Effects of 56-Kilogram Kettlebell Swing Endpoint on Total Body Mechanics.

ABSTRACT: Ross, JA, Keogh, JWL, Lorenzen, C, and Lake, J. Effects of 56-kilogram kettlebell swing endpoint on total body mechanics. J Strength Cond Res 37(12): 2333-2338, 2023-In the past 2 decades, kettlebell training popularity has increased and the range of kettlebells has expanded to 2-92 kg. However, commercially available kettlebells above 56 kg have 12 kg increments, so alternatives to load are required to provide a suitable way of increasing training stimulus until the athlete is strong enough for a load increase. This study aimed to determine the differences in the force plate-derived biomechanical characteristics of heavy kettlebell swings to 3 different heights, as altering the height of the kettlebell swing may be one way to alter the mechanical demands with the same kettlebell mass. Fifteen resistance-trained men performed the kettlebell swing to acromion process height (AH), acromion process height + 20% (AH+20), and acromion process height-20% (AH-20). Swing height significantly affected vertical braking and propulsion phase net impulse and displacement, vertical braking velocity, and braking and propulsion work but not braking and propulsion duration. Altering kettlebell swing trajectory endpoint is a method to regress/progress the demands of kettlebell training. Coaches may be able to alter the acute demands and likely chronic adaptations of kettlebell training by prescribing different swing heights and kettlebell masses to their athletes.

The influence of hypoxia on the prostate cancer proteome.

Prostate cancer accounts for around 15% of male deaths in Western Europe and is the second leading cause of cancer death in men after lung cancer. Mounting evidence suggests that prostate cancer deposits exist within a hypoxic environment and this contributes to radio-resistance thus hampering one of the major therapies for this cancer. Recent reports have shown that nitric oxide (NO) donating non-steroidal anti-inflammatory drugs (NSAIDs) reduced tumour hypoxia as well as maintaining a radio-sensitising/therapeutic effect on prostate cancer cells. The aim of this study was to evaluate the impact of hypoxia on the proteome of the prostate and to establish whether NO-NSAID treatment reverted the protein profiles back to their normoxic status. To this end an established hormone insensitive prostate cancer cell line, PC-3, was cultured under hypoxic and normoxic conditions before and following exposure to NO-NSAID in combination with selected other common prostate cancer treatment types. The extracted proteins were analysed by ion mobility-assisted data independent acquisition mass spectrometry (MS), combined with multivariate statistical analyses, to measure hypoxia-induced alterations in the proteome of these cells. The analyses demonstrated that under hypoxic conditions there were well-defined, significantly regulated/differentially expressed proteins primarily involved with structural and binding processes including, for example, TUBB4A, CIRP and PLOD1. Additionally, the exposure of hypoxic cells to NSAID and NO-NSAID agents, resulted in some of these proteins being differentially expressed; for example, both PCNA and HNRNPA1L were down-regulated, corresponding with disruption in the nucleocytoplasmic shuttling process.

Hamstring Myoelectrical Activity During Three Different Kettlebell Swing Exercises.

Del Monte, MJ, Opar, DA, Timmins, RG, Ross, JA, Keogh, JWL, and Lorenzen, C. Hamstring myoelectrical activity during three different kettlebell swing exercises. J Strength Cond Res 34(7): 1953-1958, 2020-Kettlebell exercises have become an increasingly popular form of resistance training and component of lower-body rehabilitative training programs, despite a lack of scientific literature illustrating internal mechanisms and effectiveness of these approaches. Participants (n = 14) performed 3 different styles of kettlebell swings (hip hinge, squat, and double knee extension) and were assessed for medial hamstrings (MHs) and biceps femoris (BF) myoelectrical activity through surface electromyography (sEMG). Bipolar pregelled Ag/AgCl sEMG electrodes (10 mm diameter, 20 mm interelectrode distance) were placed on the participant's dominant limb after correct skin preparation. There was a main effect for swing type (p = 0.004), where the hip hinge swing elicited a greater overall MH and BF sEMG in comparison with the squat swing (mean difference = 3.92; 95% confidence interval [CI] = 1.53-6.32; p = 0.002) and the double knee extension swing (mean difference = 5.32; 95% CI = 0.80-9.83; p = 0.020). Across all swing types, normalized percentage of MH sEMG was significantly higher compared with the BF (mean difference = 9.93; 95% CI = 1.67-18.19; p = 0.022). The hip hinge kettlebell swing produced the greatest amount of hamstring sEMG for the 3 styles of kettlebell swings assessed. These findings have implications for the application of kettlebell swing exercises in strength and conditioning, injury prevention, and rehabilitation programs.

