13C Metabolic Flux Analysis in Chondrocytes Reveals a Novel Switch in Metabolic Phenotype.

Chondrocytes are typically known for their anaerobic metabolism both in vivo and under culture conditions in vitro. However, chondrocytes have been shown to display greater biosynthetic activity when subjected to conditions that elicit aerobic metabolism. We have previously shown that tissue formation by chondrocytes can be upregulated by controlling nutrient availability and that this response arises from changes in glucose metabolism. The aim of the present study was to further characterize these changes through 13C-metabolic flux analysis (13C-MFA), as well as to determine the most optimal response. Primary bovine chondrocytes were grown in scaffold-free high-density tissue culture. [U-13C] glucose labeling experiments were combined with a tissue-specific metabolic network model to carry out 13C-MFA under varying levels of nutrient availability. 13C-MFA results demonstrated that when subjected to increasing nutrient availability, chondrocytes switch from a predominately anaerobic to a mixed aerobic-anaerobic phenotype. This metabolic switch was attributed to the saturation of the lactate fermentation pathway and metabolite overflow toward the tricarboxylic acid cycle. This effect appears to be similar to, but the inverse of, the Crabtree effect ("inverse Crabtree effect"). The relationships between metabolic flux and nutrient availability were then utilized to identify culture conditions that promote enhanced tissue formation. This novel metabolic effect presents a simple but effective approach for enhancing the biosynthetic response of chondrocytes-a key requirement to develop functional engineered cartilaginous tissue for joint resurfacing.

Exploring Nitric Oxide (NO)-Releasing Celecoxib Derivatives as Modulators of Radioresponse in Pheochromocytoma Cells.

COX-2 can be considered as a clinically relevant molecular target for adjuvant, in particular radiosensitizing treatments. In this regard, using selective COX-2 inhibitors, e.g., in combination with radiotherapy or endoradiotherapy, represents an interesting treatment option. Based on our own findings that nitric oxide (NO)-releasing and celecoxib-derived COX-2 inhibitors (COXIBs) showed promising radiosensitizing effects in vitro, we herein present the development of a series of eight novel NO-COXIBs differing in the peripheral substitution pattern and their chemical and in vitro characterization. COX-1 and COX-2 inhibition potency was found to be comparable to the lead NO-COXIBs, and NO-releasing properties were demonstrated to be mainly influenced by the substituent in 4-position of the pyrazole (Cl vs. H). Introduction of the N-propionamide at the sulfamoyl residue as a potential prodrug strategy lowered lipophilicity markedly and abolished COX inhibition while NO-releasing properties were not markedly influenced. NO-COXIBs were tested in vitro for a combination with single-dose external X-ray irradiation as well as [177Lu]LuCl3 treatment in HIF2α-positive mouse pheochromocytoma (MPC-HIF2a) tumor spheroids. When applied directly before X-ray irradiation or 177Lu treatment, NO-COXIBs showed radioprotective effects, as did celecoxib, which was used as a control. Radiosensitizing effects were observed when applied shortly after X-ray irradiation. Overall, the NO-COXIBs were found to be more radioprotective compared with celecoxib, which does not warrant further preclinical studies with the NO-COXIBs for the treatment of pheochromocytoma. However, evaluation as radioprotective agents for healthy tissues could be considered for the NO-COXIBs developed here, especially when used directly before irradiation.

Effect of nutrient metabolism on cartilaginous tissue formation.

A major shortcoming in cartilage tissue engineering is the low biosynthetic response of chondrocytes. While different strategies have been investigated, a novel approach may be to control nutrient metabolism. Although known for their anaerobic metabolism, chondrocytes are more synthetically active under conditions that elicit mixed aerobic-anaerobic metabolism. Here, we postulate this metabolic switch induces HIF-1α signaling resulting in improved growth. Transition to different metabolic states can result in the pooling of metabolites, several of which can stabilize HIF-1α by interfering with PHD2. Chondrocytes cultured under increased media availability accelerated tissue deposition with the greatest effect occurring at 2 ml/106 cells. Under higher media availability, metabolism switched from anaerobic to mixed aerobic-anaerobic. Around this transition, maximal changes in PHD2 activity, HIF-1α expression, and HIF-1 target gene expression were observed. Loss-of-function studies using YC-1 confirmed the involvement of HIF-1. Lastly, targeted metabolomic studies revealed that intracellular lactate and succinate correlated with PHD2 activity. This study demonstrates that cartilaginous tissue formation can be regulated by nutrient metabolism and that this response is mediated through changes in HIF-1α signaling. By harnessing this newly identified metabolic switch, engineered cartilage implants may be developed without the need for sophisticated methods which could aid translation to the clinic.

