Nitric oxide is a positive regulator of the Warburg effect in ovarian cancer cells

Ovarian cancer (OVCA) is among the most lethal gynecological cancers leading to high mortality rates among women. Increasing evidence indicate that cancer cells undergo metabolic transformation during tumorigenesis and growth through nutrients and growth factors available in tumor microenvironment. This altered metabolic rewiring further enhances tumor progression. Recent studies have begun to unravel the role of amino acids in the tumor microenvironment on the proliferation of cancer cells. One critically important, yet often overlooked, component to tumor growth is the metabolic reprogramming of nitric oxide (NO) pathways in cancer cells. Multiple lines of evidence support the link between NO and tumor growth in some cancers, including pancreas, breast and ovarian. However, the multifaceted role of NO in the metabolism of OVCA is unclear and direct demonstration of NO''s role in modulating OVCA cells'' metabolism is lacking. This study aims at indentifying the mechanistic links between NO and OVCA metabolism. We uncover a role of NO in modulating OVCA metabolism: NO positively regulates the Warburg effect, which postulates increased glycolysis along with reduced mitochondrial activity under aerobic conditions in cancer cells. Through both NO synthesis inhibition (using L-arginine deprivation, arginine is a substrate for NO synthase (NOS), which catalyzes NO synthesis; using L-Name, a NOS inhibitor) and NO donor (using DETA-NONOate) analysis, we show that NO not only positively regulates tumor growth but also inhibits mitochondrial respiration in OVCA cells, shifting these cells towards glycolysis to maintain their ATP production. Additionally, NO led to an increase in TCA cycle flux and glutaminolysis, suggesting that NO decreases ROS levels by increasing NADPH and glutathione levels. Our results place NO as a central player in the metabolism of OVCA cells. Understanding the effects of NO on cancer cell metabolism can lead to the development of NO targeting drugs for OVCAs.Despite recent medical and pharmaceutical advances in cancer research, ovarian cancer (OVCA) remains one of the most deadly gynecological malignancies, with most of the cancer first detected in late stages when metastasis has already occurred.¹ Only 20% of OVCA patients are diagnosed when cancer has not spread past the ovaries; in the other 80% of cases, the cancer has metastasized, most frequently to the peritoneum.² Platinum-based preoperative chemotherapy is the standard of care of early stage disease, and surgical resection along with platinum-based postoperative chemotherapy is the standard of care for late stage disease.¹ However, many platinum-based chemotherapy drugs come with unwanted side effects. Therefore, an alternative therapy for OVCA is needed.Nitric oxide (NO) shows promise either as a cancer therapeutic agent by itself or as a target of cancer therapies.³ This may be because NO can act as a signaling molecule or as a source of oxidative and nitrosative stress.⁴ NO can stimulate mitochondrial biogenesis through PGC-1-related coactivator⁵ and increase mitochondrial function.^{6, 7} In follicular thyroid carcinoma cells, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a NO donor, was shown to increase the expression of genes involved in mitochondrial biogenesis.^{8, 9} A 14-day treatment of lung carcinoma cells with dipropylenetriamine NONOate (DETA-NONOate), another NO donor, increased cell migration compared with the absence of treatment.¹⁰ In breast cancer cells, exogenous NO increased cell proliferation, as well as cyclin-D1 and ornithine decarboxylase expression.¹¹ In prostate cancer cells, NO was shown to inhibit androgen receptor-dependent promoter activity and proliferation of androgen-dependent cells, indicating that NO would select for the development of prostate cancer cells that are androgen-independent.¹² NO has even been shown to inhibit mitochondrial ATP production, and therefore inhibit apoptosis, as ATP is necessary for the apoptotic process.¹³ Moreover, inducible nitric oxide synthase (iNOS) knockout mice had less tumor formation than wild-type mice, indicating that NO promotes lung tumorigenesis.¹⁴ On the other hand, NO production, as induced by proinflammatory cytokines, induced apoptosis in OVCA cells.³ NOS overexpression by transfection of a plasmid containing NOS-3 DNA resulted in increased cell death in HepG2 cells.¹⁵ In another study, NO was implicated in N-(4-hydroxyphenyl) retinamide-mediated apoptosis.