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SUMMARYMetabolic aberrations impact the pathogenesis of multiple sclerosis (MS) and possibly can provide clues for new treatment strategies. Using untargeted metabolomics, we measured serum metabolites from 35 relapsing-remitting patients and 14 healthy age-matched controls. Out of 632 known metabolites detected, 60 were significantly altered in relapsing-remitting MS (RRMS). Bioinformatics analysis identified an altered “metabotype” in RRMS patients, represented by 4 changed metabolic pathways of glycerophospholipid, citrate cycle, sphingolipid, and pyruvate metabolism. Interestingly, the common upstream metabolic pathway feeding these 4 pathways is the glycolysis pathway. Real-time bioenergetic analysis of the patient derived peripheral blood mononuclear cells, showed enhanced glycolysis, supporting the altered metabolic state of immune cells. Experimental autoimmune encephalomyelitis mice treated with the glycolytic inhibitor, 2-deoxy-D-glucose ameliorated the disease progression and inhibited the disease pathology significantly by promoting the anti-inflammatory phenotype of monocytes/macrophage in the central nervous system. Our study suggests that targeting glycolysis offers a potential target for MS.

Gynecologic cancers, starting in the reproductive organs of females, include cancer of cervix, endometrium, ovary commonly and vagina and vulva rarely. The changes in the composition of microbiome in gut and vagina affect immune and metabolic signaling of the host cells resulting in chronic inflammation, angiogenesis, cellular proliferation, genome instability, epithelial barrier breach and metabolic dysregulation that may lead to the onset or aggravated progression of gynecologic cancers. While microbiome in gynecologic cancers is just at horizon, certain significant microbiome signature associations have been found. Cervical cancer is accompanied with high loads of human papillomavirus, Fusobacteria and Sneathia species; endometrial cancer is reported to have presence of Atopobium vaginae and Porphyromonas species and significantly elevated levels of Proteobacteria and Firmicutes phylum bacteria, with Chlamydia trachomatis, Lactobacillus and Mycobacterium reported in ovarian cancer. Balancing microbiome composition in gynecologic cancers has the potential to be used as a therapeutic target. For example, the Lactobacillus species may play an important role in blocking adhesions of incursive pathogens to vaginal epithelium by lowering the pH, producing bacteriocins and employing competitive exclusions. The optimum or personalized balance of the microbiota can be maintained using pre- and probiotics, and fecal microbiota transplantations loaded with specific bacteria. Current evidence strongly suggest that a healthy microbiome can train and trigger the body's immune response to attack various gynecologic cancers. Furthermore, microbiome modulations can potentially contribute to improvements in immuno-oncology therapies.

Objectives: The purpose of this study was to identify genes and pathways differentially expressed in platinum resistant high grade serous ovarian cancer (HGSOC) when compared to sensitive HGSOC. Methods: A total of 37 patients with HGSOC tissue samples underwent RNA sequencing performed by TEMPUS (N=37, 21 platinum sensitive, 16 resistant; 85% Stage III-IV; 58% received neoadjuvant chemotherapy). RNA gene expression data and significantly impacted pathways were analyzed using Advaita Bio's iPathwayGuide. Differentially expressed (DE) genes were identified using FDR of 0.05 and fold-change of 1.5. Genes from several impacted canonical metabolic pathways were validated by PCR against external data sets in a separate ovarian cancer sample group (n=15), platinum resistant ovarian cancer mouse tumor model, and wild-type sensitive and platinum resistant ovarian cancer cell lines. Relative gene expression was calculated using the comparative Ct method, also referred to as the “2 DDCT”, using L27 as internal control gene. Download : Download high-res image (333KB) Download : Download full-size image Results: We identified 177 differentially expressed (DE) genes out of a total of 16,607 genes (1.1%) with measured expression. 15 pathways were found to be significantly impacted. Of the 15 canonical pathways, all were up regulated in the resistant HGSOC and the majority of the most significantly altered (5/10) were related to metabolism (Retinol metabolism (p-value = 0.002); Tyrosine Metabolism (p-value = 0.005); Tryptophan Metabolism (p-value = 0.009); and Phenylalanine Metabolism (p-value = 0.012); CYP Drug Metabolism (p-value = 0.022)). A total of 3 separate genes from the CYP family and two from the Dopa Decarboxylase family of genes were validated against an external data set of human ovarian tissue samples, cell lines, mouse ovarian tumor model, and found to have similarly increased gene expression in the genes tested in the platinum resistant groups. Compilation of KEGG analysis and the common network genes revealed pathways associated with amino acid metabolism to be most significantly altered. Conclusions: We describe the identification of a unique transcriptomic profile associated with platinum resistance. Interestingly, the main pathways identified are related to metabolism, suggesting that the survival to chemotherapy demands a major metabolic adaptation. These findings also represent a first step towards the identification of biomarkers for the detection of chemo-resistant disease and metabolism-based drug targets specific for chemo-resistant tumors. Further validation of this model is required in order to determine its clinical value.

