Activation induces shift in nutrient utilization that differentially impacts cell functions in human neutrophils.

Neutrophils - the first responders in innate immunity - perform a variety of effector functions associated with specific metabolic demand. To maintain fitness and support functions, neutrophils have been found to utilize extracellular glucose, intracellular glycogen, and other alternative substrates. However, the quantitative contribution of these nutrients under specific conditions and the relative dependence of various cell functions on specific nutrients remain unclear. Here, using ex vivo and in vivo isotopic tracing, we reveal that under resting condition, human peripheral blood neutrophils, in contrast to in vitro cultured human neutrophil-like cell lines, rely on glycogen as a major direct source of glycolysis and pentose phosphate pathway. Upon activation with a diversity of stimuli, neutrophils undergo a significant and often rapid nutrient preference shift, with glucose becoming the dominant metabolic source thanks to a multi-fold increase in glucose uptake mechanistically mediated by the phosphorylation and translocation of GLUT1. At the same time, cycling between gross glycogenesis and glycogenolysis is also substantially increased, while the net flux favors sustained or increased glycogen storage. The shift in nutrient utilization impacts neutrophil functions in a function-specific manner. The activation of oxidative burst specifically depends on the utilization of extracellular glucose rather than glycogen. In contrast, the release of neutrophil traps can be flexibly supported by either glucose or glycogen. Neutrophil migration and fungal control is promoted by the shift away from glycogen utilization. Together, these results quantitatively characterize fundamental features of neutrophil metabolism and elucidate how metabolic remodeling shapes neutrophil functions upon activation.

EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression.

Exchange proteins directly activated by cAMP (EPAC) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activates RAP1 and the alternative cAMP signaling pathway. We previously showed that the differential growth response of primary and metastatic melanoma cells to cAMP is mediated by EPAC. However, the mechanisms responsible for this differential response to EPAC signaling are not understood. In this study, we show that pharmacologic inhibition or siRNA-mediated knockdown of EPAC selectively inhibits the growth and survival of primary melanoma cells by downregulation of cell-cycle proteins and inhibiting the cell-cycle progression independent of ERK1/2 phosphorylation. EPAC inhibition results in upregulation of AKT phosphorylation but a downregulation of mTORC1 activity and its downstream effectors. We also show that EPAC regulates both glycolysis and oxidative phosphorylation, and production of mitochondrial reactive oxygen species, preferentially in primary melanoma cells. Employing a series of genetically matched primary and lymph node metastatic (LNM) melanoma cells, and distant organ metastatic melanoma cells, we show that the LNM and metastatic melanoma cells become progressively less responsive and refractory to EPAC inhibition suggesting loss of dependency on EPAC signaling correlates with melanoma progression. Analysis of The Cancer Genome Atlas dataset showed that lower RAPGEF3, RAPGEF4 mRNA expression in primary tumor is a predictor of better disease-free survival of patients diagnosed with primary melanoma suggesting that EPAC signaling facilitates tumor progression and EPAC is a useful prognostic marker. These data highlight EPAC signaling as a potential target for prevention of melanoma progression. IMPLICATIONS: This study establishes loss of dependency on EPAC-mTORC1 signaling as hallmark of primary melanoma evolution and targeting this escape mechanism is a promising strategy for metastatic melanoma.

Design, synthesis and broad spectrum antibreast cancer activity of diarylindoles via induction of apoptosis in aggressive breast cancer cells.

Breast cancer is the second leading cause of cancer deaths in women with significant morbidity and mortality. Present study describes design, synthesis and detailed pharmacology of indole derivatives exhibiting remarkable broad spectrum antiproliferative activity against breast cancer cells. Detailed mechanistic evaluations confirmed induction of G0/G1 arrest, apoptosis induction, loss of mitochondrial integrity, enhanced ROS generation, autophagy, estrogen receptor ß-transactivation and increased tubulin polymerization. In in-vivo efficacy studies in rodent model, these indole derivatives induced significant regression in mice mammary tumour on 21 days daily oral dose. Moreover, compounds 19 and 23 were safe in Swiss albino mice in safety studies. These diarylindoles may further be optimized for better efficacy.

How to starve cancer cells when nutrients are abundant.

Metabolic flexibility represents a potential point of attack for novel cancer treatments. We recently described the signaling mechanism inducing a metabolic shift in response to metformin and phenformin in leukemia and lymphoma cells. Enhanced glucose utilization was critically dependent on mitochondrial stress signaling/hypoxia-inducible factor-1α representing a therapeutic vulnerability.

Molecular epidemiology and associated risk factors of Anaplasma marginale and Theileria annulata in cattle from North-western Pakistan.

