Shailendra Giri

5-Aminoimidazole-4-Carboxamide-1-β-4-Ribofuranoside Inhibits Proinflammatory Response in Glial Cells: A Possible Role of AMP-Activated Protein Kinase

AMP-activated protein kinase (AMPK) is tightly regulated by the cellular AMP:ATP ratio and plays a central role in the regulation of energy homeostasis and metabolic stress. A pharmacological activator of AMPK, 5-amino-4-imidazole carboxamide riboside (AICAR) inhibited lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines (tumor necrosis factor α, interleukin-1β, and interleukin-6) and inducible nitric oxide synthase in primary rat astrocytes, microglia, and peritoneal macrophages. AICAR attenuates the LPS-induced activation of nuclear factor κB via downregulation of IκB kinase α/β activity. It also inhibits nuclear translocation of CCAAT/enhancer-binding protein (C/EBP) transcription factor by inhibiting the expression of C/EBP-δ in brain glial cells. The dominant negative form of AMPKα2 (D157A) and its antisense documents a possible role of AMPK in the regulation of the cellular proinflammatory process. AICAR also inhibited the production of inflammatory mediators in serum and their expression in CNS of rats injected with a sublethal dose of LPS by intraperitoneal injection. These observations in cultured cells as well as in the animal model suggest that AICAR may be of therapeutic value in treating inflammatory diseases.

The 15-Deoxy-δ12,14-Prostaglandin J 2 Inhibits the Inflammatory Response in Primary Rat Astrocytes via Down-Regulating Multiple Steps in Phosphatidylinositol 3-Kinase-Akt-NF-κB-p300 Pathway Independent of Peroxisome Proliferator-Activated Receptor γ

Ligands for peroxisome proliferator-activated receptor γ (PPARγ), such as 15-deoxy-12,14-PGJ2 (15d-PGJ2), have been proposed as a new class of anti-inflammatory compounds because 15d-PGJ2 was able to inhibit the induction of inflammatory response genes such as inducible NO synthase (iNOS) and TNF (TNF-α) in a PPAR-dependent manner in various cell types. In primary astrocytes, the anti-inflammatory effects (inhibition of TNF-α, IL-1β, IL-6, and iNOS gene expression) of 15d-PGJ2 are observed to be independent of PPARγ. Overexpression (wild-type and dominant-negative forms) of PPARγ and its antagonist (GW9662) did not alter the 15d-PGJ2-induced inhibition of LPS/IFN-γ-mediated iNOS and NF-κB activation. The 15d-PGJ2 inhibited the inflammatory response by inhibiting IκB kinase activity, which leads to the inhibition of degradation of IκB and nuclear translocation of p65, thereby regulating the NF-κB pathway. Moreover, 15d-PGJ2 also inhibited the LPS/IFN-γ-induced PI3K-Akt pathway. The 15d-PGJ2 inhibited the recruitment of p300 by NF-κB (p65) and down-regulated the p300-mediated induction of iNOS and NF-κB luciferase reporter activity. Coexpression of constitutive active Akt and PI3K (p110) reversed the 15d-PGJ2-mediated inhibition of p300-induced iNOS and NF-κB luciferase activity. This study demonstrates that 15d-PGJ2 suppresses inflammatory response by inhibiting NF-κB signaling at multiple steps as well as by inhibiting the PI3K/Akt pathway independent of PPARγ in primary astrocytes.

Molecular mechanism of psychosine-induced cell death in human oligodendrocyte cell line

