Decrease In HIF-1α Research Articles

Dimethyloxalylglycine prevents bone loss in ovariectomized C57BL/6J mice through enhanced angiogenesis and osteogenesis.

Hypoxia-inducible factor 1-α (HIF-1α) plays a critical role in angiogenesis-osteogenesis coupling during bone development and bone regeneration. Previous studies have shown that 17β-estradiol activates the HIF-1α signaling pathway and that mice with conditional activation of the HIF-1α signaling pathway in osteoblasts are protected from ovariectomy (OVX)-induced bone loss. In addition, it has been shown that hypoxia facilitates the osteogenic differentiation of mesenchymal stem cells (MSCs) and modulates Wnt/β-catenin signaling. Therefore, we hypothesized that activation of the HIF-1α signaling pathway by hypoxia-mimicking agents would prevent bone loss due to estrogen deficiency. In this study, we confirmed the effect of dimethyloxalylglycine (DMOG), a hypoxia-mimicking agent, on the HIF-1α signaling pathway and investigated the effect of DMOG on MSC osteogenic differentiation and the Wnt/β-catenin signaling pathway. We then investigated the effect of DMOG treatment on OVX-induced bone loss. Female C57BL/6J mice were divided into sham, OVX, OVX+L-DMOG (5 mg/kg/day), and OVX+H-DMOG (20 mg/kg/day) groups. At sacrifice, static and dynamic bone histomorphometry were performed with micro computed tomography (micro-CT) and undecalcified sections, respectively. Bone strength was assessed with the three-point bending test, and femur vessels were reconstructed and analyzed by micro-CT. Serum vascular endothelial growth factor (VEGF), osteocalcin, and C-terminal telopeptides of collagen type(CTX) were measured by ELISA. Tartrate-resistant acid phosphatase staining was used to assess osteoclast formation. Alterations in the HIF-1α and Wnt/β-catenin signaling pathways in the bone were detected by western blot. Our results showed that DMOG activated the HIF-1α signaling pathway, which further activated the Wnt/β-catenin signaling pathway and enhanced MSC osteogenic differentiation. The micro-CT results showed that DMOG treatment improved trabecular bone density and restored the bone microarchitecture and blood vessels in OVX mice. Bone strength was also partly restored in DMOG-treated OVX mice. Dynamic bone histomorphometric analysis of the femur metaphysic revealed that DMOG increased the mineralizing surface, mineral apposition rate, and bone formation rate. The serum levels of VEGF and osteocalcin were higher in DMOG-treated OVX mice. However, there were no significant differences in serum CTX or in the number of tartrate-resistant acid phosphatase-stained cells between DMOG-treated OVX mice and OVX mice. Western blot results showed that DMOG administration partly rescued the decrease in HIF-1α and β-catenin expression following ovariectomy. Collectively, these results indicate that DMOG prevents bone loss due to ovariectomy in C57BL/6J mice by enhancing angiogenesis and osteogenesis, which are associated with activated HIF-1α and Wnt/β-catenin signaling pathways.