The circulating metabolome is a product of interactions between the genome, epigenome, exposome and microbiome. The metabolome may be altered in people with multiple sclerosis (MS); however, existing metabolomics studies were relatively small or characterized a limited number of metabolites. Herein, we performed a multi-site study profiling the circulating metabolome to obtain relative abundances for 269 metabolites in a large cohort of MS patients and healthy controls. After adjusting for batch effects and extensive quality control, we created an overall metabolic dysfunction score, defined apriori sets of metabolites using known metabolic pathways, and derived novel networks of correlated metabolites using a weighted correlation network analysis (WGCNA). We assessed whether metabolic dysfunction, individual metabolites, metabolic pathways or WGCNA-identified module scores differed between people with MS versus healthy controls (HC) after adjusting for age, sex and race using generalized estimating equations (participants could provide multiple samples). In a subset of patients, information on disability status was also available. Similar models assessed the association between metabolites and metabolite sets with measures of disability. In people with MS, we identified striking abnormalities in a WGCNA-defined module enriched in aromatic amino acid (AAA) metabolites (FDR-adjusted p-value=2.77E-18) that are also strongly associated with disability (FDR-adjusted p-value for AAA module=1.01E-4). Consistent results were obtained using apriori-defined metabolite sets or in analyses of individual metabolites. The identified abnormalities likely relate to imbalances in gut microbial metabolism of AAAs resulting in reduced production of immunomodulatory metabolites and increased production of metabotoxins (indole acetate, phenylacetylglutamine, p-cresol sulfate, p-cresol glucuronide). Single cell RNA sequencing data analysis demonstrated altered AAA metabolism in CSF and blood derived monocyte cell populations, while treatment of human peripheral blood mononuclear cells with AAA-derived metabotoxins resulted in increased production of tumor necrosis factor-α. We identify novel metabolic alterations in people with MS potentially contributing to disease pathophysiology.

Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found 2 peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on T cell immunoglobulin and mucin domain containing 4 (Tim-4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4– TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4– TAMs, promoted tumor growth in vivo. Relative to Tim-4– TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1–mediated metabolism. Furthermore, genetic deficiency of autophagy element FAK family-interacting protein of 200 kDa resulted in Tim-4+ TAM loss via ROS-mediated apoptosis and elevated T cell immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer–associated macrophages positive for complement receptor of the immunoglobulin superfamily (CRIg) were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat patients with ovarian cancer with peritoneal metastasis., Ovarian tumor-associated peritoneal macrophages have potent mitochondria activity, which enables reliance on the mitophagy pathway to alleviate oxidative stress and facilitate survival in the tumor microenvironment.

Summary The circulating metabolome provides unique insights into multiple sclerosis (MS) pathophysiology, but existing studies are relatively small or characterized limited metabolites. We test for differences in the metabolome between people with MS (PwMS; n = 637 samples) and healthy controls (HC; n = 317 samples) and assess the association between metabolomic profiles and disability in PwMS. We then assess whether metabolic differences correlate with changes in cellular gene expression using publicly available scRNA-seq data and whether identified metabolites affect human immune cell function. In PwMS, we identify striking abnormalities in aromatic amino acid (AAA) metabolites (p = 2.77E−18) that are also strongly associated with disability (p = 1.01E−4). Analysis of scRNA-seq data demonstrates altered AAA metabolism in CSF and blood-derived monocyte cell populations in PwMS. Treatment with AAA-derived metabolites in vitro alters monocytic endocytosis and pro-inflammatory cytokine production. We identify shifts in AAA metabolism resulting in the reduced production of immunomodulatory metabolites and increased production of metabotoxins in PwMS., Graphical abstract, Highlights • Significant alterations in the circulating metabolome are noted in multiple sclerosis • Aromatic amino acid (AAA) metabolite levels are linked to disease severity • Expression of AAA metabolism genes is altered in MS blood and CSF immune cells • AAA metabolites alter human monocyte cytokine production and endocytosis, Fitzgerald et al. identify alterations in circulating aromatic amino acid (AAA) metabolites in multiple sclerosis that are related to disease severity. They also demonstrate changes in AAA metabolism-related gene expression in blood and cerebrospinal fluid immune cells, and that treatment with AAA metabolites alters monocyte pro-inflammatory cytokine production and endocytosis.

