29±3% to 95±2%. In a rat hind limb I (30 min)/R (60 min) model, blood hydrogen peroxide levels serves as an indicator of blood oxidative stress and was measured in real‐time via a hydrogen peroxide microsensor (100 μm) inserted into both femoral veins (one served as sham, the other as I/R). We found that in the control group, I/R significantly increased blood hydrogen peroxide levels to 2.1±0.8 μM relative to the sham limb at 60 minutes reperfusion when saline was given at the beginning of reperfusion (n=5). By contrast, CAPE when given at reperfusion (40 μM, n=5) significantly reduced blood hydrogen peroxide levels from 30 min reperfusion and throughout the rest of experiment (p<0.05). The relative change was −0.4±0.3 μM between the I/R and sham limbs at 60 minutes reperfusion. In H9C2 rat myoblast cells, cobalt chloride simulated hypoxia and induced cell damage. Tetrazolium, which can differentiate metabolically active and inactive cells/tissues, was used to measure cell damage by measuring absorbance at 450 nm. We found that incubation of cobalt chloride (800 μM, n=8) for 24 hr induced 55±5% cell death. By contrast, pretreatment of CAPE (0.5 μM–10 μM; n=1–3) for 24 hr dose‐dependently improved cell survival up to 30±9% relative to cobalt chloride treatment. In summary, this study suggests that CAPE attenuates oxidative stress from neutrophils and I/R, which may contribute to improvement in cell survival under hypoxia conditions.Support or Funding InformationThis study was supported by Division of Research and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

Cobalt chloride treatment induces autophagic apoptosis in human glioma cells via a p53-dependent pathway.

Malignant glioma is the most aggressive brain tumor. Hypoxic condition has been explored for killing cancer stem cells or drug-resistant tumor cells. This study investigated the effects of hypoxia on autophagic death and the possible mechanisms. Exposure of human malignant glioma U87-MG cells to cobalt chloride (CoCl2) increased cellular hypoxia-inducible factor-1α levels and concurrently decreased cell viability concentration- and time-dependently. In parallel, treatment with CoCl2 suppressed proliferation of human U87-MG cells. Autophagic cells and levels of LC3-II were concentration- and time-dependently induced in human U87-MG cells after exposure to CoCl2. However, pretreatment with 3-mehyladenine (3-MA) and chloroquine, inhibitors of cell autophagy, caused significant alleviations in CoCl2-induced cell autophagy. In contrast, exposure to rapamycin, an inducer of cell autophagy, synergistically induced hypoxia-induced autophagy of U87-MG cells. Administration of human U87-MG cells with CoCl2 triggered caspase-3 activation and cell apoptosis. Interestingly, pretreatment with 3-MA and chloroquine remarkably suppressed CoCl2-induced caspase-3 activation and cell apoptosis. Application of p53 small interference (si)RNA into human U87-MG cells downregulated levels of this protein and simultaneously lowered hypoxia- and 3-MA-induced alterations in cell autophagy, apoptosis, and death. The hypoxia-induced autophagy and apoptosis of DBTRG-05MG cells were significantly lowered by 3-MA pretreatment and p53 knockdown. Therefore, the present study shows that CoCl2 treatment can induce autophagy of human glioma cells and subsequent autophagic apoptosis via a p53-dependent pathway. Hypoxia-induced autophagic apoptosis may be applied as a therapeutic strategy for treatment of glioma patients.

Degenerative effects of cobalt-chloride treatment on neurons and microglia in a porcine retina organ culture model

In order to understand the pathological processes of retinal diseases, experimental models are necessary. Cobalt, as part of the vitamin B12 complex, is important for neuronal integrity. However, it is known that high quantities of cobalt induce cytotoxic mechanisms via hypoxia mimicry. Therefore, we tested the degenerative effect of cobalt chloride (CoCl2) on neurons and microglia in a porcine retina organ culture model.Organotypic cultures of porcine retinas were cultured and treated with different concentrations of CoCl2 (0, 100, 300 and 500 μM) for 48 h. After four and eight days, CoCl2 induced a strong degeneration of the porcine retina, starting at 300 μM. A loss of retinal ganglion cells (RGCs, Brn-3a), amacrine cells (calretinin) and bipolar cells (PKCα) was observed. Additionally, a high expression of hypoxia induced factor-1a (HIF-1a) and heat shock protein 70 (HSP70) was noted at both points in time. Also, the Caspase 3 protein was activated and P21 expression was induced. However, only at day four, the Bax/Bcl-2 ratio was increased. The effect of CoCl2 was not restricted to neurons. CoCl2 concentrations reduced the microglia amount (Iba1) and activity (Iba1 + Fcγ-Receptor) at both points in time. These damaging effects on microglia were surprising, since CoCl2 causes hypoxia and a pro-inflammatory environment. However, high concentrations of CoCl2 also seem to be toxic to these cells. Similar degenerative mechanisms as in comparison to retinal ischemia animal models were observed. In summary, an effective and reproducible hypoxia-mimicking organotypic model for retinal degeneration was established, which is easy to handle and ready for drug studies.

Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.

Lutein Attenuates Both Apoptosis and Autophagy upon Cobalt (II) Chloride-Induced Hypoxia in Rat Műller Cells.

Retinal ischemia/reperfusion injury is a common feature of various retinal diseases such as glaucoma and diabetic retinopathy. Lutein, a potent anti-oxidant, is used to improve visual function in patients with age-related macular degeneration (AMD). Lutein attenuates apoptosis, oxidative stress and inflammation in animal models of acute retinal ischemia/hypoxia. Here, we further show that lutein improved Műller cell viability and enhanced cell survival upon hypoxia-induced cell death through regulation of intrinsic apoptotic pathway. Moreover, autophagy was activated upon treatment of cobalt (II) chloride, indicating that hypoxic injury not only triggered apoptosis but also autophagy in our in vitro model. Most importantly, we report for the first time that lutein treatment suppressed autophagosome formation after hypoxic insult and lutein administration could inhibit autophagic event after activation of autophagy by a pharmacological approach (rapamycin). Taken together, lutein may have a beneficial role in enhancing glial cell survival after hypoxic injury through regulating both apoptosis and autophagy.

Prolyl hydroxylase 2 (PHD2) inhibition protects human renal epithelial cells and mice kidney from hypoxia injury.

Prolyl hydroxylase domain protein 2 (PHD2) is a key oxygen sensor, setting low steady-state level of hypoxia-inducible factor-α (HIF-α). Here, we showed that treatment of cobalt chloride (CoCl2), a hypoxia mimic, in HK-2 tubular epithelial cells induced PHD2 and HIF-1/2α expression as well as cell apoptosis and autophagy activation. Three methyladenine (3-MA), the autophagy inhibitor, blocked autophagy and protected HK-2 cells from CoCl2. Significantly, siRNA knockdown of PHD2 also protected HK-2 cells from CoCl2, possibly via increasing HIF-1α expression. Reversely, HIF-1α siRNA knockdown almost abolished cytoprotection by PHD2 siRNA in CoCl2-treated HK-2 cells. In vivo, pretreatment with a PHD inhibitor L-mimosine remarkably attenuated mice renal ischemia-reperfusion injuries. Molecularly, L-mimosine inhibited apoptosis and inflammatory responses in injured mice kidneys. Together, our results suggest that PHD2 silence or inhibition protects human renal epithelial cells and mice kidney from hypoxia injuries.

Cobalt Chloride Treatment Used to Ablate the Lateral Line System Also Impairs the Olfactory System in Three Freshwater Fishes.

Fishes use multimodal signals during both inter- and intra-sexual displays to convey information about their sex, reproductive state, and social status. These complex behavioral displays can include visual, auditory, olfactory, tactile, and hydrodynamic signals, and the relative role of each sensory channel in these complex multi-sensory interactions is a common focus of neuroethology. The mechanosensory lateral line system of fishes detects near-body water movements and is implicated in a variety of behaviors including schooling, rheotaxis, social communication, and prey detection. Cobalt chloride is commonly used to chemically ablate lateral line neuromasts, thereby eliminating water-movement cues to test for mechanosensory-mediated behavioral functions. However, cobalt acts as a nonspecific calcium channel antagonist and could potentially disrupt function of all superficially located sensory receptor cells, including those for chemosensing. Here, we examined whether CoCl2 treatment used to ablate the lateral line system also impairs olfaction in three freshwater fishes, the African cichlid fish Astatotilapia burtoni, goldfish Carassius auratus, and the Mexican blind cavefish Astyanax mexicanus. To examine the impact of CoCl2 on the activity of peripheral receptors, we quantified DASPEI fluorescence intensity of the olfactory epithelium from fish exposed to control and CoCl2 solutions. In addition, we examined brain activation in olfactory processing regions of A. burtoni immersed in either control or cobalt solutions. All three species exposed to CoCl2 had decreased DASPEI staining of the olfactory epithelium, and in A. burtoni, cobalt treatment caused reduced neural activation in olfactory processing regions of the brain. To our knowledge this is the first empirical evidence demonstrating that the same CoCl2 treatment used to ablate the lateral line system also impairs olfactory function. These data have important implications for the use of CoCl2 in future research and suggest that previous studies using CoCl2 should be reinterpreted in the context of both impaired mechanoreception and olfaction.