Abstract A76: Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma

Abstract Aim: Ewing sarcoma (ES) is the second most common and aggressive primary malignant bone tumor in children and adolescents. The hallmarks of the tumor are specific translocations; the most frequent involves the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11. Our aim was to identify microRNAs (miRs) and genes involved in the aggressiveness of ES. Out of the miRs located on these chromosomes, 3 belong to the let-7 family. Methods: We evaluated the expression levels of hsa-let-7a and hsa-let-7b by RQ-PCR in 57 primary ES tumors. We measured the protein levels of p-ERK, HIF-1a and EWS-FLI-1 following transfection with let-7a, let-7b or HIF-1a . The effect of a Ras inhibitor was evaluated in vitro and in vivo. Results: In the group of patients with localized disease, a significant correlation with outcome was observed. Progression free survival (PFS) at ten years for patients with high hsa-let-7b expression was 62% versus 29% for those with low hsa-let-7b expression level (p=0.029). Multivariate Cox regression analysis identified expression of let-7b as an independent prognostic factor in ES, with a 4.4 fold increased risk of relapse (p=0.021). hsa-let-7 is known to be a negative regulator of the RAS oncogene. Positive p-ERK immuno-staining was detected in 31 out of the 45 (69%) tumors analyzed, and a significant inverse correlation was identified between hsa-let-7a and p-ERK (p=0.037). One of the target genes of RAS is Hypoxia inducible factor 1 (HIF-1). We detected a high level of HIF-1α protein in ES cell lines, under normoxic conditions, which did not change following induced hypoxia. This implies for a mechanism that mimics hypoxia and probably contributes to the high frequency of metastasis and thereby aggressiveness of this tumor. Following transfection with hsa-let-7a and hsa-let-7b mimics (overexpression) the levels of RAS, p-ERK, HIF-1α and EWS-FLI-1 proteins were significantly reduced (60%, p=0.0019, 52%, p=0.003, 45%, p=0.0028 and 38%, p=0.006, respectively). Silencing of HIF-1α resulted in a decrease of 61% of EWS-FLI-1, suggesting that HIF-1α regulates EWS-FLI-1, and we were able to show that HIF-1α directly binds EWS promoter. This was accompanied by a reduction of 32% (p=0.009) in cell proliferation rate and by a significant decrease of 36% (p=0.0013) in cell migration ability. Using a RAS inhibitor, Salirasib, we observed a significant decrease of HIF-1α and EWS-FLI-1 protein levels in both in-vitro and in-vivo settings. In addition, a significant inhibition of tumor growth was demonstrated in the treated animals. Conclusions: These results imply that Ras and HIF-1α might contribute to the high frequency of metastasis and thereby aggressiveness of ES tumors. The reduction in tumor burden in a mouse model of ES following Salirasib treatment, demonstrates the therapeutic potential of this RAS inhibitor in ES. Citation Format: Michal Hameiri-Grossman, Adi Porat-Klein, Ian J. Cohen, Shifra Ash, Yoel Kloog, Ronit Haklai, Galit Elad-Sfadia, Avraham Weizman, Isaac Yaniv, Smadar Avigad. Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A76.

Ginsenoside 20(S)-Rg3 targets HIF-1α to block hypoxia-induced epithelial-mesenchymal transition in ovarian cancer cells.

The prognosis of patients with ovarian cancer has remained poor mainly because of aggressive cancer progression. Since epithelial-mesenchymal transition (EMT) is an important mechanism mediating invasion and metastasis of cancer cells, targeting the EMT process with more efficacious and less toxic compounds to inhibit metastasis is of great therapeutic value for the treatment of ovarian cancer. We have found for the first time that the ginsenoside 20(S)-Rg3, a pharmacologically active component of the traditional Chinese herb Panax ginseng, potently blocks hypoxia-induced EMT of ovarian cancer cells in vitro and in vivo. Mechanistic studies confirm the mode of action of 20(S)-Rg3, which reduces the expression of hypoxia-inducible factor 1α (HIF-1α) by activating the ubiquitin-proteasome pathway to promote HIF-1α degradation. A decrease in HIF-1α in turn leads to up-regulation, via transcriptional suppression of Snail, of the epithelial cell-specific marker E-cadherin and down-regulation of the mesenchymal cell-specific marker vimentin under hypoxic conditions. Importantly, 20(S)-Rg3 effectively inhibits EMT in nude mouse xenograft models of ovarian cancer, promising a novel therapeutic agent for anticancer therapy.

Recurrent low-dose chemotherapy to inhibit and oxygenate head and neck tumors.

A lack of strategy to counteract hypoxia (pO₂ < 10-15 mmHg) and technique to repeatedly measure tumor pO₂ has restricted therapeutic optimization. We report the results obtained with an innovative anti-angiogenic strategy of recurrent low-dose (metronomic) chemotherapy to modulate hypoxia and growth of the Head and Neck tumor xenografts.The FaDu tumors were established in the flank of immune deficient mice and EPR oximetry with lithium phthalocyanine crystals was used to follow the temporal changes in tumor pO₂ on treatment with gemcitabine including controls for three weeks. The FaDu tumors were hypoxic with a baseline (pre-treatment) pO₂ of 2-8 mmHg. A transient increase in the tumor pO₂ was evident on day 3 on treatment with a conventional schedule of gemcitabine (150 mg/kg, d1, d8, d15). No significant change in the tumor pO₂ on treatment with metronomic gemcitabine (25 mg/kg on d1, d3, d5 for 3 weeks) was observed. However, tumor pO₂ increased significantly on d15-d18 during treatment with a metronomic schedule of 15 mg/kg gemcitabine (d1, d3, d5 for 3 weeks). A modest decrease in the tumor growth was evident on treatment with conventional gemcitabine. Notably, tumor growth was significantly inhibited by metronomic (25 and 15 mg/kg) gemcitabine treatment. The immunohistochemistry (IHC) analyses of the tumor samples indicate a decrease in HIF-1α and TSP-1 on treatment with metronomic gemcitabine.In conclusion, a significant inhibition of tumor growth on treatment with metronomic gemcitabine was observed; however, the increase in pO₂ was dose dependent. EPR oximetry can be used to follow the temporal changes in tumor pO₂ to identify a therapeutic window on treatment with metronomic chemotherapy for potential combination with radiotherapy.