Objective Low-grade serous ovarian cancer (LGSOC) constitutes 5–8% of epithelial ovarian cancers and is refractory to chemotherapy. We and others have shown metformin to cause significant growth inhibition in high-grade ovarian cancer both in vitro and in vivo. Here, we aimed to analyze if metformin was effective in inhibiting proliferation of LGSOC alone and in combination with MEK inhibitor. Methods Three LGSOC lines (VOA1056, VOA1312 and VOA5646) were treated with metformin, trametinib or 2-deoxyglucose (2DG) alone or in combination with metformin. Proliferation was measured by MTT assay over a period of four days. Protein expression was measured by western blotting. Seahorse Analyzer was used to measure effect of metformin on glycolysis and mitochondrial respiration. Results All LGSOC cell lines showed significant inhibition with metformin in a dose- and time-dependent manner. Trametinib significantly inhibited the growth of Ras mutated LGSOC lines (VOA1312 and VOA1056), while VOA5646 cells without RAS mutation did not show any response. Metformin and trametinib combination showed synergistic inhibition of RAS mutated VOA1312 and VOA1056 cells, but not for non-Ras mutated VOA5646 cells. Metformin and trametinib increased phosphorylated AMPK expression in LGSOC lines with combination showing stronger expression. Trametinib decreased 42/44 mitogen activated kinase phosphorylation in all cell lines, while metformin and combination had no significant effect. 2-DG significantly inhibited glycolysis in all LGSOC lines and combination with metformin showed synergistic inhibitory effect. Conclusions Metformin alone or in combination with MEK and glycolytic inhibitors may be a potential therapy for LGSOC, a cancer that is indolent but chemo-resistant.

Purpose Age, sex, and genetics are important biological variables in determining an individual's susceptibility or response to infectious agents; however, their role has not been evaluated in intraocular infections. In this study, we comprehensively examined the impact of these host biological factors in the pathogenesis of experimental bacterial endophthalmitis. Methods Endophthalmitis was induced by intravitreal injection of bacteria (Staphylococcus aureus) in the eyes of male and female C57BL/6 mice of different ages: group I (young, 6-8 weeks), group II (mid-age, 18-20 weeks), and group III (old, 1 year). Highly heterogeneous outbred J:DO mice were used for genetic diversity analysis. Eyes were subjected to clinical examination, retinal function testing using electroretinography (ERG), histopathological analysis (hematoxylin and eosin staining), and bacterial burden estimation. The levels of inflammatory mediators were measured using qPCR and ELISA, and the infiltration of neutrophils was determined by flow cytometry. Results Both inbred C57BL/6 and diversity outbred (J:DO) mice were equally susceptible to S. aureus endophthalmitis, as evidenced by a time-dependent increase in clinical scores, bacterial burden, intraocular inflammation, and retinal tissue damage, in addition to decreased retinal function. However, no significant differences were observed in disease severity and innate responses in male versus female mice. Older mice (group III) exhibited higher clinical scores coinciding with increased bacterial proliferation and intraocular inflammation, resulting in enhanced disease severity. Moreover, bone-marrow-derived macrophages from old mice exhibited reduced phagocytic activity but increased inflammatory response toward S. aureus challenge. Conclusions Age, but not sex, is an important biological variable in bacterial endophthalmitis. Identification of pathways underlying altered innate immunity and impaired bacterial clearance in aging eyes could provide new insights into the pathobiology of intraocular infections in elderly patients.

BRCA mutated ovarian cancers show increased responsiveness to PARP inhibitors. PARP inhibitors target DNA repair and provide a second hit to BRCA mutated tumors, resulting in "synthetic lethality". We investigated a combination of metformin and olaparib to provide "synthetic lethality" in BRCA intact ovarian cancer cells.Ovarian cancer cell lines (UWB1.289, UWB1.289.BRCA, SKOV3, OVCAR5, A2780 and C200) were treated with a combination of metformin and olaparib. Cell viability was assessed by MTT and colony formation assays. Flow cytometry was used to detect cell cycle events. In vivo studies were performed in SKOV3 or A2780 xenografts in nude mice. Animals were treated with single agent, metformin or olaparib or combination. Molecular downstream effects were examined by immunohistochemistry.Compared to single drug treatment, combination of olaparib and metformin resulted in significant reduction of cell proliferation and colony formation (p0.001) in ovarian cancer cells. This treatment was associated with a significant S-phase cell cycle arrest (p0.05). Combination of olaparib and metformin significantly inhibited SKOV3 and A2780 ovarian tumor xenografts which were accompanied with decreased Ki-index (p0.001). Metformin did not affect DNA damage signaling, while olaparib induced adenosine monophosphate activated kinase activation; that was further potentiated with metformin combination in vivo.Combining PARP inhibitors with metformin enhances its anti-proliferative activity in BRCA mutant ovarian cancer cells. Furthermore, the combination showed significant activity in BRCA intact cancer cells in vitro and in vivo. This is a promising treatment regimen for women with epithelial ovarian cancer irrespective of BRCA status.