Stem cell-derived models to improve mechanistic understanding and prediction of human drug-induced liver injury.

Current preclinical drug testing does not predict some forms of adverse drug reactions in humans. Efforts at improving predictability of drug-induced tissue injury in humans include using stem cell technology to generate human cells for screening for adverse effects of drugs in humans. The advent of induced pluripotent stem cells means that it may ultimately be possible to develop personalized toxicology to determine interindividual susceptibility to adverse drug reactions. However, the complexity of idiosyncratic drug-induced liver injury means that no current single-cell model, whether of primary liver tissue origin, from liver cell lines, or derived from stem cells, adequately emulates what is believed to occur during human drug-induced liver injury. Nevertheless, a single-cell model of a human hepatocyte which emulates key features of a hepatocyte is likely to be valuable in assessing potential chemical risk; furthermore, understanding how to generate a relevant hepatocyte will also be critical to efforts to build complex multicellular models of the liver. Currently, hepatocyte-like cells differentiated from stem cells still fall short of recapitulating the full mature hepatocellular phenotype. Therefore, we convened a number of experts from the areas of preclinical and clinical hepatotoxicity and safety assessment, from industry, academia, and regulatory bodies, to specifically explore the application of stem cells in hepatotoxicity safety assessment and to make recommendations for the way forward. In this short review, we particularly discuss the importance of benchmarking stem cell-derived hepatocyte-like cells to their terminally differentiated human counterparts using defined phenotyping, to make sure the cells are relevant and comparable between labs, and outline why this process is essential before the cells are introduced into chemical safety assessment. (Hepatology 2017;65:710-721).

Efficient episomal reprogramming of blood mononuclear cells and differentiation to hepatocytes with functional drug metabolism.

The possibility of converting cells from blood mononuclear cells (MNC) to liver cells provides promising opportunities for the study of diseases and the assessment of new drugs. However, clinical applications have to meet GMP requirements and the methods for generating induced pluripotent cells (iPCs) have to avoid insertional mutagenesis, a possibility when using viral vehicles for the delivery of reprogramming factors. We have developed an efficient non-integration method for reprogramming fresh or frozen blood MNC, maintained in an optimised cytokine cocktail, to generate induced pluripotent cells. Using electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) in a feeder-free system, without any requirement for small molecules, we achieved a reprogramming efficiency of up to 0.033% (65 colonies from 2×10(5) seeded MNC). Applying the same cytokine cocktail and reprogramming methods to cord blood or fetal liver-derived CD34(+) cells, we obtained 148 iPS colonies from 10(5) seeding cells (0.148%). The iPS cell lines we generated maintained typical characteristics of pluripotent cells and could be successfully differentiated into hepatocytes with drug metabolic function.

SUMOylation of HNF4α regulates protein stability and hepatocyte function.

The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4α. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4α in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types.

Synthesis and biological evaluation of nitric oxide-donating analogues of sulindac for prostate cancer treatment.