ROS-dependent HIF1α activation under forced lipid catabolism entails glycolysis and mitophagy as mediators of higher proliferation rate in cervical cancer cells.

BACKGROUND: In the last decades, the concept of metabolic rewiring as a cancer hallmark has been expanded beyond the "Warburg effect" and the importance of other metabolic routes, including lipid metabolism, has emerged. In cancer, lipids are not only a source of energy but are also required for the formation of membranes building blocks, signaling and post-translational modification of proteins. Since lipid metabolism contributes to the malignancy of cancer cells, it is an attractive target for therapeutic strategies. METHODS: Over-expression of the adipose triglyceride lipase (ATGL) was used to boost lipid catabolism in cervical cancer cells. The cervical cancer cell line HeLa was employed as the primary experimental model for all subsequent studies. The lipolytic activity of ATGL was mimicked by caproate, a short-chain fatty acid that is efficiently oxidized in mitochondria. RESULTS: Here, we provide evidence of the association between boosted lipid catabolism and the increased proliferation and migration capability of cervical cancer cells. These pro-tumoral effects were ascribed to the reactive oxygen species (ROS)-mediated induction of hypoxia-inducible factor-1α (HIF1α) triggered by the increased mitochondrial fatty acids (FAs) oxidation. HIF1α activation increases glycolytic flux and lactate production, promoting cell proliferation. At the same time, HIF1α increases protein and mRNA levels of its known target BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), which in turn activates mitophagy as a pro-survival process, as demonstrated by the induction of apoptosis upon inhibition of mitophagy. These effects were mimicked by the short-chain fatty acid caproate, confirming that forcing lipid catabolism results in HIF1α induction. CONCLUSIONS: Boosting lipid catabolism by ATGL over-expression has a pro-tumor role in cervical cancer cells, dependent on ROS production and HIF1α induction. Together with the bioinformatics evidence of the correlation of ATGL activity with the aggressiveness of cervical cancer cells, our data suggest that ATGL could be a promising prognostic marker for cervical cancer and highlight the need of further investigations on the role of this lipase in cancer cells. This evidence could be exploited to develop new personalized therapy, based on the functionality of the antioxidant equipment of cancer cells, considering that ROS content could affect ATGL role.

A prospective evaluation of thiamine and magnesium status in relation to clinicopathological characteristics and 1-year mortality in patients with alcohol withdrawal syndrome.

BACKGROUND: Alcohol withdrawal syndrome (AWS) is routinely treated with B-vitamins. However, the relationship between thiamine status and outcome is rarely examined. The aim of the present study was to examine the relationship between thiamine and magnesium status in patients with AWS. METHODS: Patients (n = 127) presenting to the Emergency Department with AWS were recruited to a prospective observational study. Blood samples were drawn to measure whole blood thiamine diphosphate (TDP) and serum magnesium concentrations. Routine biochemistry and haematology assays were also conducted. The Glasgow Modified Alcohol Withdrawal Score (GMAWS) measured severity of AWS. Seizure history and current medications were also recorded. RESULTS: The majority of patients (99%) had whole blood TDP concentration within/above the reference interval (275-675 ng/gHb) and had been prescribed thiamine (70%). In contrast, the majority of patients (60%) had low serum magnesium concentrations (< 0.75 mmol/L) and had not been prescribed magnesium (93%). The majority of patients (66%) had plasma lactate concentrations above 2.0 mmol/L. At 1 year, 13 patients with AWS had died giving a mortality rate of 11%. Male gender (p < 0.05), BMI < 20 kg/m2 (p < 0.01), GMAWS max ≥ 4 (p < 0.05), elevated plasma lactate (p < 0.01), low albumin (p < 0.05) and elevated serum CRP (p < 0.05) were associated with greater 1-year mortality. Also, low serum magnesium at time of recruitment to study and low serum magnesium at next admission were associated with higher 1-year mortality rates, (84% and 100% respectively; both p < 0.05). CONCLUSION: The prevalence of low circulating thiamine concentrations were rare and it was regularly prescribed in patients with AWS. In contrast, low serum magnesium concentrations were common and not prescribed. Low serum magnesium was associated more severe AWS and increased 1-year mortality.