The Promoting Effect of Radiation on Glucose Metabolism in Breast Cancer Cells under the Treatment of Cobalt Chloride.

We aimed to investigate the influence of radiation on hypoxia-treated breast cancers cells and its underlying mechanism. We mimicked the hypoxic response in MCF-7 cells by the treatment of CoCl2. Meanwhile, hypoxic MCF-7 cells induced by CoCl2 or untreated MCF-7 cells were treated with or without radiation, and then treated with or without hypoxia inducible factors-1α (HIF-1α) inhibitor. Subsequently, glucose update and lactate release rate were determined by commercial kits, as well as the expressions of HIF-1α and the glucose metabolic pathway related genes, including fructose biphoshatase 1 (FBP1), glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), and isocitrate dehydrogenase 2 (IDH20) were detected by western blotting and/or RT-PCR. The results showed that glucose uptake rate and lactate release rate were increased in cells under hypoxia and/or radiation condition compared with untreated cells (p<0.05), while the addition of HIF-1α inhibitor decreased these rates in hypoxia + radiation treated cells (p<0.05). In addition, compared with untreated cells, the mRNA and protein levels of HIF-1α were significantly increased under hypoxia and radiation condition (p<0.05), while which decreased after the addition of HIF-1α inhibitor (p<0.05). Similar content changing trends (all p<0.05) were observed in FBP1, IDH2, GLUT1, and LDHA but not HK2. In conclusion, the combination of radiation and hypoxia could promote the glucose metabolism. Furthermore, HIF-1α might inhibit the promoting effect of radiation on glycolysis in hypoxic MCF-7 cells by regulating the glucose metabolic pathway.

Rnd3/RhoE Modulates Hypoxia-Inducible Factor 1α/Vascular Endothelial Growth Factor Signaling by Stabilizing Hypoxia-Inducible Factor 1α and Regulates Responsive Cardiac Angiogenesis.

The insufficiency of compensatory angiogenesis in the heart of patients with hypertension contributes to heart failure transition. The hypoxia-inducible factor 1α-vascular endothelial growth factor (HIF1α-VEGF) signaling cascade controls responsive angiogenesis. One of the challenges in reprograming the insufficient angiogenesis is to achieve a sustainable tissue exposure to the proangiogenic factors, such as HIF1α stabilization. In this study, we identified Rnd3, a small Rho GTPase, as a proangiogenic factor participating in the regulation of the HIF1α-VEGF signaling cascade. Rnd3 physically interacted with and stabilized HIF1α, and consequently promoted VEGFA expression and endothelial cell tube formation. To demonstrate this proangiogenic role of Rnd3 in vivo, we generated Rnd3 knockout mice. Rnd3 haploinsufficient (Rnd3(+/-)) mice were viable, yet developed dilated cardiomyopathy with heart failure after transverse aortic constriction stress. The poststress Rnd3(+/-) hearts showed significantly impaired angiogenesis and decreased HIF1α and VEGFA expression. The angiogenesis defect and heart failure phenotype were partially rescued by cobalt chloride treatment, a HIF1α stabilizer, confirming a critical role of Rnd3 in stress-responsive angiogenesis. Furthermore, we generated Rnd3 transgenic mice and demonstrated that Rnd3 overexpression in heart had a cardioprotective effect through reserved cardiac function and preserved responsive angiogenesis after pressure overload. Finally, we assessed the expression levels of Rnd3 in the human heart and detected significant downregulation of Rnd3 in patients with end-stage heart failure. We concluded that Rnd3 acted as a novel proangiogenic factor involved in cardiac responsive angiogenesis through HIF1α-VEGFA signaling promotion. Rnd3 downregulation observed in patients with heart failure may explain the insufficient compensatory angiogenesis involved in the transition to heart failure.