A series of analogues of the non-steroidal anti-inflammatory drug (NSAID) sulindac 1 were synthesised tethered to nitric oxide (NO) donating functional groups. Sulindac shows antiproliterative effects against immortal PC3 cell lines. It was previously demonstrated that the effect can be enhanced when tethered to NO releasing groups such as nitrate esters, furoxans and sydnonimines. To explore this approach further, a total of fifty-six sulindac-NO analogues were prepared and they were evaluated as NO-releasing cytotoxic agents against prostate cancer (PCa) cell lines. Compounds 1k and 1n exhibited significant cytotoxic with IC50 values of 6.1±4.1 and 12.1±3.2µM, respectively, coupled with observed nitric oxide release.

Heat-shock proteins and acute ischaemic kidney injury.

The incidence of acute kidney injury due to ischaemia-reperfusion injury (IRI) is rising but effective treatments and preventative approaches are currently lacking. IRI is also an inevitable consequence of kidney transplantation and significantly contributes to delayed graft function. Heat-shock proteins (Hsps) are highly conserved and ubiquitously expressed molecular chaperones that help maintain and restore normal cellular function in the kidney following IRI. Hsp70 is one of the most frequently studied Hsps because of potential cytoprotective properties and attractiveness as a therapeutic target. However, the protective properties of Hsp70 in renal IRI are not fully understood and putative modes of protection include correction of protein conformation, cytoskeletal stabilisation, anti-inflammatory effects, requirement in autophagy, anti-apoptotic properties, influence over macrophage phenotype and stimulation of regulatory T cells. Significant clinical interest has been generated about the possibility of applying pharmacological agents to induce Hsp70 and prevent renal IRI, but prior to this, an increased mechanistic understanding of the protective nature of Hsp70 is needed. In particular, further investigation of Hsp expression on inflammatory cell behaviour is required as this could lead to the development of new therapeutic strategies for enhancing recovery following renal IRI and broaden the range of these therapies to a wider group of patients.

Liver tissue engineering and cell sources: issues and challenges.

Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.

Oxidative stress and docosahexaenoic acid injury lead to increased necroptosis and ferroptosis in retinal pigment epithelium.

Age-related macular degeneration (AMD) is a complex disease caused by different genetic and environmental risk factors leading to loss of cells in the central part of the retina. Oxidative stress appears to be an important environmental risk factor that contributes to both the initiation and progression of AMD. Retinal pigment epithelium (RPE) plays an important role in regulating oxidative stress in the retina and is one of the main retinal cell types affected in AMD. A main function of RPE is to phagocytose photoreceptor outer segments (POS) which are rich in the polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA), making this cell type potentially more susceptible to oxidative stress-induced lipid peroxidation which can lead to cell death. RPE is known to undergo necrotic cell death in response to oxidative stress. The aim of this study was to determine if DHA in POS can increase oxidative damage to RPE. It was found that RPE undergo increased lipid peroxidation and decreased cell viability when stressed with hydrogen peroxide in combination with DHA or POS. H2O2-induced oxidative stress was found to cause both ferroptosis and necroptosis. However, the ferroptosis regulator acyl-CoA synthetase long-chain family member 4 (ACSL4) was found to be downregulated in RPE exposed to H2O2 and this effect was exacerbated when the RPE cells were simultaneously treated with DHA. Together, these results show a response of RPE when stressed which will likely be overwhelmed under disease conditions such as AMD resulting in cell death.

Heat-shock protein 60 translocates to the surface of apoptotic cells and differentiated megakaryocytes and stimulates phagocytosis.

Heat-shock protein 60 (Hsp60) is a highly conserved stress protein which has chaperone functions in prokaryotes and mammalian cells. Hsp60 is associated with the mitochondria and the plasma membrane through phosphorylation by protein kinase A, and is incorporated into lipid membranes as a protein-folding chaperone. Its diverse intracellular chaperone functions include the secretion of proteins where it maintains the conformation of precursors and facilitates their translocation through the plasma membrane. We report here that Hsp60 is concentrated in apoptotic membrane blebs and translocates to the surface of cells undergoing apoptosis. Hsp60 is also enriched in platelets derived from terminally differentiated megakaryocytes and expressed at the surface of senescent platelets. Furthermore, the exposure of monocytic U937 cells to Hsp60 enhanced their phagocytic activity. Our results suggests that externalized Hsp60 in apoptotic cells and senescent platelets influences events subsequent to apoptosis, such as the clearance of apoptotic cells by phagocytes.