Abstract 5422: Human GBM-derived brain tumor stem cells resist glycolysis inhibition through Glucose 6 phosphatase: a potential clinical implication in the treatment of recurrent brain tumors.

Abstract Glioblastomas (GBMs) are characterized by a very aggressive behavior and a heterogeneous presentation that includes proliferative, invasive, and necrotic areas. The rapid expansion of GBMs frequently outgrowths the blood supply, originating hypoxic and necrotic regions, which induces metabolic changes in the tumor cells. In a survival adaptation response, tumor cells adjust their glucose metabolism to an anaerobic glycolysis, even in the presence of oxygen. This is known as the Warburg effect and it is enhanced in Brain Tumor Stem Cells (BTSCs), the cellular sub-population considered to be responsible for the tumor origin and recurrence. Glycolysis inhibition can be achieved using glucose analogues like 2-deoxyglucose (2DG). We have previously demonstrated that 2DG treatment of BTSCs induces a decrease in cell proliferation and an increase in cell death and neuronal differentiation. Nevertheless, some BTSC cells are able to survive glycolysis inhibition and recover their aggressive phenotype which might be related to brain tumor recurrence. Here we studied the tumorigenic behavior of human GBM-derived primary BTSCs after recovery from glycolysis inhibition. We observed that cells which were pretreated with 2DG and subsequently allowed to recover for 72h in glucose-containing media, exhibited a more aggressive phenotype; including an increased migration, invasion, and proliferation capability; as well as MMP2 and nestin overexpression. Some of these features recapitulate what occurs in recurrent brain tumors. In studying the potential molecular mechanisms responsible for this behavior, we observed an increased expression of glucose-6-phosphatase isoform α (G6PC) in our primary GBM cells when compared to normal brain. Moreover, 2DG treatment induced an increase in the expression of this isoform. G6PC is a key glucose homeostasis enzyme not previously reported in brain tissue. In addition, when G6PC was inhibited, either pharmacologically (with chlorogenic acid) or genetically (with shRNA), we observed that the recovery capacity of GBM cells was significantly reduced. This was evident by a decrease in their proliferation, migration, and invasion ability, as well as by a decrease in HIF1α, pSTAT3 protein, MMP2 gene expression. In summary, we report for the first time the expression of G6PC in GBM cells. This specific isoform is not expressed in normal brain parenchyma, which makes it a very attractive target for anticancer therapy. Our results suggest a role of G6PC in promoting a mechanism of GBM cell recovery from glycolysis inhibition. By unraveling these mechanisms we describe an important therapeutic target that could impact the recurrence ability of brain tumors. Citation Format: Sara Abbadi, Hugo Guerrero-Cazares, Ameer Abutaleb, Chris L. Smith, William Ruff, Jennifer Schiller, Andre Levchenko, Alfredo Quinones-Hinojosa. Human GBM-derived brain tumor stem cells resist glycolysis inhibition through Glucose 6 phosphatase: a potential clinical implication in the treatment of recurrent brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5422. doi:10.1158/1538-7445.AM2013-5422

Gambogic acid inhibits angiogenesis through inhibiting PHD2–VHL–HIF-1α pathway

Our previous studies revealed that gambogic acid (GA), the major active ingredient of gamboge, possessed antiangiogenic activities. In this study, we further explored the mechanism of inhibition effects of GA in tumor angiogenesis. The results of luciferase, RT-PCR, and ELISA assays indicated that GA significantly decreased transcription activation, mRNA expression, and secretion of VEGF in hypoxia. We detected that GA had no effect on mRNA level of HIF-1α which targets VEGF gene, but the increase of HIF-1α protein expression in hypoxia was repressed by GA, which can be reversed by proteasomal inhibitor MG132 and siRNA of VHL. But GA exhibited no effect on expression of VHL both in normoxia and hypoxia. HIF prolyl hydroxylases (PHD enzymes) act as oxygen sensors regulating HIF, and hence angiogenesis. Our results showed that GA potentially enhanced level of PHD2, the most important HIF hydroxylase, and showed no effect on PHD1 and PHD3. Transient transfection of siRNA of PHD2 could eliminate GA-induced VEGF secretion increase. Growth of HepG2 xenografts in BALB/cA nude mice was inhibited by GA and angiogenesis was repressed significantly in tumor xenografts by immunohistochemical staining of CD-31, a vascular endothelial marker, accompanied with decrease of HIF-1α and increase of PHD2 expression in tissue extracts. This work provides the demonstration that GA shows anti-angiogenic effects via inhibiting PHD2–VHL–HIF-1α pathway.