Hypoxia, pseudohypoxia and cellular differentiation.

Tumor hypoxia correlates to aggressive disease, and while this is explained by a variety of factors, one clue to understand this phenomena was the finding that hypoxia induces a de-differentiated, stem cell-like phenotype in neuroblastoma and breast tumor cells. The hypoxia inducible transcription factors (HIFs) are regulated at the translational level by fluctuating oxygen concentrations, but emerging data reveal that both HIF-1α and HIF-2α expression can be induced by aberrantly activated growth factor signaling independently of oxygen levels. Furthermore, HIF-2α is regulated by hypoxia also at the transcriptional level in neuroblastoma and glioma cells. In cultured tumor cells, HIF-2α is stabilized at physiological oxygen concentrations followed by induced expression of classical hypoxia-driven genes, resulting in a pseudohypoxic phenotype. In addition, in neuroblastoma and glioma specimens, a small subset of HIF-2α positive, HIF-1α negative, tumor cells is found adjacent to blood vessels, i.e. in areas with presumably adequate oxygenation. These tumor niches are thus pseudohypoxic, and the HIF-2α expressing cells present immature features. We have postulated that this niche in neuroblastomas encompass the tumor stem cells. Oncogenes or tumor suppressor genes associated with pseudohypoxia are frequently mutated or deleted in the germline, implicating that the pseudohypoxic phenotype indeed is tumorigenic. In summary, the hypoxic and pseudohypoxic phenotypes of solid tumors are attractive therapeutic targets.

Heritable Cancer Syndromes Related to the Hypoxia Pathway.

Families of tumor-suppressor genes, such as those involved in homologous recombination or mismatch repair, contain individual genes implicated in hereditary cancer syndromes. Collectively, such groupings establish that inactivating germline changes in genes within pathways related to genomic repair can promote carcinogenesis. The hypoxia pathway, whose activation is associated with aggressive and resistant sporadic tumors, is another pathway in which tumor-suppressor genes have been identified. von Hippel-Lindau disease, some of the hereditary paraganglioma-pheochromocytoma (PGL/PCC) syndromes, and the syndrome of hereditary leiomyomatosis and renal cell carcinoma are heritable conditions associated with genes involved or associated with the hypoxia pathway. This review links these heritable cancer syndromes to the hypoxia pathway while also comparing the relative aggression and treatment resistance of syndrome-associated tumors to similar, sporadic tumors. The reader will become aware of shared phenotypes (e.g., PGL/PCC, renal cell carcinoma) among these three hypoxia-pathway-associated heritable cancer syndromes as well as the known associations of tumor aggressiveness and treatment resistance within these pathways.

HIF1-α-mediated gene expression induced by vitamin B1 deficiency.

It is well established that thiamine deficiency results in an excess of metabolic intermediates such as lactate and pyruvate, which is likely due to insufficient levels of cofactor for the function of thiamine-dependent enzymes. When in excess, both pyruvate and lactate can increase the stabilization of the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, resulting in the trans-activation of HIF-1α regulated genes independent of low oxygen, termed pseudo-hypoxia. Therefore, the resulting dysfunction in cellular metabolism and accumulation of pyruvate and lactate during thiamine deficiency may facilitate a pseudo-hypoxic state. In order to investigate the possibility of a transcriptional relationship between hypoxia and thiamine deficiency, we measured alterations in metabolic intermediates, HIF-1α stabilization, and gene expression. We found an increase in intracellular pyruvate and extracellular lactate levels after thiamine deficiency exposure to the neuroblastoma cell line SK-N-BE. Similar to cells exposed to hypoxia, there was a corresponding increase in HIF-1α stabilization and activation of target gene expression during thiamine deficiency, including glucose transporter-1 (GLUT1), vascular endothelial growth factor (VEGF), and aldolase A. Both hypoxia and thiamine deficiency exposure resulted in an increase in the expression of the thiamine transporter SLC19A3. These results indicate thiamine deficiency induces HIF-1α-mediated gene expression similar to that observed in hypoxic stress, and may provide evidence for a central transcriptional response associated with the clinical manifestations of thiamine deficiency.