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.

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.

Signal Transducers and Activators of Transcription 3 (STAT3) is a transcription factor that is known to play a key role in cancer progression. In ovarian cancer, STAT3 overexpression leads to increased cancer cell proliferation and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. It is constitutively activated in patient-derived ovarian cancer cells and a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to translocate to mitochondria, facilitated by phosphorylation at S727 and modulate mitochondrial function to promote carcinogenesis. Our study aimed to investigate if mitochondrial-STAT3, rather than nuclear-STAT3, plays a major role in modulating cellular metabolism and promoting chemoresistance in ovarian cancer cells. We generated stable clones overexpressing STAT3 in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 resulted in increased proliferation, colony formation ability, and chemoresistance in vitro and led to large and aggressive ovarian tumors compared to parental and vector controls in the xenograft mouse model. STAT3 overexpressing clones exhibited higher mitochondrial respiration and glycolysis placing them in the “metabolically active” phenotype compared to parental and vector clones (metabolically less active phenotype). A STAT3 inhibitor, Stattic, inhibited both nuclear and mitochondrial STAT3 and also attenuated the STAT3-mediated growth of overexpressing clones both in vitro and in vivo. Stattic treatment also reversed the STAT3-mediated chemoresistance. In contrast, a selective inhibitor of STAT3-Y705, cryptotanshinone, was relatively less effective. Also, Stattic treatments reversed the “metabolically active” state of STAT3 overexpressing clones to a “lower metabolic state,” as the control cells. Stattic also inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200, and OVCAR3) that display a metabolically active phenotype, suggesting STAT3 plays a vital role in attaining a metabolically active phenotype by cancer cells. Further, silencing STAT3-Y705 (nuclear-STAT3) did not significantly affect the chemoresistance, while silencing STAT3-S727 (mitochondrial-STAT3) reversed chemoresistance. Although an increase in mitochondrial function was observed in overexpressing A2780 clones as evident from enhanced oxidative phosphorylation, there was no change in the mitochondrial mass or number, indicating the critical role of STAT3 in mitochondrial function rather than mitochondrial biogenesis in ovarian cancer cells. Thus, further evaluation of expression and function of mitochondrial STAT3 in ovarian cancers is warranted. Overall, mitochondrial-STAT3 can induce metabolic changes in ovarian cancer cells and enhance their cellular fitness by promoting chemoresistance. Citation Format: Vaishnavi Raja, Shailendra Giri, Suhail Hamid, Thomas Buekers, Adnan Munkarah, Ramandeep Rattan. Role of mitochondrial-STAT3 in promoting chemoresistance by modulating the energy metabolism in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A69.