This study delineates the molecular mechanism underlying psychosine‐induced oligodendroglial cell death. An immortalized human oligodendroglial cell line, MO3.13, was treated with exogenous psychosine (β‐galactosylsphingosine), a toxic metabolite that accumulates in the tissues of patients with Krabbes disease. The mode of cell death induced by psychosine was found to be apoptotic, as revealed by different apoptotic markers viz., TUNEL, DNA fragmentation and caspase cleavage/activation. The action of psychosine was redox sensitive, as measured by changes in mitochondrial membrane potential (ψΔ), and this effect of psychosine could be reversed by pre‐treatment with the antioxidant molecules N‐acetyl‐l‐cysteine or pro‐cysteine. Psychosine directly affects the mitochondria as revealed by the activation of caspase 9 but not caspase 8. Up‐regulation of the c‐jun/c‐jun N‐terminal kinase pathway by psychosine leads to the induction of AP‐1 and, at the same time, psychosine also down‐regulates the lipopolysaccharide‐induced NF‐κB transactivation. These observations indicate that the mechanism of action of psychosine is, through the up‐regulation of AP‐1, a pro‐apoptotic pathway as well as, through the down‐regulation of the NF‐κB pathway, an antiapoptotic pathway.

PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples

BACKGROUND Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer. METHODS PG545s anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models. RESULTS PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response. CONCLUSION Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

AMP-Activated Protein Kinase Suppresses Autoimmune Central Nervous System Disease by Regulating M1-Type Macrophage-Th17 Axis.

The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood–brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10lowIL-23/IL-1β/IL-6high), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35–55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35–55–primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4+ T cells were due to an autocrine effect of IL-1β/IL-23–mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype–Th17 axis in an animal model of multiple sclerosis.

Absence of IFN-γ Increases Brain Pathology in Experimental Autoimmune Encephalomyelitis–Susceptible DRB1*0301.DQ8 HLA Transgenic Mice through Secretion of Proinflammatory Cytokine IL-17 and Induction of Pathogenic Monocytes/Microglia into the Central Nervous System

Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)–induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP91–110–induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ−/−). Immunization with PLP91–110 peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ−/− mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.

Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer

BackgroundLoss of the endosulfatase HSulf-1 is common in ovarian cancer, upregulates heparin binding growth factor signaling and potentiates tumorigenesis and angiogenesis. However, metabolic differences between isogenic cells with and without HSulf-1 have not been characterized upon HSulf-1 suppression in vitro. Since growth factor signaling is closely tied to metabolic alterations, we determined the extent to which HSulf-1 loss affects cancer cell metabolism.ResultsIngenuity pathway analysis of gene expression in HSulf-1 shRNA-silenced cells (Sh1 and Sh2 cells) compared to non-targeted control shRNA cells (NTC cells) and subsequent Kyoto Encyclopedia of Genes and Genomics (KEGG) database analysis showed altered metabolic pathways with changes in the lipid metabolism as one of the major pathways altered inSh1 and 2 cells. Untargeted global metabolomic profiling in these isogenic cell lines identified approximately 338 metabolites using GC/MS and LC/MS/MS platforms. Knockdown of HSulf-1 in OV202 cells induced significant changes in 156 metabolites associated with several metabolic pathways including amino acid, lipids, and nucleotides. Loss of HSulf-1 promoted overall fatty acid synthesis leading to enhance the metabolite levels of long chain, branched, and essential fatty acids along with sphingolipids. Furthermore, HSulf-1 loss induced the expression of lipogenic genes including FASN, SREBF1, PPARγ, and PLA2G3 stimulated lipid droplet accumulation. Conversely, re-expression of HSulf-1 in Sh1 cells reduced the lipid droplet formation. Additionally, HSulf-1 also enhanced CPT1A and fatty acid oxidation and augmented the protein expression of key lipolytic enzymes such as MAGL, DAGLA, HSL, and ASCL1. Overall, these findings suggest that loss of HSulf-1 by concomitantly enhancing fatty acid synthesis and oxidation confers a lipogenic phenotype leading to the metabolic alterations associated with the progression of ovarian cancer.ConclusionsTaken together, these findings demonstrate that loss of HSulf-1 potentially contributes to the metabolic alterations associated with the progression of ovarian pathogenesis, specifically impacting the lipogenic phenotype of ovarian cancer cells that can be therapeutically targeted.