Reliability of kettlebell swing one and five repetition maximum.

Background: Research into the kettlebell swing has increased in the last decade. There has been a paucity of literature assessing an individual's ability to perform the kettlebell swing exercise. The purpose of this study was to determine the test-retest reliability of the one and five repetition maximum (1RM and 5RM) kettlebell swing. Materials & Methods: Twenty four recreational resistance-trained participants performed an isometric mid-thigh pull (IMTP) and two familiarization sessions followed by three test sessions for each RM load approximately one week apart, using a custom-built plate-loaded kettlebell. On each test occasion, subjects completed a series of warm-up sets followed by 3-4 progressively heavier kettlebell swings to a standardized height until 1RM or 5RM was reached. Test-retest reliability was calculated using the intra-class correlation (ICC) and typical error was represented as the coefficient of variation (CV%) with 90% confidence limits (90% CL). The smallest worthwhile change (SWC%) representing the smallest change of practical importance, was calculated as 0.2 × between-subject standard deviation. The relationship of kettlebell swing performance and maximum strength was determined by Pearson correlation with ±90% CL between the absolute peak force recorded during IMTP and 1RM or 5RM. Results: Results demonstrated a high test-retest reliability for both the 1RM (ICC = 0.97, 90% CL [0.95-0.99]; CV = 2.7%, 90% CL [2.2-3.7%]) and 5RM (ICC = 0.98, 90% CL [0.96-0.99]; CV = 2.4%, 90% CL [1.9-3.3%]), respectively. The CV% was lower than the SWC for both the 1RM (SWC = 2.8%, 90% CL [1.9-3.5]) and 5RM (SWC = 2.9%, 90% CL [1.9-3.6]) kettlebell swing. The correlation between IMTP absolute peak force and the 1RM (r = 0.69, 90% CL 0.43-0.83) was large and very large for the 5RM (r = 0.75, 90% CL [0.55-0.87]). Conclusions: These results demonstrate the stability of 1RM and 5RM kettlebell swing performance after two familiarization sessions. Practitioners can be confident that changes in kettlebell swing 1RM and 5RM performance of >3.6 kg represent a practically important difference, which is the upper limit of the 90% CL.

Generation of functional human hepatic endoderm from human induced pluripotent stem cells.

UNLABELLED: With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. CONCLUSION: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models.

Portal venous endothelium in developing human liver contains haematopoietic and epithelial progenitor cells.

Future treatments for chronic liver disease are likely to involve manipulation of liver progenitor cells (LPCs). In the human, data characterising the regenerative response is limited and the origin of adult LPCs is unknown. However, these remain critical factors in the design of cell-based liver therapies. The developing human liver provides an ideal model to study cell lineage derivation from progenitors and to understand how foetal haematopoiesis and liver development might explain the nature of the adult LPC population. In 1st trimester human liver, portal venous endothelium (PVE) expressed adult LPC markers and markers of haematopoietic progenitor cells (HPCs) shared with haemogenic endothelium found in the embryonic dorsal aorta. Sorted PVE cells were able to generate hepatoblast-like cells co-expressing CK18 and CK19 in addition to Dlk/pref-1, E-cadherin, albumin and fibrinogen in vitro. Furthermore, PVE cells could initiate haematopoiesis. These data suggest that PVE shares phenotypical and functional similarities both with adult LPCs and embryonic haemogenic endothelium. This indicates that a temporal relationship might exist between progenitor cells in foetal liver development and adult liver regeneration, which may involve progeny of PVE.