¹⁶ Finally, iNOS expression in p53-depleted mice increased apoptosis of lymphoma cells compared with p53-deficient mice without iNOS expression.¹⁷ Therefore, NO has been seen to have both an anti-tumorigenic as well as a pro-tumorigenic effect.Arginine, a conditionally essential amino acid used to produce NO, is also a potential target for cancer therapy. L-arginine is normally produced by the body; however, in some diseased states, more arginine than what the body normally produces is required.¹⁸ Arginine sources include protein breakdown or directly from the diet, in addition to de novo synthesis.¹⁹ In the de novo production of L-arginine, citrulline and aspartate are first converted to argininosuccinate by arginase, which is then split into arginine and fumarate by argininosuccinate lyase.²⁰ L-arginine can also be converted to citrulline and NO through NO synthase (NOS).¹⁹ Some cancer cells, including melanoma and hepatocellular carcinoma, do not express argininosuccinate synthase (ASS), an enzyme involved in arginine production and thus rely on exogenous arginine.¹⁹ For these cancers, arginine-deprivation therapy is being heavily explored as a treatment.^{21, 22} OVCA cells have been shown to express ASS.²³ In fact, OVCA cells were shown to have increased expression of ASS compared with normal ovarian surface epithelium.²⁴ As OVCA can synthesize arginine de novo, strategies which target arginine''s conversion into citrulline are needed for regulating OVCA tumor growth.Recent studies suggest that cancer cells undergo metabolic reprogramming, which drives cancer cells'' growth and progression.^{25, 26, 27, 28, 29, 30, 31, 32, 33} One critically important, yet often overlooked, component to tumor growth is the metabolic rewiring of NO pathways in OVCA cells. Despite considerable investigation on NO''s regulation of cancer cell proliferation and growth, mechanistic details regarding the effect of NO on cancer cell metabolism is still lacking: specifically, how NO affects glycolysis, TCA cycle flux, and ROS production. Studies on the effects of NO on cancer cell metabolism have mainly focused on the effect of NO on mitochondrial respiration.^{34, 35, 36, 37} NO has been shown to inhibit cytochrome c oxidase (COX) in the mitochondria of breast cancer cells, as well as decrease oxygen consumption rate.^{37, 38, 39} Moncada and colleagues studied the effect of NO on the metabolism of rat cortical astrocytes and neurons, two cells with different glycolytic capacities. They showed that NO decreased ATP concentration, which led to an increase in glycolysis in astrocytes, but not in neurons, indicating that glycolytic capacity affects the metabolic response of these cells to NO.⁴⁰ NO was shown to reduce ATP production via OXPHOS in rat reticulocytes, cells that produce 90% of their ATP from OXPHOS.⁴¹ Endothelial NOS (eNOS) was shown to have a role in the upregulation of GLUT4 transporters by AMPK and AICAR in the heart muscle.⁴² Additionally, NO can serve to stabilize HIF-1α in hypoxic conditions through S-nitrosylation of PHD2,⁴ and as HIF-1α upregulates GLUT transporters and glycolysis,⁴³ NO may affect the metabolism of cancer cells.Although NO is found to affect glycolysis of normal cells, how NO modulates glycolysis of OVCA cells is less understood. The multifaceted role of NO in the metabolism of OVCA is unclear, and direct demonstration of NO''s role in modulating the metabolism of OVCA cells is lacking. This study aims at understanding the mechanistic links between NO and the overall cancer metabolism – specifically, its effects on glycolysis, TCA cycle, OXPHOS, and ROS production – of OVCA cells. Our results show that NO decreases mitochondrial respiration, forcing OVCA cells to undergo higher glycolytic rates to maintain ATP production levels. Our work is the first to illustrate the central role of NO on OVCA metabolism – specifically, showing how NO (i) positively regulates the Warburg effect in OVCA cell, (ii) maintains low ROS levels by upregulating NADPH generation, and (ii) negatively alters mitochondrial respiration, thus promoting cancer growth and proliferation. Our work is also unique in that it is the first to explore the effects of NO on TCA cycle flux and glutaminolysis, potentially also affecting ROS levels by affecting antioxidant levels. In conclusion, by elucidating the effects of NO on cancer metabolism and ROS levels, we have a better understanding of the different mechanisms by which NO affects cancer cell growth. This understanding may lead to potentially useful therapies to halt cancer progression.     

Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro

Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (F_cytosol/F_nucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells.     

Urban Options for Psychological Restoration: Common Strategies in Everyday Situations

Objectives

Given the need for knowledge on the restorative potential of urban settings, we sought to estimate the effects of personal and contextual factors on preferences and restoration likelihood assessments for different urban activities-in-environments. We also sought to study the generality of these effects across different countries.

Methods

We conducted a true experiment with convenience samples of university students in the Netherlands (n = 80), Sweden (n = 100), and the USA (n = 316). In each country, the experiment had a mixed design with activities-in-environments (sitting in a park, sitting in a cafe, walking in a shopping mall, walking along a busy street) manipulated within-subjects and the need for restoration (attentional fatigue, no attentional fatigue) and immediate social context (in company, alone) manipulated between-subjects. The manipulations relied on previously tested scenarios describing everyday situations that participants were instructed to remember and imagine themselves being in. For each imagined situation (activity-in-environment with antecedent fatigue condition and immediate social context), subjects provided two criterion measures: general preference and the likelihood of achieving psychological restoration.

Results

The settings received different preference and restoration likelihood ratings as expected, affirming that a busy street, often used in comparisons with natural settings, is not representative of the restorative potential of urban settings. Being with a close friend and attentional fatigue both moderated ratings for specific settings. Findings of additional moderation by country of residence caution against broad generalizations regarding preferences for and the expected restorative effects of different urban settings.

Conclusions

Preferences and restoration likelihood ratings for urban activity-environment combinations are subject to multiple personal and contextual determinants, including level of attentional fatigue, being alone versus in company, and broader aspects of the urban context that vary across cities and countries. Claims regarding a lack of restorative quality in urban environments are problematic.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Soft shoreline engineering survey of ecological effectiveness

Historically, many urban waterfront shorelines were stabilized using hard shoreline engineering to protect developments from flooding and erosion, or to accommodate commercial navigation or industry. Today, there is growing interest in developing shorelines using ecological principles and practices that enhance habitat and improve aesthetics, while at the same time reducing erosion, providing stability, and ensuring shoreline safety (i.e., soft shoreline engineering). In 2008-2009, a survey of 38 soft shoreline engineering projects in the Detroit River-western Lake Erie watershed was conducted. In total, $17.3 million (combined U.S. and Canadian currency) was spent on these projects. Of the 38 projects implemented, six (16%) had some quantitative assessment of ecological effectiveness, while the remaining 32 lacked monitoring or only had qualitative assessment through visual inspection. Key lessons learned include: involve habitat experts at the initial stages of waterfront planning; establish broad-based goals with quantitative targets to measure project success; ensure multidisciplinary project support; start with demonstration projects and attract partners; treat habitat modification projects as experiments that promote learning; involve citizen scientists, volunteers, and universities in monitoring, and obtain post-project monitoring commitments up front in project planning; measure benefits and communicate successes; and promote education and outreach, including public events that showcase results and communicate benefits.     

Peroxisome targeting signal 1: is it really a simple tripeptide?

Originally, the peroxisomal targeting signal 1 (PTS1) was defined as a tripeptide at the C-terminus of proteins prone to be imported into the peroxisomal matrix. The corresponding receptor PEX5 initiates the translocation of proteins by identifying potential substrates via their C-termini and trapping PTS1s through remodeling of its TPR domain. Thorough studies on the interaction between PEX5 and PTS1 as well as sequence-analytic tools revealed the influence of amino acid residues further upstream of the ultimate tripeptide. Altogether, PTS1s should be defined as dodecamer sequences at the C-terminal ends of proteins. These sequences accommodate physical contacts with both the surface and the binding cavity of PEX5 and ensure accessibility of the extreme C-terminus. Knowledge-based approaches in applied Bioinformatics provide reliable tools to accurately predict the peroxisomal location of proteins not yet determined experimentally.