Redox Metabolism and Vascular Calcification in Chronic Kidney Disease.

Vascular calcification (VC) is a common complication in patients with chronic kidney disease which increases their mortality. Although oxidative stress is involved in the onset and progression of this disorder, the specific role of some of the main redox regulators, such as catalase, the main scavenger of H2O2, remains unclear. In the present study, epigastric arteries of kidney transplant recipients, a rat model of VC, and an in vitro model of VC exhibiting catalase (Cts) overexpression were analysed. Pericalcified areas of human epigastric arteries had increased levels of catalase and cytoplasmic, rather than nuclear runt-related transcription factor 2 (RUNX2). In the rat model, advanced aortic VC concurred with lower levels of the H2O2-scavenger glutathione peroxidase 3 compared to controls. In an early model of calcification using vascular smooth muscle cells (VSMCs), Cts VSMCs showed the expected increase in total levels of RUNX2. However, Cts VMSCs also exhibited a lower percentage of the nucleus stained for RUNX2 in response to calcifying media. In this early model of VC, we did not observe a dysregulation of the mitochondrial redox state; instead, an increase in the general redox state was observed in the cytoplasm. These results highlight the complex role of antioxidant enzymes as catalase by regulation of RUNX2 subcellular location delaying the onset of VC.

Photoacoustic Tomography Detects Response and Resistance to Bevacizumab in Breast Cancer Mouse Models.

Angiogenesis is an established prognostic factor in advanced breast cancer, yet response to antiangiogenic therapies in this disease remains highly variable. Noninvasive imaging biomarkers could help identify patients that will benefit from antiangiogenic therapy and provide an ideal tool for longitudinal monitoring, enabling dosing regimens to be altered with real-time feedback. Photoacoustic tomography (PAT) is an emerging imaging modality that provides a direct readout of tumor hemoglobin concentration and oxygenation. We hypothesized that PAT could be used in the longitudinal setting to provide an early indication of response or resistance to antiangiogenic therapy. To test this hypothesis, PAT was performed over time in estrogen receptor-positive and estrogen receptor-negative breast cancer xenograft mouse models undergoing treatment with the antiangiogenic bevacizumab as a single agent. The cohort of treated tumors, which were mostly resistant to the treatment, contained a subset that demonstrated a clear survival benefit. At endpoint, the PAT data from the responding subset showed significantly lower oxygenation and higher hemoglobin content compared with both resistant and control tumors. Longitudinal analysis revealed that tumor oxygenation diverged significantly in the responding subset, identifying early treatment response and the evolution of different vascular phenotypes between the subsets. Responding tumors were characterized by a more angiogenic phenotype when analyzed with IHC, displaying higher vessel density, yet poorer vascular maturity and elevated hypoxia. Taken together, our findings indicate that PAT shows promise in providing an early indication of response or resistance to antiangiogenic therapy. SIGNIFICANCE: Photoacoustic assessment of tumor oxygenation is a noninvasive early indicator of response to bevacizumab therapy, clearly distinguishing between control, responding, and resistant tumors within just a few weeks of treatment.

Evaluation of Label-Free Confocal Raman Microspectroscopy for Monitoring Oxidative Stress In Vitro in Live Human Cancer Cells.

Understanding the impact of free radicals and antioxidants in cell biology is vital; however, noninvasive nonperturbative imaging of oxidative stress remains a challenge. Here, we evaluated the ability of label-free Raman spectroscopy to monitor redox biochemical changes in antioxidant (N-acetyl-l-cysteine, NAC) and pro-oxidant (tert-butyl hydroperoxide, TBHP) environments. Cellular changes were compared to fluorescence microscopy using CellROX Orange as a marker of oxidative stress. We also investigated the influence of cell media with and without serum. Incubation of cells with NAC increased the Raman signal at 498 cm-1 from S-S disulphide stretching mode, one of the most important redox-related sensors. Exposure of cells to TBHP resulted in decreased Raman spectral signals from DNA/proteins and lipids (at 784, 1094, 1003, 1606, 1658 and 718, 1264, 1301, 1440, 1746 cm-1). Using partial least squares-discriminant analysis, we showed that Raman spectroscopy can achieve sensitivity up to 96.7%, 94.8% and 91.6% for control, NAC and TBHP conditions, respectively, with specificity of up to 93.5, 90.1% and 87.9%. Our results indicate that Raman spectroscopy can directly measure the effect of NAC antioxidants and accurately characterize the intracellular conditions associated with TBHP-induced oxidative stress, including lipid peroxidation and DNA damage.