Metformin‐induced mitochondrial function and ABCD2 up‐regulation in X‐linked adrenoleukodystrophy involves AMP‐activated protein kinase

X‐linked adrenoleukodystrophy (X‐ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP‐activated protein kinase α1 (AMPKα1) in ALD patient‐derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro‐inflammatory response. The FDA approved anti‐diabetic drug Metformin, a well‐known AMPK activator, induces mitochondrial biogenesis and is documented for its anti‐inflammatory role. We observed a dose‐dependent activation of AMPKα1 in metformin‐treated X‐ALD patient‐derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X‐ALD patient‐derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro‐inflammatory cytokine gene expressions in X‐ALD patient‐derived cells. Abcd2 [adrenoleukodystrophy protein‐related protein] levels were increased in metformin‐treated X‐ALD patient‐derived fibroblasts and Abcd1‐KO mice primary mixed glial cells. Abcd2 induction was AMPKα1‐dependent since metformin failed to induce Abcd2 levels in AMPKα1‐KO mice‐derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1‐KO mice. Taken together, these results provide proof‐of‐principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X‐ALD by targeting AMPK.

Bioenergetic Adaptations in Chemoresistant Ovarian Cancer Cells

Earlier investigations have revealed that tumor cells undergo metabolic reprogramming and mainly derive their cellular energy from aerobic glycolysis rather than oxidative phosphorylation even in the presence of oxygen. However, recent studies have shown that certain cancer cells display increased oxidative phosphorylation or high metabolically active phenotype. Cellular bioenergetic profiling of 13 established and 12 patient derived ovarian cancer cell lines revealed significant bioenergetics diversity. The bioenergetics phenotype of ovarian cancer cell lines correlated with functional phenotypes of doubling time and oxidative stress. Interestingly, chemosensitive cancer cell lines (A2780 and PEO1) displayed a glycolytic phenotype while their chemoresistant counterparts (C200 and PEO4) exhibited a high metabolically active phenotype with the ability to switch between oxidative phosphorylation or glycolysis. The chemosensitive cancer cells could not survive glucose deprivation, while the chemoresistant cells displayed adaptability. In the patient derived ovarian cancer cells, a similar correlation was observed between a high metabolically active phenotype and chemoresistance. Thus, ovarian cancer cells seem to display heterogeneity in using glycolysis or oxidative phosphorylation as an energy source. The flexibility in using different energy pathways may indicate a survival adaptation to achieve a higher ‘cellular fitness’ that may be also associated with chemoresistance.

Targeting of free fatty acid receptor 1 in EOC: A novel strategy to restrict the adipocyte-EOC dependence

OBJECTIVES Adipocyte derived free fatty acids (FFA) promote epithelial ovarian cancer (EOC) by acting as a fuel source to support the energy requirement of the cancer cells. FFA may also exert biological effects through signaling pathways. Recently, a family of FFA activated G-protein coupled receptors (FFAR/GPCRs) was identified. Our objective was to investigate the role of FFAR/GPCRs in EOC and assess their potential as therapeutic targets. METHODS The mRNA (RT-PCR) expression of FFAR/GPCR family members (FFAR1/GPR40; FFAR2/GPR43, FFAR3/GPR41, FFAR4/GPR120 and GPR84) was examined in: (1) a syngeneic mouse model of EOC fed high energy diet (60% fat) or regular diet (30% fat), (2) EOC cell lines exposed to free fatty acids and (3) specimens from 13 histologically normal ovaries and 28 high grade ovarian serous carcinomas. The GPR 40 antagonist, GW1100, was used to inhibit FFAR1/GPR40 and cell survival was assayed by MTT in various cell lines. RESULTS High Grade Serous carcinoma specimens expressed significantly increased GPR40 compared to normal ovaries (p=0.0020). Higher expression was noted in advanced stage disease. ID8 ovarian tumors from mice fed with high fat diet also showed higher GPR40 expression. Exposing EOC cells to FFAs, increased GPR40 expression. Treatment of EOC cell lines with GW100 resulted in growth inhibition and was associated with an alteration in their energy metabolism. CONCLUSION FFA-induced cancer cell growth may be partly mediated through FFAR1/GPR40. Targeting of FFAR1/GPR40 may be an attractive treatment strategy in EOC, and possibly offers a targeted treatment for a subset of EOC patients.