Interaction of gonadal status with systemic inflammation and opioid use in determining nutritional status and prognosis in advanced pancreatic cancer.

PURPOSE: Hypogonadism has been linked with systemic inflammation and opioid use in males with advanced cancer. We aimed to investigate the interaction of gonadal status with systemic inflammation and opioids in determining nutritional status and prognosis in advanced pancreatic cancer. METHODS: One hundred and seventy-five patients (92 males, 83 postmenopausal females) with unresectable pancreatic cancer were studied. Serum sex steroids (total testosterone [TT], calculated free testosterone [cFT], oestradiol, sex hormone binding globulin), gonadotropins (follicle-stimulating hormone and luteinising hormone) and pro-inflammatory mediators (C-reactive protein [CRP], interleukin-6 [IL-6], soluble tumour necrosis factor receptor 75 [sTNFR75]) were measured, and nutritional assessment and opioid use recorded. RESULTS: Seventy-three percent of males were hypogonadal (by cFT definition). cFT correlated positively with BMI (r (2) =0.349; p< 0.001) and grip strength (r (2) = 0.229; p = 0.034) and inversely with weight loss (r (2) = -0.287; p = 0.007), CRP (r (2) = -0.426; p < 0.001) and IL-6 (r (2) = -0.303; p = 0.004). CRP (p= 0.007), opioid dosage (p = 0.009) and BMI (p = 0.005) were independent determinants of cFT on ANOVA. Hypogonadal males demonstrated worsened survival compared with eugonadal patients (TT: OR of death = 2.87; p < 0.001; cFT: OR = 2.26; p = 0.011). Furthermore, male opioid use was associated with decreased TT (p < 0.001) and cFT (p < 0.001) and worsened survival (OR = 1.96; p = 0.012). In contrast, 18% of postmenopausal females exhibited premenopausal ("hyperoestrogenic") oestradiol levels. Oestradiol correlated positively with sTNFR75 (r (2) = 0.299; p = 0.008). CRP (p < 0.001) was an independent determinant of oestradiol. Hyperoestrogenic females demonstrated worsened survival compared with eugonadal patients (OR = 2.43; p = 0.013). CONCLUSIONS: In males with pancreatic cancer, systemic inflammation and opioid use are associated with hypogonadism. Male hypogonadism and female hyperoestrogenism are associated with shortened survival in advanced pancreatic cancer.

Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling.

Human embryonic stem cells (hESCs) are a valuable source of pluripotential primary cells. To date, however, their homogeneous cellular differentiation to specific cell types in vitro has proven difficult. Wnt signaling has been shown to play important roles in coordinating development, and we demonstrate that Wnt3a is differentially expressed at critical stages of human liver development in vivo. The essential role of Wnt3a in hepatocyte differentiation from hESCs is paralleled by our in vitro model, demonstrating the importance of a physiologic approach to cellular differentiation. Our studies provide compelling evidence that Wnt3a signaling is important for coordinated hepatocellular function in vitro and in vivo. In addition, we demonstrate that Wnt3a facilitates clonal plating of hESCs exhibiting functional hepatic differentiation. These studies represent an important step toward the use of hESC-derived hepatocytes in high-throughput metabolic analysis of human liver function.

Liver development, regeneration, and carcinogenesis.

The identification of putative liver stem cells has brought closer the previously separate fields of liver development, regeneration, and carcinogenesis. Significant overlaps in the regulation of these processes are now being described. For example, studies in embryonic liver development have already provided the basis for directed differentiation of human embryonic stem cells and induced pluripotent stem cells into hepatocyte-like cells. As a result, the understanding of the cell biology of proliferation and differentiation in the liver has been improved. This knowledge can be used to improve the function of hepatocyte-like cells for drug testing, bioartificial livers, and transplantation. In parallel, the mechanisms regulating cancer cell biology are now clearer, providing fertile soil for novel therapeutic approaches. Recognition of the relationships between development, regeneration, and carcinogenesis, and the increasing evidence for the role of stem cells in all of these areas, has sparked fresh enthusiasm in understanding the underlying molecular mechanisms and has led to new targeted therapies for liver cirrhosis and primary liver cancers.