Androgen-Dependent Prostate Cancer Cells Reprogram Their Metabolic Signature upon GLUT1 Upregulation by Manganese Superoxide Dismutase.

Prostate cancer is the second leading cause of cancer in men across the globe. The prostate gland accounts for some unique glycolytic metabolic characteristics, which causes the metabolic features of prostate tumor initiation and progression to remain poorly characterized. The mitochondrial superoxide dismutase (SOD2) is one of the major redox metabolism regulators. This study points out SOD2 as one major regulator for both redox and glycolytic metabolism in prostate cancer. SOD2 overexpression increases glucose transporter GLUT-1 and glucose uptake. This is not an insulin-mediated effect and seems to be sex-dependent, being present in male mice only. This event concurs with a series of substantial metabolic rearrangements at cytoplasmic and mitochondrial level. A concomitant decrease in glycolytic and pentose phosphate activity, and an increase in electron transfer in the mitochondrial electronic chain, were observed. The Krebs Cycle is altered to produce amino-acid intermediates by decreasing succinate dehydrogenase. This in turn generates a 13-fold increase in the oncometabolite succinate. The protein energy sensor AMPK is decreased at basal and phosphorylated levels in response to glucose deprivation. Finally, preliminary results in prostate cancer patients indicate that glandular areas presenting high levels of SOD2 show a very strong correlation with GLUT-1 protein levels (R2 = 0.287 p-value < 0.0001), indicating that in patients there may exist an analogous phenomenon to those observed in cell culture and mice.

Photoacoustics resolves species-specific differences in hemoglobin concentration and oxygenation.

SIGNIFICANCE: Photoacoustic imaging (PAI) enables the detection of blood hemoglobin (HB) concentration and oxygenation (sO2) with high contrast and resolution. Despite the heavy use of photoacoustically determined total hemoglobin (THb) and oxygenation (sO2) biomarkers in PAI research, their relationship with underlying biochemical blood parameters and the impact of intra- and interspecies genetic variability have yet to be established. AIM: To explore the relationship between THb and sO2 photoacoustic biomarkers and the underlying biochemical blood parameters in a species-specific manner. APPROACH: Experiments were performed on blood in vitro using tissue-mimicking agar phantoms. Blood was extracted from mouse, rat, human, and naked mole-rat (Heterocephalus glaber), anticoagulated in ethylenediaminetetraacetic acid, and measured within 48 h. THb and sO2 were measured using a commercial photoacoustic tomography system (InVision 128, iThera Medical GmBH). Biochemical blood parameters such as HB concentration (g/dL), hematocrit (HCT, %), and red blood cell (RBC) count (µL - 1) were assessed using a hematology analyzer (Mythic 18 Vet, Woodley Equipment). RESULTS: A significant correlation was observed between THb and biochemical HB, HCT, and RBC in mouse and rat blood. Moreover, PAI accurately recapitulated interspecies variations in HB and HCT between mouse and rat blood and resolved differences in the oxygen dissociation curves measured using sO2 between human, mouse, and rat. With these validation data in hand, we applied PAI to studies of blood obtained from naked mole-rats and could confirm the high oxygen affinity of this species in comparison to other rodents of similar size. CONCLUSIONS: Our results demonstrate the high sensitivity of photoacoustically determined hemoglobin biomarkers toward species-specific variations in vitro.