Roles and Mechanism of miR-199a and miR-125b in Tumor Angiogenesis

MicroRNAs (miRNAs) have been shown to be involved in different aspects of cancer biology including tumor angiogenesis. In this study, we identified that two miRNAs, miR-199a and miR-125b were downregulated in ovarian cancer tissues and cell lines. Overexpression of miR-199a and miR-125b inhibited tumor-induced angiogenesis associated with the decrease of HIF-1α and VEGF expression in ovarian cancer cells. Moreover, the levels of miR-199a and miR-125b were negatively correlated with VEGF mRNA levels in ovarian tissues. We further showed that direct targets of miR-199a and miR-125b HER2 and HER3 were functionally relevant. Forced expression of HER2 and HER3 rescued miR-199a- and miR-125b-inhibiting angiogenesis responses and Akt/p70S6K1/HIF-1α pathway. This study provides a rationale for new therapeutic approach to suppress tumor angiogenesis using miR-199a, miR-125b, or their mimics for ovarian cancer treatment in the future.

Phenazine Methosulfate Decreases HIF-1α Accumulation during the Exposure of Cells to Hypoxia

In HEK293 cells, exposure to various NAD(P)H oxidants, including phenazine methosulfate (PMS), that non-enzymatically oxidize intracellular NAD(P)H to NAD(P), decreased hypoxia-induced hypoxia-inducible factor 1 (HIF-1α) accumulation. RT-PCR and cycloheximide inhibition experiments indicated that PMS-induced HIF-1α decrease is involved in post-translational degradation during hypoxia. The decrease in HIF-1α caused by PMS was not eliminated by proteasome inhibitor MG132. Moreover, the increase in HIF-1α induced by exposure to MG132 alone in normoxia was diminished by PMS. In contrast, calpastatin peptide, a calpain inhibitor, fully prevented PMS-induced reduction in HIF-1α in hypoxic cells. These data suggest that the decreased stability of HIF-1α induced by PMS is due to the activation by PMS of a protein degradation system that is independent of the ubiquitin-proteasome pathway.

Perfluorocarbon Emulsions Prevent Hypoxia of Pancreatic β-Cells

As oxygen carriers, perfluorocarbon emulsions might be useful to decrease hypoxia of pancreatic islets before transplantation. However, their hydrophobicity prevents their homogenisation in culture medium. To increase the surface of contact between islets and Perfluorooctyl bromide (PFOB), and consequently oxygen delivery, we tested effect of a PFOB emulsion in culture medium on β-cell lines and rat pancreatic islets. RINm5F β-cell line or pancreatic rat islets were incubated for 3 days in the presence of PFOB emulsion in media (3.5% w/v). Preoxygenation of the medium was performed before culture. Cell viability was assessed by apoptotic markers (Bax and Bcl-2) and by staining (fluoresceine diacetate and propidium iodide). β-Cell functionality was determined by insulin release during a glucose stimulation test and. Hypoxia markers, HIF-1α and VEGF, were studied at days 1 and 3 using RT-PCR, Western blotting, and ELISA. PFOB emulsions preserved viability and functionality of RINm5F cells with a decrease of HIF-1α and VEGF expression. Islets viability was preserved during 3 days of culture. Secretion of VEGF was higher in untreated control (0.09 ± 0.041 μg VEGF/mg total protein) than in PFOB emulsion incubated islets (0.02 ± 0.19 μg VEGF/mg total protein, n = 4, p < 0.05) at day 1. At day 3, VEGF secretion was increased as compared to day 1 in control (0.23 ± 0.04 μg VEGF/mg total protein) but it was imbalance by the presence of PFOB emulsion (0.09 ± 0.03 μg VEGF/mg total protein, n = 5, p < 0.05). While insulin secretion was maintained in response to a glucose stimulation test until day 3 when islets were incubated in the presence of PFOB emulsion preoxygenated (0.81 ± 0.16 at day 1 vs. 0.75 ± 0.24 at day 3), the ability to secrete insulin in the presence of high glucose concentration was lost in islets controls (0.51 ± 0.18 at day 1 vs. 0.21 ± 0.13 at day 3). Atmospheric oxygen delivery by PFOB emulsion might be sufficient to decrease islets hypoxia. However, to improve islets functionality, overoxygenation is needed. Finally, maintenance of islet viability and functionality for several days after isolation could improve the outcome of islets transplantation.