The relevance of a hypoxic tumour microenvironment in prostate cancer.

Research into the hypoxic tumour microenvironment is accelerating and the reversal of hypoxia is increasingly being suggested as a mechanism for improving cancer treatment. Recent studies have suggested that hypoxia is also a feature in prostate cancer and is associated with a poor prognosis. Hypoxia has been shown to cause radio-resistance and hence hamper one of the major treatments for prostate cancer. However, unlike other solid tumours, such as cervical and head-and-neck cancer, there are inconsistencies and unanswered questions about the relevance of hypoxia in prostate cancer. This review outlines the role of low-oxygen conditions in prostate cancer and the areas where further studies are required.

Side population cells in developing human liver are primarily haematopoietic progenitor cells.

Side population (SP) cells have recently been identified in a number of tissues although their phenotype and functional abilities are poorly understood. Surface marker characterisation and functional assessment of developing liver SP cells might allow for their isolation and manipulation using clinically relevant techniques. It was hypothesised that SP cells are present early during human liver development and contribute to haematopoietic and epithelial lineage generation. Whilst the SP population remained positive for CD34 during the 1st and 2nd trimester, 1st trimester SP cells were more highly enriched for haematopoietic and epithelial progenitor activity than those from the 2nd trimester in vitro. Marker expression and functional similarities indicate that SP cells in developing human liver may share a temporal relationship with oval/progenitor cells, responsible for liver regeneration after massive or chronic hepatic injury. Furthermore, modification of SP integrin expression during development suggests a potential adaptive interaction with niche components such as fibronectin. Improved understanding of developing human liver SP cells will contribute to the generation of novel cell-based therapies for liver disease.

NO-sulindac inhibits the hypoxia response of PC-3 prostate cancer cells via the Akt signalling pathway.

Nitric oxide-donating non-steroidal anti-inflammatory drugs are safer than traditional NSAIDs and inhibit the growth of prostate cancer cells with greater potency than NSAIDs. In vivo, prostate cancer deposits are found in a hypoxic environment which induces resistance to chemotherapy. The aim of this study was to assess the effects and mechanism of action of a NO-NSAID called NO-sulindac on the PC-3 prostate cancer cell line under hypoxic conditions. NO-sulindac was found to have pro-apoptotic, cytotoxic, and anti-invasive effect on PC-3 cells under normoxia and hypoxia. NO-sulindac was significantly more cytotoxic than sulindac at all oxygen levels. The sulindac/linker and NO-releasing subunits both contributed to the cytotoxic effects of NO-sulindac. Resistance of PC-3 cells to NO-sulindac was induced as the oxygen concentration declined. Hypoxia-induced chemoresistance was reversed by knocking-down hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA using RNAi. Nuclear HIF-1alpha levels were upregulated at 0.2% oxygen but reduced by treatment with NO-sulindac, as was Akt phosphorylation. NO-sulindac treatment of hypoxic PC-3 cells transfected with a reporter construct, downregulated activation of the hypoxia response element (HRE) promoter. Co-transfection of PC-3 cells with the HRE promoter reporter construct and myr-Akt (constitutively active Akt) plasmids reversed the NO-sulindac induced reduction in HRE activation. Real-time polymerase chain reaction analysis of hypoxic, NO-sulindac treated PC-3 cells showed downregulation of lysyl oxidase and carbonic anhydrase IX mRNA expression. Collectively, these novel findings demonstrate that NO-sulindac directly inhibits the hypoxia response of PC-3 prostate cancer cells by inhibiting HIF-1alpha translation via the Akt signalling pathway. The ability of NO-sulindac to inhibit tumour adaption to hypoxia has considerable relevance to the future management of prostate cancer with the same cellular properties as PC-3.