Melatonin-Induced Cytoskeleton Reorganization Leads to Inhibition of Melanoma Cancer Cell Proliferation.

Neuroindole melatonin, a hormone synthesized during the night mainly-but not exclusively-by the pineal gland of all vertebrates, functions as an adapting signal to the light-dark cycle. Its antioxidant, neuroprotective, anti-inflammatory, and antitumor properties are all well-known and widely reported. Melanoma is one of the most common carcinomas among developed countries and a type of tumor particularly difficult to fight back in medium/advanced stages. In contrast to other types of cancer, influence of melatonin on melanoma has been scarcely investigated. Thus, we have chosen the murine melanoma model B16-F10 cell line to study antiproliferative and antitumoral actions of melatonin. For this purpose, we combined both, cell culture and in vivo models. Melatonin reduced either, growth rate or migration of B16-F10 cells. Furthermore, melanin synthesis was altered by melatonin, promoting its synthesis. Melatonin also induced a G2/M cell cycle arrest and altered the cytoskeletal organization. To corroborate these results, we tested the effect of melatonin in the in vivo model of B16-F10 cell injection in the tail vein, which causes numerous lung metastases. Two different strategies of melatonin administration were used, namely, in drinking water, or daily intraperitoneal injection. However, contrary to what occurred in cell culture, no differences were observed between control and melatonin treated groups. Results obtained led us to conclude that melatonin exerts an antiproliferative and anti-migrating effect on this melanoma model by interfering with the cytoskeleton organization, but this pharmacological effect cannot be translated in vivo as the indole did not prevent metastasis in the murine model, suggesting that further insights into the effects of the indole in melanoma cells should be approached to understand this apparent paradox.

Oxygen-Enhanced and Dynamic Contrast-Enhanced Optoacoustic Tomography Provide Surrogate Biomarkers of Tumor Vascular Function, Hypoxia, and Necrosis.

Measuring the functional status of tumor vasculature, including blood flow fluctuations and changes in oxygenation, is important in cancer staging and therapy monitoring. Current clinically approved imaging modalities suffer long procedure times and limited spatiotemporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that may overcome these challenges. By acquiring data at multiple wavelengths, OT can interrogate hemoglobin concentration and oxygenation directly and resolve contributions from injected contrast agents. In this study, we tested whether two dynamic OT techniques, oxygen-enhanced (OE) and dynamic contrast-enhanced (DCE)-OT, could provide surrogate biomarkers of tumor vascular function, hypoxia, and necrosis. We found that vascular maturity led to changes in vascular function that affected tumor perfusion, modulating the DCE-OT signal. Perfusion in turn regulated oxygen availability, driving the OE-OT signal. In particular, we demonstrate for the first time a strong per-tumor and spatial correlation between imaging biomarkers derived from these in vivo techniques and tumor hypoxia quantified ex vivo Our findings indicate that OT may offer a significant advantage for localized imaging of tumor response to vascular-targeted therapies when compared with existing clinical DCE methods.Significance: Imaging biomarkers derived from optoacoustic tomography can be used as surrogate measures of tumor perfusion and hypoxia, potentially yielding rapid, multiparametric, and noninvasive cancer staging and therapeutic response monitoring in the clinic.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5980/F1.large.jpg Cancer Res; 78(20); 5980-91. ©2018 AACR.

Melatonin Uptake by Cells: An Answer to Its Relationship with Glucose?