Signal Transducers and Activators of Transcription 3 (STAT3) is a transcription factor that is known to play a key role in cancer progression. In ovarian cancer, STAT3 overexpression leads to increased cancer cell proliferation and confers resistance to chemotherapy-induced apoptosis in epithelial malignancies. It is constitutively activated in patient-derived ovarian cancer cells and a predictor of poor prognosis. Apart from its function as a transcription factor, recently STAT3 has been shown to translocate to mitochondria facilitated by phosphorylation at S727 and modulate mitochondrial function to promote carcinogenesis. Our study aimed to investigate if STAT3 overexpression can modulate cellular metabolism and promote the growth of ovarian cancer cells. We generated stable clones overexpressing STAT3 in A2780 ovarian cancer cells, along with empty vector clones. Ectopic expression of STAT3 in A2780 resulted in increased proliferation, colony formation ability and chemoresistance in vitro and led to large and aggressive ovarian tumors compared to parental and vector controls in xenograft mouse model. STAT3 overexpressing clones exhibited higher mitochondrial respiration and glycolysis placing them in the ‘metabolically active' phenotype compared to parental and vector clones (metabolically less active phenotype). A selective inhibitor of STAT3, Stattic, inhibited both nuclear and mitochondrial STAT3 and also attenuated the STAT3 mediated growth of over-expressing clones both in vitro and in vivo. Stattic treatment also reversed the STAT3-mediated chemoresistance. In contrast, a selective inhibitor of STAT3-Y705, Cryptotanshinone was relatively less effective. Also, Stattic treatments reversed the ‘metabolically active’ state of STAT3 overexpressing clones to a ‘lower metabolic state,’ as the control cells. Stattic also inhibited the cell proliferation and modulated bioenergetic phenotype of other ovarian cancer cells lines (PEO4, C200, and OVCAR3) that display a metabolically active phenotype suggesting STAT3 plays a vital role in attaining a metabolically active phenotype by cancer cells. Although, an increase in mitochondrial function was observed in overexpressing A2780 clones as evident from enhanced oxidative phosphorylation, there is no change in the mitochondrial mass or number in overexpressing clones compared to parental A2780 and vector clones indicating the critical role of STAT3 in mitochondrial functions rather than mitochondrial biogenesis in ovarian cancer cells. Further, evaluation of expression and function of mitochondrial STAT3 in ovarian cancers is warranted. Overall, STAT3 can induce metabolic changes in ovarian cancer cells by facilitating mitochondrial function, maybe as a survival mechanism and enhances the cellular fitness of the ovarian cancer cell resulting in increased oncogenic abilities. Citation Format: Vaishnavi Raja, Shailendra Giri, Suhail Hamid, Adnan R Munkarah and Ramandeep Rattan. STAT3 PROMOTES OVARIAN CANCER GROWTH AND DRUG RESISTANCE BY MODULATING THE ENERGY METABOLISM [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-049.

Identification of non-invasive biomarkers of disease progression in multiple sclerosis (MS) is critically needed for monitoring the disease progression and for effective therapeutic interventions. Urine is an attractive source for non-invasive biomarkers because it is easily obtained in the clinic. In search of a urine metabolite signature of progression in chronic experimental autoimmune encephalomyelitis (EAE), we profiled urine at the chronic stage of the disease (day 45 post immunization) by global untargeted metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found 105 metabolites (P 0.05) significantly altered at the chronic stage, indicating a robust alteration in the urine metabolite profile during disease. Assessment of altered metabolites against the Kyoto Encyclopedia of Genes and Genomes revealed distinct non-overlapping metabolic pathways and revealed phenylalanine-tyrosine and associated metabolism being the most impacted. Combined with previously performed plasma profiling, eight common metabolites were significantly altered in both of the biofluids. Metaboanalyst analysis of these common metabolites revealed that phenylalanine metabolism and Valine, leucine, and isoleucine biosynthetic pathways are central metabolic pathways in both bio-fluids and could be analyzed further, either for the discovery of therapeutics or biomarker development. Overall, our study suggests that urine and plasma metabolomics may contribute to the identification of a distinct metabolic fingerprint of EAE disease discriminating from the healthy control which may aid in the development of an objective non-invasive monitoring method for progressive autoimmune diseases like MS. Graphical Abstract Untargeted urinary metabolomics of a chronic mouse model of multiple sclerosis identified Phenylalanine, tyrosinetryptophan metabolism as the significantly altered metabolic pathway. Eight common metabolites were identified when we combined urinary and plasma metabolic signature, which revealed a perturbation of Phenylalanine metabolism and valine, leucineisoleucine metabolic pathways, involved in CNS dysfunction during diseases. The identified eight metabolic signature of urine and plasma may be of clinical relevance as potential biomarkers and guide towards the identification of specific metabolic pathways as novel drug targets.

Recently, microRNAs (miRNAs) have been implicated in regulating neuroinflammatory and demyelinative responses in multiple sclerosis (MS) and its mouse model of experimental autoimmune encephalomyelitis (EAE). miRNAs have also been studied as biomarkers of disease pathology and drug-response in MS. However, no complete miRNA profiling at various stages of EAE disease has been examined, especially in the urine. We carried out a systematic analysis of miRNAs in the urine exosomes as well as in the plasma and spinal cord at pre-onset, onset and peak stages of EAE established in the chronic B6 mice model. For the first time, we provide evidence that urine exosomes can be a specific and sensitive source of miRNA biomarkers for all 3 stages of EAE disease. In a significant observation, we observed that miR-155-5p expression increased in urine exosomes, plasma and spinal cord 6 days before the onset of disease, suggesting its early involvement in the pathology of EAE disease. We also analyzed the effect of Glatiramer acetate (GA; copaxone) treatment, an approved treatment for MS patients, in modulating miRNA expression at the peak of EAE disease. We identified miR-155-5p, miR-27a-3p, miR-9-5p and miR-350-5p as putative GA-treatment responsive miRNA biomarkers. Since, EAE is a mainly CD4 cells mediated disease, we also examined the above set of miRNAs and found to be significantly altered in T cells polarized to Th1 and Th17 phenotype, similar to urine exosomes. Thus, urine exosome miRNAs hold the potential to be defined as novel accessible stage-specific biomarkers of EAE (MS) disease as well as treatment response.