Ticks and tick-borne pathogens are one of the major threats to livestock production worldwide. The aim of present study is to specify the molecular epidemiology and its associated risk factors of two well-distributed tick-borne pathogens Anaplasma marginale and Theileria annulata in cattle population from North-western Pakistan. Blood samples were Collected from 60 (32.6 %) local breed cattle, 101 (54.9 %) from crossbreed, and 24 (13.0 %) from exotic breed with total of 184 blood samples. Species-specific PCR assays were performed to detect the presence of A. marginale and T. annulata based on 16S rRNA and 18S rRNA genetic makers respectively. PCR results showed that A. marginale was 16.3 % prevalent and T. annulata was 29.9 % prevalent in the study area with a total prevalence rate of 46.2 % (85/184) of the tested blood samples. District wise analysis revealed that both pathogens were highly prevalent in district Dir Upper (13.6 %) and least prevalent in district Chitral (10.3 %). Univariable analysis of risk factors showed that only breed and acaricidal treatment were significant determinants (P < 0.05) for A. marginale infection, however, in case of T. annulata infection; breed, age, gender, grazing practice, and acaricidal treatment were potential determinants (P < 0.05). Multivariable analysis specified that breed and acaricidal treatment were considered as significant risk factors for A. marginale infection (P < 0.05) whereas acaricidal treatment was found to be a significant determinant for T. annulata infection (P < 0.05). Phylogenetic analysis indicated that A. marginale 16S rRNA and T. annulata 18S rRNA isolates showed similarities and shared phylogeny with same isolates reported from Asia. This is the first molecular report on the epidemiology and risk factors analysis of A. marginale and T. annulata infections in cattle population from NW Pakistan. Further large scale study is required to investigate molecular, epidemiological and genotypic aspects as well as potential risk factors analysis from the country to facilitate designing strategies to control tick-borne pathogen and reduce losses to cattle industry.

Metabolic Rewiring in Response to Biguanides Is Mediated by mROS/HIF-1a in Malignant Lymphocytes.

Metabolic flexibility allows cells to adapt to various environments and limits the efficacy of metabolic drugs. Therapeutic targeting of cancer metabolism relies on defining limiting requirements and vulnerabilities in the highly dynamic metabolic network. Here, we characterize the metabolic reprogramming and identify cancer-specific metabolic vulnerabilities in response to the pharmacological inhibition of mitochondrial complex I. Our work reveals the adaptation mechanism in malignant lymphocytes providing resistance against the biguanides phenformin and metformin by transcriptionally reprogramming glucose metabolism. Metabolic adaptation to complex I inhibition is mediated by mitochondrial reactive oxygen species (mROS) serving as a mitochondrial stress signal activating hypoxia-inducible factor-1a (HIF-1a). Inhibition of the mROS/HIF-1a axis through antioxidants or direct suppression of HIF-1a selectively disrupts metabolic adaptation and survival during complex I dysfunction in malignant lymphocytes. Our results identify HIF-1a signaling as a critical factor in resistance against biguanide-induced mitochondrial dysfunction, allowing selective targeting of metabolic pathways in leukemia and lymphoma.

A study of oxidative stress, cytokines and glutamate in Wilson disease and their asymptomatic siblings.

BACKGROUND: Free copper in Wilson disease (WD) is toxic and may reduce antioxidant, increase oxidative stress marker and thereby cytokine release and excitotoxic injury, but there is paucity of studies in humans. We report oxidative stress markers, cytokines and glutamate in neurologic WD and correlate these with their clinical severity, laboratory findings and extent of Magnetic resonance imaging (MRI) changes. METHODS: 29 patients with neurologic WD and 9 asymptomatic WD siblings were included and their clinical, treatment history, disease severity, biochemical findings and MRI changes were noted. Glutathione (GSH), total antioxidant capacity (TAC) and malonodialdehyde (MDA) were measured by spectrophotometer, cytokines by cytokine bead array and glutamate by the fluorometer. RESULTS: In WD patients, the glutathione (mean±SEM, 2.20±0.06 vs. 2.73±0.04mg/dl, P<0.001) and TAC (1.70±0.03 vs. 2.29±0.02 Trolox_Eq_mmol/l, P<0.001) were reduced, and MDA and glutamate (23.93±0.54 vs. 19.96±0.27µmol/l; P<0.001) were increased (4.7±0.11 vs. 3.03±0.52nmol/ml, P<0.001) compared to controls. The serum IL6 {median (IQRs), 9.42(10.92) vs. 5.2(5.34) pg/ml; P=0.001}, IL8 {12.37(10.92) vs. 5.63(5.52) pg/ml; P<0.001}, IL10 {8.33(8.3) vs. 2.05(1.37) pg/ml; P=0.001} and TNFα {6.14(8.95) vs. 3.61(3.58) pg/ml; P<0.001} were also increased in WD patients compared to controls. These changes were more marked in the neurologic WD compared to asymptomatic WD and in the untreated compared to treated patients. TAC correlated with duration of illness, serum free copper, 24hour urinary copper and serum ceruloplasmin, and glutamate with MDA, TNFα, ceruloplasmin and 24-hour urinary copper. CONCLUSIONS: In WD patients, antioxidants are reduced and MDA, cytokines and glutamate are increased which are more marked in symptomatic neurologic WD than asymptomatic patients.