Melatonin, N-acetyl-5-methoxytryptamine, is an indole mainly synthesized from tryptophan in the pineal gland and secreted exclusively during the night in all the animals reported to date. While the pineal gland is the major source responsible for this night rise, it is not at all the exclusive production site and many other tissues and organs produce melatonin as well. Likewise, melatonin is not restricted to vertebrates, as its presence has been reported in almost all the phyla from protozoa to mammals. Melatonin displays a large set of functions including adaptation to light: dark cycles, free radical scavenging ability, antioxidant enzyme modulation, immunomodulatory actions or differentiation⁻proliferation regulatory effects, among others. However, in addition to those important functions, this evolutionary 'ancient' molecule still hides further tools with important cellular implications. The major goal of the present review is to discuss the data and experiments that have addressed the relationship between the indole and glucose. Classically, the pineal gland and a pinealectomy were associated with glucose homeostasis even before melatonin was chemically isolated. Numerous reports have provided the molecular components underlying the regulatory actions of melatonin on insulin secretion in pancreatic beta-cells, mainly involving membrane receptors MTNR1A/B, which would be partially responsible for the circadian rhythmicity of insulin in the organism. More recently, a new line of evidence has shown that glucose transporters GLUT/SLC2A are linked to melatonin uptake and its cellular internalization. Beside its binding to membrane receptors, melatonin transportation into the cytoplasm, required for its free radical scavenging abilities, still generates a great deal of debate. Thus, GLUT transporters might constitute at least one of the keys to explain the relationship between glucose and melatonin. These and other potential mechanisms responsible for such interaction are also discussed here.

Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry.

BACKGROUND: Optoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures. METHODS: Multispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO2MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation. RESULTS: MCF-7 tumours show SO2MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO2MSOT with a significant rim-core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry. CONCLUSIONS: OT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Fluorescence hyperspectral imaging (fHSI) using a spectrally resolved detector array.

The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyperspectral imaging (HSI) facilitates the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for HSI is typically complex, bulky and expensive. We sought to overcome these limitations using a novel robust and low cost HSI camera based on a spectrally resolved detector array (SRDA). We integrated this HSI camera into a wide-field reflectance-based imaging system operating in the near-infrared range to assess the suitability for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence HSI (fHSI) system, we were able to accurately resolve the presence and concentration of at least 7 fluorescent dyes in solution. We also demonstrate high spectral unmixing precision, signal linearity with dye concentration and at depth in tissue mimicking phantoms, and delineate 4 fluorescent dyes in vivo. Our approach, including statistical background removal, could be directly generalised to broader spectral ranges, for example, to resolve tissue reflectance or autofluorescence and in future be tailored to video rate applications requiring snapshot HSI data acquisition.

Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects.

Optoacoustic tomography (OT) is now widely used in preclinical imaging; however, the precision (repeatability and reproducibility) of OT has yet to be determined. Methods: We used a commercial small-animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, and frame averaging. Variables due to animal handling and physiology, such as anatomic placement and anesthesia conditions, were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and in vivo. Sensitivity to small-molecule dyes was determined in phantoms and in vivo; precision was assessed in vivo using IRDye800CW. Results: OT COV in a stable phantom was less than 2.8% across all wavelengths over 30 d. The factors with the greatest impact on signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4% at 700 nm. Anatomic region-of-interest size showed the highest variation, at 12% and 18% COV in the kidney and spleen, respectively; however, functional SO2 measurements based on a standard operating procedure showed an exceptional reproducibility of less than 4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for in vivo data included respiration rate, degree of user experience, and animal placement. Conclusion: Data acquired with our small-animal OT system were highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.

IGFBP3 and MAPK/ERK signaling mediates melatonin-induced antitumor activity in prostate cancer.