Caloric restriction (CR) was recently demonstrated by us to restrict ovarian cancer growth in vivo. CR resulted in activation of energy regulating enzymes adenosine monophosphate activated kinase (AMPK) and sirtuin 1 (SIRT1) followed by downstream inhibition of Akt-mTOR. In the present study, we investigated the effects of metformin on ovarian cancer growth in mice fed a high energy diet (HED) and regular diet (RD) and compared them to those seen with CR in an immunocompetent isogeneic mouse model of ovarian cancer. Mice either on RD or HED diet bearing ovarian tumors were treated with 200 mg/kg metformin in drinking water. Metformin treatment in RD and HED mice resulted in a significant reduction in tumor burden in the peritoneum, liver, kidney, spleen and bowel accompanied by decreased levels of growth factors (IGF-1, insulin and leptin), inflammatory cytokines (MCP-1, IL-6) and VEGF in plasma and ascitic fluid, akin to the CR diet mice. Metformin resulted in activation of AMPK and SIRT1 and inhibition of pAkt and pmTOR, similar to CR. Thus metformin can closely mimic CR's tumor suppressing effects by inducing similar metabolic changes, providing further evidence of its potential not only as a therapeutic drug but also as a preventive agent.

BACKGROUND: Preclinical studies have demonstrated antitumor effects of bisphosphonates. The objective of the current study was to determine the effect of exposure to bisphosphonate on the incidence of endometrial cancer. METHODS: The authors used data from the National Cancer Institute’s Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial, which collected data on all cancers. In year 5, all participants were asked to complete a self-administered supplemental questionnaire (SQX) that included questions regarding bone medication use. For women without a cancer diagnosis at the time of the SQX, the authors identified whether a woman reported current or former use of a nitrogenous bisphosphonate (NBP), defined as ever-use, and compared them with women never exposed to an NBP. Women with missing information were excluded as were women who reported undergoing a hysterectomy. Incidence rates and rate ratios were calculated with 95% confidence intervals (95% CIs). Cox proportional hazard ratios were also calculated and adjusted for covariates. RESULTS: A total of 29,254 women were included in the current analysis; an additional 77 cases of endometrial cancer have been diagnosed since the SQX. The incidence rate for endometrial cancer among women exposed to NBPs was 8.7 per 10,000 person-years versus 17.7 per 10,000 person-years among never-exposed women (rate ratio, 0.49; 95% CI, 0.30-0.80). The effect was similar after adjusting for all the covariates in the Cox proportional hazards analysis, with a hazard ratio of 0.56 (95% CI, 0.34-0.93). CONCLUSIONS: The results of the current study suggest that use of NBPs may have a protective effect on the incidence of endometrial cancer. However, additional studies are needed that include other potential confounders and a larger sample. Cancer 2014;000:000-000. V C 2014 American Cancer Society.

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.

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey’s analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small cell lung cancer cells (IC50 = 1.1 μM). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 μM, and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

A high energy balance, or caloric excess, accounts as a tumor promoting factor, while a negative energy balance via caloric restriction, has been shown to delay cancer progression. The effect of energy balance on ovarian cancer progression was investigated in an isogeneic immunocompetent mouse model of epithelial ovarian cancer kept on a regimen of regular diet, high energy diet (HED) and calorie restricted diet (CRD), prior to inoculating the animals intraperitoneally with the mouse ovarian surface epithelial ID8 cancer cells. Tumor evaluation revealed that mice group on HED displayed the most extensive tumor formation with the highest tumor score at all organ sites (diaphragm, peritoneum, bowel, liver, kidney, spleen), accompanied with increased levels of insulin, leptin, insulin growth factor-1 (IGF-1), monocyte chemoattractant protein-1 (MCP-1), VEGF and interleukin 6 (IL-6). On the other hand, the mice group on CRD exhibited the least tumor burden associated with a significant reduction in levels of insulin, IGF-1, leptin, MCP-1, VEGF and IL-6. Immunohistochemistry analysis of tumors from HED mice showed higher activation of Akt and mTOR with decreased adenosine monophosphate activated kinase (AMPK) and SIRT1 activation, while tumors from the CRD group exhibited the reverse profile. In conclusion, ovarian cancer growth and metastasis occurred more aggressively under HED conditions and was significantly curtailed under CRD. The suggested mechanism involves modulated secretion of growth factors, cytokines and altered regulation of AMPK and SIRT1 that converges on mTOR inhibition. While the role of a high energy state in ovarian cancer has not been confirnmed in the literature, the current findings support investigating the potential impact of diet modulation as adjunct to other anticancer therapies and as possible individualized treatment strategy of epithelial ovarian cancer.