Pseudolaric acid B-driven phosphorylation of c-Jun impairs its role in stabilizing HIF-1alpha: a novel function-converter model

We have recently discovered that c-Jun executes a non-transcriptional function to stabilize hypoxia inducible factor 1α (HIF-1α) and that pseudolaric acid B (PAB) accelerates HIF-1α degradation and phosphorylates c-Jun at Ser63/73. In this study, PAB was used as a probe to investigate whether and how the Ser63/73 phosphorylation of c-Jun regulates its functions. The PAB-induced reduction of HIF-1α protein was rescued through supplying additional non-phosphorylated c-Jun. However, c-Jun siRNA, which reduced both the PAB-driven phosphorylated c-Jun and the total c-Jun protein, did not prevent the PAB-induced decrease in HIF-1α. HIF-1α was revealed to be co-immunoprecipitated only with the non-phosphorylated c-Jun. PAB increased the phosphorylated c-Jun while reducing the non-phosphorylated c-Jun at Ser63/73, which impaired its function in stabilizing HIF-1α. Consequently, PAB led to the degradation of HIF-1α, thus resulting in the decreased HIF-1α-dependent expression of mdr-1 and VEGF. We accordingly propose a function-converter model of c-Jun: the Ser63/73 phosphorylation serves as a function converter to convert c-Jun from its non-transcriptional function to its transcriptional function.

Hypoxic preconditioning involves system Xc − regulation in mouse neural stem cells

In animals, hypoxic preconditioning has been used as a form of neuroprotection. The exact mechanism involved in neuroprotective hypoxic preconditioning has not been described, yet could be valuable for possible neuroprotective strategies. The overexpression of the cystine-glutamate exchanger, system Xc −, has been demonstrated as being neuroprotective (Shih, Erb et al. 2006). Here, using immunohistochemistry, we demonstrate that C57BL/6 mice exposed to hypoxia showed an increase in system Xc − expression, with the highest level of intensity in the hippocampus. Western Blot analysis also showed an almost 2-fold increase in system Xc − protein in hypoxia-exposed versus control mice. The mRNA for the regulatory subunit of system Xc −, xCT, and the xCT/actin ratio were also increased under hypoxic conditions. Experiments using hypoxia-inducible factor (HIF-1α) siRNA showed a statistically significant decrease in HIF-1α and system Xc − expression. Under hypoxic conditions, system Xc − activity, as determined by cystine uptake, increased 2-fold. Importantly, hypoxic preconditioning was attenuated in neural stem cells by pharmacological inhibition of system Xc − activity with S4-carboxyphenylglycine. These data provide the first evidence of hypoxic regulation of the cystine glutamate exchanger system Xc −.

Antitumor agent PX-12 inhibits HIF-1α protein levels through an Nrf2/PMF-1-mediated increase in spermidine/spermine acetyl transferase

Thioredoxin-1 (Trx-1) redox signaling regulates multiple aspects of cell growth and survival, and elevated tumor levels of Trx-1 have been associated with decreased patient survival. PX-12, an inhibitor of Trx-1 currently in clinical development, has been found to decrease tumor levels of the HIF-1α transcription factor. SSAT1 has been reported to bind to HIF-1α and RACK1, resulting in oxygen-independent HIF-1 ubiquitination and degradation. SSAT2, a related protein, stabilizes the interaction of the VHL protein and elongin C with HIF-1 leading to oxygen-dependent HIF-1α ubiquitination and degradation. We investigated the effects of PX-12 and Trx-1 on SSAT1, SSAT2, and inhibition of HIF-1α. A panel of cell lines was treated with PX-12 to investigate its effects on SSAT1 and SSAT2 expression, and on HIF-1α protein levels. We also evaluated the regulation of SSAT1 through the Nrf2 and PMF-1, two trans-acting transcription factors. We found that PX-12 increased nuclear Nrf2 activity and antioxidant response element binding. PX-12 also increased the expression of SSAT1 but not SSAT2 in a PMF-1-dependent manner that was independent of Trx-1. Inhibition of Nrf2 or PMF-1 prevented the increase in SSAT1 caused by PX-12. The results show that PX-12, acting independently of Trx-1, increases nuclear Nrf2, which interacts with PMF-1 to increase the expression of SSAT1. The degradation of HIF-1α that results from binding with SSAT1 may explain the decrease in HIF-1α caused by PX-12 and could contribute to the antitumor activity of PX-12.