Treatment of prostate cancer (PCa), a leading cause of cancer among males, lacks successful strategies especially in advanced, hormone-refractory stages. Some clinical studies have shown an increase in neuroendocrine-like cells parallel to the tumor progression but their exact role is a matter of debate. The prostate is a well-known target for melatonin, which reduces PCa cells proliferation and induces neuroendocrine differentiation. To evaluate the mechanisms underlying the indole effects on neuroendocrine differentiation and its impact on PCa progression, we used a cell culture model (LNCaP) and a murine model (TRAMP). Persistent ERK1/2 activation was found in both, melatonin and androgen-deprived cells. Melatonin blocked nuclear translocation of androgen receptor (AR), thus confirming anti-androgenic actions of the indole. However, using a comparative genome microarray to check the differentially expressed genes in control, melatonin, or androgen-deprived cells, some differences were found, suggesting a more complex role of the indole. By comparing control cells with those treated with melatonin or depleted of androgen, a cluster of 26 differentially expressed genes (±2.5-fold) was found. Kallikreins (KLK)2 and KLK3 (PSA) were dramatically downregulated by both treatments whereas IGFBP3 and IGF1R were up- and downregulated, respectively, in both experimental groups, thus showing a role for IGF in both scenarios. Finally, melatonin prolonged the survival of TRAMP mice by 33% when given at the beginning or at advances stages of the tumor. Serum IGFBP3 was significantly elevated by the indole in early stages of the tumor, confirming in vivo the role of the IGF signaling in the oncostatic action of the indole.

Lamin A is involved in the development of vascular calcification induced by chronic kidney failure and phosphorus load.

Vascular calcification remains one of the main factors associated to morbidity and mortality in both ageing and chronic kidney disease. Both hyperphosphataemia, a well-known promoter of vascular calcification, and abnormal processing defects of lamin A/C have been associated to ageing. The main aim of this study was to analyse the effect of phosphorus load in the differential expression pattern of genes and proteins, particularly of lamin A/C, which are involved in phenotypic change of the vascular smooth muscle cells to osteoblast-like cells. The in vivo study of the calcified abdominal aortas from nephrectomized rats receiving a high phosphorus diet showed among others, a repression of muscle related proteins and overexpression of lamin A/C. Similar results were observed in vitro, where primary vascular smooth muscle cells cultured in calcifying medium showed increased expression of prelamin A and lamin A and abnormalities in the nuclear morphology. Co-immunoprecipitation assays showed novel and important physical interactions between lamin A and RUNX2 during the process of calcification. In fact, the knockdown of prelamin A and lamin A inhibited the increase of Runx2, osteocalcin and osteopontin gene expression, calcium deposition, nuclear abnormalities and the RUNX2 protein translocation into the nucleus of the cell. These in vivo and in vitro results highlight the important role played by lamin A in the process of vascular calcification.

Melatonin uptake through glucose transporters: a new target for melatonin inhibition of cancer.

Melatonin is present in a multitude of taxa and it has a broad range of biological functions, from synchronizing circadian rhythms to detoxifying free radicals. Some functions of melatonin are mediated by its membrane receptors but others are receptor-independent. For the latter, melatonin must enter into the cell. Melatonin is a derivative of the amino acid tryptophan and reportedly easily crosses biological membranes due to its amphipathic nature. However, the mechanism by which melatonin enters into cells remains unknown. Changes in redox state, endocytosis pathways, multidrug resistance, glycoproteins or a variety of strategies have no effect on melatonin uptake. Herein, it is demonstrated that members of the SLC2/GLUT family glucose transporters have a central role in melatonin uptake. When studied by docking simulation, it is found that melatonin interacts at the same location in GLUT1 where glucose does. Furthermore, glucose concentration and the presence of competitive ligands of GLUT1 affect the concentration of melatonin into cells. As a regulatory mechanism, melatonin reduces the uptake of glucose and modifies the expression of GLUT1 transporter in prostate cancer cells. More importantly, glucose supplementation promotes prostate cancer progression in TRAMP mice, while melatonin attenuated glucose-induced tumor progression and prolonged the lifespan of tumor-bearing mice. This is the first time that a facilitated transport of melatonin is suggested. In fact, the important role of glucose transporters and glucose metabolism in cell fate might explain some of the diverse functions described for melatonin.

Central genes, pathways and modules that regulate bone mass.