// Calvin Tebbe 1 , Jasdeep Chhina 1 , Sajad A. Dar 1 , Kalli Sarigiannis 1 , Shailendra Giri 2 , Adnan R. Munkarah 1 , and Ramandeep Rattan 1 1 Division of Gynecology Oncology, Department of Women’s Health, Obstetrics and Gynecology, Henry Ford Hospital, Detroit, MI 48202, USA. 2 Department of Neurology, Henry Ford Hospital, Detroit, MI, USA. Correspondence: Ramandeep Rattan, email: // Keywords : ovarian cancer, 3T3L1 adipocytes, metformin, AMPK, ID8 cells, adipogenesis Received : March 6, 2014 Accepted : May 26, 2014 Published : May 26, 2014 Abstract Omental adipocytes promote ovarian cancer by secretion of adipokines, cytokines and growth factors, and acting as fuel depots. We investigated if metformin modulates the ovarian cancer promoting effects of adipocytes. Effect of conditioned media obtained from differentiated mouse 3T3L1 preadipoctes on the proliferation and migration of a mouse ovarian surface epithelium cancer cell line (ID8) was estimated. Conditioned media from differentiated adipocytes increased the proliferation and migration of ID8 cells, which was attenuated by metformin. Metformin inhibited adipogenesis by inhibition of key adipogenesis regulating transcription factors (CEBPα, CEBPs, and SREBP1), and induced AMPK. A targeted Cancer Pathway Finder RT-PCR (real-time polymerase chain reaction) based gene array revealed 20 up-regulated and 2 down-regulated genes in ID8 cells exposed to adipocyte conditioned media, which were altered by metformin. Adipocyte conditioned media also induced bio-energetic changes in the ID8 cells by pushing them into a highly metabolically active state; these effects were reversed by metformin. Collectively, metformin treatment inhibited the adipocyte mediated ovarian cancer cell proliferation, migration, expression of cancer associated genes and bio-energetic changes. Suggesting, that metformin could be a therapeutic option for ovarian cancer at an early stage, as it not only targets ovarian cancer, but also modulates the environmental milieu.

Design, synthesis, and evaluation of α-methylene-γ-butyrolactone analogues and their evaluation as anticancer agents is described. SAR identified a spirocyclic analogue 19 that inhibited TNFα-induced NF-κB activity, cancer cell growth and tumor growth in an ovarian cancer model. A second iteration of synthesis and screening identified 29 which inhibited cancer cell growth with low-μM potency. Our data suggest that an isatin-derived spirocyclic α-methylene-γ-butyrolactone is a suitable core for optimization to identify novel anticancer agents.

We performed untargeted metabolomics in plasma of B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation, and membrane stability were observed to be significantly altered between EAE and control (p < 0.05, false discovery rate

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that are increased in tumors and create an immunosuppressive environment by inhibiting the T-cell function. In addition, MDSCs promote angiogenesis, tumor invasion, and metastasis. Increased MDSC accumulation in epithelial ovarian cancer (EOC) has been associated with poor prognosis. Our study investigated whether depletion of MDSCs will influence EOC progression and enhance the therapeutic response of programmed death-1 (PD1) immunotherapy. The intraperitoneal ID8 syngeneic mouse epithelial ovarian cancer cell model in B6 mice was used for the study. The ID8 tumor-bearing mice were treated once a week with either the anti-Gr1 specific monoclonal antibody (Ly6G/Ly6C mAb) that targets and depletes MDSCs, or its isotype IgG2b mAb as control (100µg/dose/mouse). Mice were sacrificed at day 60 for tumor burden evaluation. Quantification of various immune cells and their effector cytokines in blood, spleen, bone marrow, ascites, and tumor were performed by fluorescence-activated cell sorter (FACS) using specific cell surface and intracellular markers, ELISA, and by immunohistochemistry (IHC). The preclinical trial of anti-GR1 and anti-PD1 Ab combination was also carried out. ID8-bearing mice exhibited significantly higher levels of MDSCs (CD11bGr1+) (p Citation Format: Sharif Sakr, Sajad Dar, Shailendra Giri, Adnan Munkarah, Ramandeep Rattan. Myeloid-derived suppressor cell depletion augments antitumor activity in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B32.

