Androgen deprivation results in time-dependent hypoxia in LNCaP prostate tumours; informed scheduling of the bioreductive drug AQ4N improves treatment response

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment hypoxia is known to drive malignant progression. This study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold chambers (DSF) were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide and vehicle-only treated tumours were re-established in vitro and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2mg/kg/day) decreased tumour oxygenation by 45% within 24h, reaching a nadir of 0.09% oxygen (0.67±0.06 mmHg) by day 7; this persisted until day 14 when it increased up to day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at days 7 and 14 with revascularization occurring by day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50mg/kg; single dose) caused greater tumour growth delay than bicalutamide alone. This study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.

Rod photoreceptor loss in Rho(-/-) mice reduces retinal hypoxia and hypoxia-regulated gene expression

PURPOSE. This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression.

METHODS. Retinas from rhodopsin knockout (Rho(-/-)) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with L-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density.

RESULTS. Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho (-/-) mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho(-/-) or with L-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1 alpha DNA-binding and VEGF mRNA expression in Rho(-/-) retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho(-/-) mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas.

CONCLUSIONS. Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

The effect of hypoxia on propranolol clearance during antegrade and retrograde flow in the isolated perfused rat liver preparation

We investigated, using the single-pass isolated perfused rat liver preparation, whether the centrilobular location of hepatic oxidative drug metabolism could be a contributing factor to the marked sensitivity of drug oxidation to hypoxia. Livers (N = 7) were each perfused for 130 min with 2 micrograms/mL (+)-propranolol, a drug metabolized almost entirely by oxidation in the rat. The direction of flow was reversed after 60 min, the order of flow direction being randomized. Normal oxygenation was used during the first 30 min of antegrade and of retrograde perfusion, but in the second 30 min perfusate was equilibrated with a N2/O2 mixture designed to reduce hepatic oxygen delivery by half. During normal oxygenation there was no significant difference between antegrade and retrograde perfusion in hepatic oxygen delivery and physiological parameters such as oxygen consumption and extraction, perfusion pressure and bile flow. During hypoxia, mean oxygen delivery was slightly lower with retrograde perfusion (retrograde: mean = 2.37 mumol/min/g liver, range = 1.56-3.17; antegrade: mean = 2.90 mumol/min/g liver, range = 1.96-4.08; P = 0.04), but there was no significant difference in physiological parameters within each liver (P > 0.05). Propranolol clearance during normal oxygenation was similar to the perfusion rate (10 mL/min) and was the same for both directions of perfusion (antegrade 9.88 +/- 0.07 mL/min, retrograde 9.88 +/- 0.13 mL/min, P > 0.05). Hypoxia reduced propranolol clearance substantially, but the decrease was significantly greater with antegrade perfusion (5.65 +/- 1.89 mL/min) than with retrograde perfusion (6.76 +/- 1.95 mL/min, P = 0.014). Oxidative drug metabolism is located primarily in the centrilobular zone and sinusoidal oxygen concentration is lowest in the "downstream" zone with both antegrade and retrograde perfusion. These findings suggest that the centrilobular location of propranolol metabolism may influence the effect of hypoxia on propranolol elimination, but is not a major contributor to the marked sensitivity of propranolol elimination to hypoxia antegrade perfusion.

MAD2 downregulation in hypoxia is independent of promoter hypermethylation

Aberrant expression of the MAD2 protein has been linked to chromosomal instability, malignant transformation and chemoresistance. Although reduced MAD2 expression is well recognised in human cancer cell lines, the mechanism(s) underlying its downregulation remain elusive. The objective of this study was to establish the impact of hypoxia on MAD2 expression and to investigate the potential role of aberrant promoter methylation as a possible mechanism of MAD2 downregulation. For this purpose, three ovarian cancer cell lines, displaying differing levels of MAD2, were treated with chromatin modifying drugs, pre and post-hypoxia exposure and a DHPLC analysis of DNA promoter methylation carried out. We show that hypoxia induces downregulation of MAD2 expression, independently of MAD2 promoter methylation. We also show no evidence of MAD2 promoter methylation in breast and prostate cancer cells or in breast cancer clinical material. While our findings provide no evidence for MAD2 promoter methylation, we show a concomitant upregulation of p21 with downregulation of MAD2 in hypoxia. Our in vitro results were also confirmed in an ovarian cancer tissue microarray (TMA), where a reciprocal staining of MAD2 and CAIX was found in 21/60 (35%) of tumours. In summary, MAD2 downregulation may be a crucial mechanism by which hypoxic cells become chemorefractory. This stems from our previous work where we demonstrated that MAD2 downregulation induces cellular senescence, a viable cellular fate, with resultant cellular resistance to paclitaxel. Moreover, MAD2 downregulation could play a central role in the induction of chemoresistance in hypoxia, a key tumour microenvironment associated with chemoresistance.