Bones are structures that give the shape and defined features to vertebrates, protect several soft organs and perform multiple endocrine influences on other organs. To achieve these functions bones are first modeled early during life and then constantly remodeled throughout life. The process of bone (re)modeling happens simultaneously at multitude of locations in the skeleton and ensures that vertebrates have a mechanically strong yet a flexible skeleton to the most part of their life. Given the extent of its occurrence in the body, bone remodeling is a highly energy demanding process and is co-ordinated with other physiological processes as diverse as energy metabolism, sleep-wake cycle and reproduction. Neuronal circuits in the brain play a very important role in the coordination of bone remodeling with other organ system functions, and perform this function in sync with environmental and peripheral hormonal cues. In this review, we will focus on the roles of hormonal signals and neural circuits that originate in, or impinge on, the brain in the regulation of bone mass. We will provide herein an updated view of how advances in molecular genetics have refined the neural circuits involved in the regulation of bone mass, from the whole brain level to the specific neuronal populations and their neurotransmitters. This will help to understand the mechanisms whereby vertebrate brain regulates bone mass by fine-tuning metabolic signals that originate in the brain or elsewhere in the body.

Vitamin B12-dependent taurine synthesis regulates growth and bone mass.

Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass.

Phenotypic changes caused by melatonin increased sensitivity of prostate cancer cells to cytokine-induced apoptosis.

Melatonin has antiproliferative properties in prostate cancer cells. Melatonin reduces proliferation without increasing apoptosis, and it promotes cell differentiation into a neuroendocrine phenotype. Because neuroendocrine cells displayed an androgen-independent growth and high resistance to radiotherapy and chemotherapy, the role of molecules that induce neuroendocrine differentiation was questioned in terms of their usefulness as oncostatic agents. By using human epithelial androgen-dependent and androgen-independent prostate cancer cells, the role of melatonin in drug-induced apoptosis was studied after acute treatments. In addition to cytokines such as hrTNF-alpha and TRAIL, chemotherapeutic compounds, including doxorubicin, docetaxel, or etoposide, were employed in combination with melatonin to promote cell death. Melatonin promotes cell toxicity caused by cytokines without influencing the actions of chemotherapeutic agents. In addition, antioxidant properties of melatonin were confirmed in prostate cancer cells. However, its ability to increase cell death caused by cytokines was independent of the redox changes. Finally, phenotypic changes caused by chronic treatment with the indole, that is, neuroendocrine differentiation, make cells significantly more sensitive to cytokines and slightly more sensitive to some chemotherapeutic compounds. Thus, melatonin is a good inhibitor of the proliferation of prostate cancer cells, promoting phenotypic changes that do not increase survival mechanisms and make cells more sensitive to cytokines such as TNF-alpha or TRAIL.

MnSOD drives neuroendocrine differentiation, androgen independence, and cell survival in prostate cancer cells.

An increase in neuroendocrine (NE) cell number has been associated with progression of prostate tumor, one of the most frequent cancers among Western males. We previously reported that mitochondrial manganese superoxide dismutase (MnSOD) increases during the NE differentiation process. The goal of this study was to find whether MnSOD up-regulation is enough to induce NE differentiation. Several human prostate cancer LNCaP cell clones stably overexpressing MnSOD were characterized and two were selected (MnSOD-S4 and MnSOD-S12). MnSOD overexpression induces NE morphological features as well as coexpression of the NE marker synaptophysin. Both MnSOD clones exhibit lower superoxide levels and higher H(2)O(2) levels. MnSOD-overexpressing cells show higher proliferation rates in complete medium, but in steroid-free medium MnSOD-S12 cells are still capable of proliferation. MnSOD up-regulation decreases androgen receptor and prevents its nuclear translocation. MnSOD also induces up-regulation of Bcl-2 and prevents docetaxel-, etoposide-, or TNF-induced cell death. Finally, MnSOD-overexpressing cells enhance growth of androgen-independent PC-3 cells but reduce growth of androgen-dependent cells. These results indicate that redox modulation caused by MnSOD overexpression explains most NE-like features, including morphological changes, NE marker expression, androgen independence, inhibition of apoptosis, and enhancement of cell growth. Many of these events can be associated with the androgen dependent-independent transition during prostate cancer progression.