Neuroprotection of deferoxamine on rotenone-induced injury via accumulation of HIF-1α and induction of autophagy in SH-SY5Y cells

Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that activates the transcription of genes and is responsible for progression of cell survival and proliferation. The synthesis of HIF-1α can be stimulated via oxygen (O 2)-independent mechanisms; whereas, the degradation of HIF-1α is regulated via Fe 2+ and/or O 2-dependent enzyme prolyl hydroxylase (PHD). Aberrant iron accumulation, mitochondrial dysfunction and impairment of protein degradation system, such as autophagy, have been implicated in the pathogenesis of Parkinson's disease, among which, iron and mitochondrial dysfunction may enhance the enzyme activity of prolyl hydroxylase and cause the decrease of HIF-1α. Recent reports have indicated that HIF-1α may induce autophagy under hypoxic condition. Considering the metabolic characteristics of HIF-1α under the pathogenesis of Parkinson's disease, we speculated that compounds that might stabilize HIF-1α could prevent neuronal injury caused by excessive iron or mitochondrial injury under normoxic condition. Deferoxamine is one of iron chelators that may accumulate HIF-1α due to the decreased degradation of HIF-1α via inhibition of prolyl hydroxylase activity. In this study, we showed that the protein level of HIF-1α was decreased in rotenone or MPP +-treated SH-SY5Y cell models of Parkinson's disease. We demonstrated that deferoxamine caused accumulation of HIF-1α accompanied by the enhancement of autophagy in SH-SY5Y cells. When HIF-1α gene was inhibited, deferoxamine-induced autophagy was suppressed accordingly, indicating that deferoxamine-induced autophagy was dependent on the expression of HIF-1α. Our results also showed that deferoxamine attenuated rotenone-induced apoptosis, which was blocked when HIF-1α or autophagy related gene Beclin 1 was suppressed. In summary, the present study indicated that the level of HIF-1α was decreased under the situation when mitochondrial complex I was inhibited, and the neuroprotective role of deferoxamine in rotenone-induced apoptosis could be partially explained by its effects on the accumulation of HIF-1α and HIF-1α-mediated induction of autophagy.

Early and late administration of MnTE-2-PyP 5+ in mitigation and treatment of radiation-induced lung damage

Chronic production of reactive oxygen and nitrogen species is an underlying mechanism of irradiation (IR)-induced lung injury. The purpose of this study was to determine the optimum time of delivery of an antioxidant and redox-modulating Mn porphyrin, MnTE-2-PyP 5+, to mitigate and/or treat IR-induced lung damage. Female Fischer-344 rats were irradiated to their right hemithorax (28 Gy). Irradiated animals were treated with PBS or MnTE-2-PyP 5+ (6 mg /kg/24 h) delivered for 2 weeks by sc-implanted osmotic pumps (beginning after 2, 6, 12, 24, or 72 h or 8 weeks). Animals were sacrificed 10 weeks post-IR. Endpoints were body weight, breathing frequency, histopathology, and immunohistochemistry (8-OHdG, ED-1, TGF-β, HIF-1α, VEGF A). A significant radioprotective effect on functional injury, measured by breathing frequency, was observed for all animals treated with MnTE-2-PyP 5+. Treatment with MnTE-2-PyP 5+ starting 2, 6, and 12 h but not after 24 or 72 h resulted in a significant decrease in immunostaining for 8-OHdG, HIF-1α, TGF-β, and VEGF A. A significant decrease in HIF-1α, TGF-β, and VEGF A, as well as an overall reduction in lung damage (histopathology), was observed in animals beginning treatment at the time of fully developed lung injury (8 weeks post-IR). The catalytic manganese porphyrin antioxidant and modulator of redox-based signaling pathways MnTE-2-PyP 5+ mitigates radiation-induced lung injury when given within the first 12 h after IR. More importantly, this is the first study to demonstrate that MnTE-2-PyP 5+ can reverse overall lung damage when started at the time of established lung injury 8 weeks post-IR. The radioprotective effects are presumably mediated through its ability both to suppress oxidative stress and to decrease activation of key transcription factors and proangiogenic and profibrogenic cytokines.