Renal replacement therapy in multiple myeloma and systemic amyloidosis

Renal failure frequently complicates both multiple myeloma and systemic amyloidosis. Renal replacement therapy (RRT) may be poorly tolerated and its role in such patients is not clearly defined. Of fifty patients (26 males and 24 females) referred to a single centre because of renal failure associated with multiple myeloma or systemic amyloidosis 37 progressed to end-stage renal failure and 30 of these patients received RRT. Nine patients have been treated by CAPD, 13 by haemodialysis, and 8 patients have required both forms of dialysis. Overall one year and two year survival rates were 66% and 57% respectively. The median duration on RRT was 7.5 months (range 1-96 months) with a 51% one year, and a 46% two year survival rate. Of 7 patients with amyloidosis who underwent renal transplantation, 3 died within 6 months of transplantation. Undiagnosed cardiac involvement contributed to this early mortality. We conclude that renal replacement therapy is appropriate for some patients with multiple myeloma and systemic amyloidosis who develop endstage renal failure. Careful assessment and selection of patients is necessary prior to renal transplantation.

The effect of hypobaric hypoxia on misonidazole binding in normal and tumour-bearing mice

The effect of hypobaric hypoxia on the in vivo binding of misonidazole was investigated in normal mice and mice bearing T50/80 or CA NT mammary carcinomas. After the intraperitoneal injection of radiolabelled misonidazole, mice were randomised to breathe either room air or air at 0.5 atmospheres. The distribution of misonidazole in liver, kidney, heart, spleen and tumour tissue, 24 h later, was studied by scintillation counting and by autoradiography. Significantly higher misonidazole binding occurred in the livers (x2.5), kidneys (x2.4), spleens (x2.9) and hearts (x1.8) of hypoxic mice compared to controls. Hypobaric hypoxia was associated with a greater than four-fold increase in misonidazole binding within T50/80 tumours. However, significantly higher binding was not demonstrated within CA NT tumours after exposure of tumour-bearing animals to hypoxic conditions. In autoradiographs of hypoxic liver, labelling was intense in regions near to hepatic veins but sparse in areas surrounding portal tracts. This pattern was striking and consistent. In hypoxic kidney, labelling was most intense over tubular cells, less intense over glomeruli and sparse in the renal medulla. It is likely that the hepatic and renal cortical distributions of misonidazole binding reflect local oxygen gradients.

KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1a (HIF-1a). HIF-1a is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1a. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1a in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1a levels in KNK437-treated cells. This suggested that the absence of HIF-1a in hypoxic cells was not due to the enhanced protein degradation. HIF-1a is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1a mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1a levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P

Local and systemic immune responses in mice to intranasal delivery of peptides representing bovine respiratory syncytial virus epitopes encapsulated in poly (dl-lactide-co-glycolide) microparticles

The potential of a microparticulate vaccine delivery system in eliciting a specific mucosal antibody response in the respiratory tract of mice was evaluated. Two vaccine candidate peptides representing epitopes from the G attachment and F fusion antigens from bovine respiratory syncytial virus (BRSV) were encapsulated into poly(dl- lactide co-glycolide) biodegradable microparticles. The encapsulation process did not denature the entrapped peptides as verified by detection of peptide-specific antibodies in mucosal secretions by ELISA using peptide as antigen. Following intranasal immunisation, the encapsulated peptides induced stronger upper and lower respiratory tract specific-IgA responses, respectively, than the soluble peptide forms. Moreover, a strong peptide-specific cell-mediated immune response was measured in splenocytes in vitro from the mice inoculated with the encapsulated peptides compared to their soluble form alone indicating that migration of primed T cells had taken place from the site of mucosal stimulation in the upper respiratory tract to the spleen. These results act as a foundation for vaccine efficacy studies in large animal BRSV challenge models.

Cultures of Rioting and Anti-Systemic Politics in Southern Europe

The article investigates why, despite similar background conditions, Greece has been the site of frequent, highly visible, fringe, anti-system politics and street riots, while similar phenomena are rare in Spain. Although the article's focal point is the eruption of the December 2008 riots in Athens, it sheds light on the two countries' diverse social reactions to the sovereign debt crisis. Deploying the tool of media framing, it argues that historical legacies and political cultures matter. In the Greek case, the transition to democracy shaped a political ‘culture of sympathy’ towards acts of resistance to the state, a culture that has been institutionalised since the mid-1970s.

Misonidazole binding in murine liver tissue: a marker for cellular hypoxia in vivo

The hepatic microcirculation is believed to cause variable cellular oxygenation within the organ. In this study a marker of cellular hypoxia was used to demonstrate liver oxygen tension gradients in vivo. Covalent binding of misonidazole adducts to cellular macromolecules is enhanced by hypoxia. Autoradiographs of liver from mice treated with radiolabeled misonidazole demonstrated enhanced binding of adducts within hepatocytes surrounding hepatic veins. Livers from both hypoxic and normal mice had characteristic autoradiographic grain patterns reflecting regional oxygen tension variation in vivo. Differential binding of misonidazole adducts formed in hypoxic cells could have an application in studies of liver physiology and biochemistry.