Different Expressions of HIF-1α, Bcl-2 and Baxin DU145 Prostate Cancer Cells Transplanted in Nude Mouse between X-Ray and Neutron Irradiation

†Purpose: To investigate the radiobiologic effects of neutron and X-ray irradiation on DU-145 prostate carcinoma cells by identifying the differences of HIF-1α expression and apoptosis. Materials and Methods: Nude mice were injected with the human prostate cancer cell line, DU-145, and then irradiated with 2 Gy and 10 Gy X-rays, or 0.6 Gy and 3.3 Gy neutrons, respectively. The mice were sacrificed at 24 hours and 120 hours after irradiation. The expression levels of HIF-1α, Bcl-2 and Bax were compared with immunohistochemical staining and western blotting. The apoptotic indexes were compared with the Terminal deoxynucleotidyl biotin-dUTP nick and labeling (TUNEL) assay. Results : At day 1, HIF-1α and Bcl-2 expression decreased, while Bax expression and the number of TUNEL positive cells increased in neutron irradiated groups for the control and X-ray irradiated groups. The Bcl-2/Bax ratio was significantly lower in the neutron irradiated groups regardless of dose (p=0.001). The same pattern of the differences in the expressions of the HIF-1α , Bcl-2, Bax, Bcl-2/Bax ratio, and apoptotic indexes were indentified at day 5. HIF-1α expression was related with Bcl-2 (p=0.031), Bax (p=0.037) expressions and the apoptotic indexes (p=0.016) at day 5. Conclusion : Neutron irradiation showed a decrease in HIF-1α, Bcl-2 expression, and Bcl-2/Bax ratio, but increased Bax expression regardless of dose. This study suggests that the differences radiobiological responses between photon and neutron irradiation may be related to different HIF-1α expression and subsequent apoptotic protein expressions.

Cerebral adaptations to chronic anemia in a model of erythropoietin-deficient mice exposed to hypoxia

Anemia and hypoxia in rats result in an increase in factors potentially involved in cerebral angiogenesis. Therefore, the aim of this study was to assess the effect of chronic anemia and/or chronic hypoxia on cerebral cellular responses and angiogenesis in wild-type and anemic transgenic mice. These studies were done in erythropoietin-deficient mice (Epo-TAg(h)) in normoxia and following acute (one day) and chronic (14 days, barometric pressure = 420 mmHg) hypoxia. In normoxia, Epo-TAg(h) mice showed an increase in transcript and protein levels of hypoxia-inducible factor 1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), erythropoietin receptors (EpoR), phospho-STAT-5/STAT-5 ratio, and neuronal neuronal nitric oxide synthase (nNOS) along with a higher cerebral capillary density. In wild-type (WT) mice, acute hypoxia increased all of the studied factors, while in chronic hypoxia, HIF-1alpha, EpoR, phospho-STAT-5/STAT-5 ratio, nNOS, and inducible NOS remained elevated, with an increase in capillary density. Surprisingly, in Epo-TAg(h) mice, chronic hypoxia did not further increase any factor except the nitric oxide metabolites, while HIF-1alpha, EpoR, and phospho-STAT-5/STAT-5 ratio were reduced. Normoxic Epo-TAg(h) mice developed cerebral angiogenesis through the HIF-1alpha/VEGF pathway. In acute hypoxia, WT mice up-regulated all of the studied factors, including cerebral NO. Polycythemia and angiogenesis occurred with acclimatization to chronic hypoxia only in WT mice. In Epo-TAg(h), the decrease in HIF-1alpha, VEGF proteins, and phospho-STAT-5 ratio in chronic hypoxia suggest that neuroprotective and angiogenesis pathways are altered.

P53 regulates renal expression of HIF-1α and pVHL under physiological conditions and after ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) is a common cause of acute kidney injury (AKI) and is characterized by widespread tubular and microvascular damage. The tumor suppressor p53 is upregulated after IRI and contributes to renal injury in part by promoting apoptosis. Acute, short-term inhibition of p53 with pifithrin-alpha conveys significant protection after IRI. The hypoxia-inducible factor-1 (HIF-1) pathway is also activated after IRI and has opposing effects to those promoted by p53. The balance between the HIF-1 and p53 responses can determine the outcome of IRI. In this manuscript, we investigate whether p53 regulates the HIF-1 pathway in a rodent model of IRI. HIF-1alpha is principally expressed in the collecting tubules (CT) and thick ascending limbs (TAL) under physiological conditions. However, inhibition of p53 with pifithrin-alpha increases the faint expression of HIF-1alpha in proximal tubules (PT) under physiological conditions. Twenty-four hours after IRI, HIF-1alpha expression is decreased in both CT and TAL. HIF-1alpha expression in the PT is not significantly altered after IRI. Acute inhibition of p53 significantly increases HIF-1alpha expression in the PT after IRI. Additionally, pifithrin-alpha prevents the IRI-induced decrease in HIF-1alpha in the CT and TAL. Parallel changes are observed in the HIF-1alpha transcriptive target, carbonic anhydrase-9. Finally, inhibition of p53 prevents the dramatic changes in Von Hippel-Lindau protein morphology and expression after IRI. We conclude that activation of p53 after IRI mitigates the concomitant activation of the protective HIF-1 pathway. Modulating the interactions between the p53 and HIF-1 pathway can provide novel options in the treatment of AKI.

Complex Interactions Between Hypoxia Inducible Factors, Insulin-Like Growth Factor-II and Oxygen in Early Murine Trophoblasts

The human first trimester placenta experiences a low oxygen environment. The hypoxia inducible factors (HIFs) mediate the response to low oxygen, inducing genes such as insulin-like growth factor (IGF)-II. Interestingly, IGF-II has been shown to promote placental growth and function. Currently, the interaction between oxygen, IGF-II and HIFs in the regulation of trophoblast behaviour are unclear. Murine implantation sites from days 5.5–10.5 were collected for immunohistochemical analyses. Use of the hypoxia marker pimonidazole indicated that the early mouse implantation site is exposed to low oxygen levels similar to those seen in the early human placenta. HIF-1α protein immunostaining was also observed in the implantation site. Culturing murine ectoplacental cones in decreasing oxygen concentrations (20%, 5% and 1% O 2), either with or without the addition of IGF-II, induced complex responses by trophoblasts in terms of their migration and differentiation. Following 3 days exposure to low oxygen there was reduced EPC outgrowth, reduced Igf2 and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. In addition, Hif-1α mRNA levels were decreased, whilst Hif-2α mRNA was unchanged. This decrease in Hif-1α may be due to the observed increase in antisense (as) Hif-1α mRNA levels in 1% cultures. Furthermore, expression of Hif-2α and the HIF target genes: asHif-1α, Vegf and Slc2a1 were reduced under low oxygen with the addition of IGF-II. In conclusion, Hif-1α and Hif-2α are differentially regulated by oxygen and IGF-II in cultured trophoblast cells and asHif-1α may mediate the response to prolonged hypoxia in murine trophoblasts.

Regulation of hypoxia-inducible factor-1alpha by nitric oxide through mitochondria-dependent and -independent pathways.

Nitric oxide (NO) has been reported both to promote and to inhibit the activity of the transcription factor hypoxia-inducible factor-1 (HIF-1). In order to avoid the pitfalls associated with the use of NO donors, we have developed a human cell line (Tet-iNOS 293) that expresses the inducible NO synthase (iNOS) under the control of a tetracycline-inducible promoter. Using this system to generate finely controlled amounts of NO, we have demonstrated that the stability of the alpha-subunit of HIF-1 is regulated by NO through two separate mechanisms, only one of which is dependent on a functional respiratory chain. HIF-1alpha is unstable in cells maintained at 21% O(2), but is progressively stabilized as the O(2) concentration decreases, resulting in augmented HIF-1 DNA-binding activity. High concentrations of NO (>1 microM) stabilize HIF-1alpha at all O(2) concentrations tested. This effect does not involve the respiratory chain, since it is preserved in cells lacking functional mitochondria (rho(0)-cells) and is not reproduced by other inhibitors of the cytochrome c oxidase. By contrast, lower concentrations of NO (<400 nM) cause a rapid decrease in HIF-1alpha stabilized by exposure of the cells to 3% O(2). This effect of NO is dependent on the inhibition of mitochondrial respiration, since it is mimicked by other inhibitors of mitochondrial respiration, including those not acting at cytochrome c oxidase. We suggest that, although stabilization of HIF-1alpha by high concentrations of NO might have implications in pathophysiological processes, the inhibitory effect of lower NO concentrations is likely to be of physiological relevance.