Myeloid Derived Suppressor Cells (MDSCs) are a heterogeneous population of immature myeloid cells that are increased in tumors and create an immunosuppressive environment by inhibiting the T-cell function. In addition, MDSCs promote angiogenesis, tumor invasion, and metastasis. Increased MDSC accumulation in epithelial ovarian cancer (EOC) has been associated with poor prognosis. Our study investigated whether depletion of MDSCs will influence EOC progression and enhance the therapeutic response of programmed death-1 (PD1) immunotherapy. The intraperitoneal ID8-luciferase syngeneic mouse epithelial ovarian cancer cell model in B6 mice was used for the study ID8 bearing mice exhibited significantly higher levels of MDSCs (CD11bGr1+) (p Citation Format: Ramandeep Rattan, Sharif Sakr, Raymond Quiles, Robert Morris, Thomas Buekers, Adnan Munkarah, Shailendra Giri. Role of myeloid derived suppressor cells in promoting ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4735.

Lung cancer is the leading cause of cancer-related deaths worldwide. Here we show for the first time that HtrA3 is a mitochondrial stress-response factor that promotes cytotoxicity to etoposide and cisplatin in lung cancer cell lines. Exogenous expression of wild type HtrA3 domain variants significantly attenuated cell survival with etoposide and cisplatin treatment in lung cancer cell lines H157 and A549 compared with expression of protease inactive mutants (S305A) or vector control. Conversely, HtrA3 suppression promoted cell survival with etoposide and cisplatin treatment in lung cancer cell lines Hop62 and HCC827. Survival was attenuated by re-expression of wild type HtrA3 variants during treatment but not by protease inactive mutants or vector control. HtrA3 also co-fractionated and co-localized with mitochondrial markers with both endogenous and exogenous expression in normal lung and lung cancer cell lines but was translocated from mitochondria following etoposide treatment. Moreover, HtrA3 translocation from mitochondria correlated with an increase in cell death that was attenuated by either HtrA3 suppression or Bcl-2 overexpression. Taken together, these results suggest that HtrA3 may be a previously uncharacterized mitochondrial cell death effector whose serine protease function may be crucial to modulating etoposide- and cisplatin-induced cytotoxicity in lung cancer cell lines.

We have previously shown that a frequently downregulated gene, transcription elongation factor A-like 7 (TCEAL7), promoted anchorage-independent growth and modulated Myc activity in ovarian surface epithelial cells immortalized with temperature-sensitive large T antigen and human telomerase reverse transcriptase (OSEtsT/hTERT). Analysis of protein/DNA array showed that TCEAL7 downregulation resulted in an approximately twofold increase in nuclear factor (NF)-kappaB binding to its target DNA sequence. In this study we showed that short hairpin RNA (shRNA)-mediated downregulation of TCEAL7 in two different immortalized OSE cells showed higher NF-kappaB activity, as determined using reporter and gel-shift assays. Transient transfection of TCEAL7 inhibited the activation of NF-kappaB in TCEAL7-downregulated clones, IOSE-523 and in other ovarian cancer cell lines (OVCAR8, SKOV3ip and DOV13), suggesting that TCEAL7 negatively regulates NF-kappaB pathway. Consistent with this observation, TCEAL7-downregulated clones showed higher levels of NF-kappaB targets, such as pro-proliferative (cyclin-D1 and cMyc), pro-angiogenic (interleukin (IL)-6, IL-8 and vascular endothelial growth factor (VEGF)), inflammatory (intercellular adhesion molecule 1 (ICAM-1) and cyclooxygenase-2 (Cox-2)) and anti-apoptotic (B-cell lymphoma-extra large (Bcl-xl)) genes when compared with vector controls. Inhibition of NF-kappaB by IkappaB kinase (IKK) inhibitor (BMS 345541) attenuated cell survival and proliferation of TCEAL-knockdown clones. Although TCEAL7 inhibited p65 transcriptional activity, it did not modulate the cytoplasmic signaling of the NF-kappaB pathway, by itself or by tumor necrosis factor-alpha (TNF-alpha). Chromatin immunoprecipitation (ChIP) assays revealed increased recruitment of p65 and p300 to the promoters of IL-8 and IL-6 in TCEAL7-downregulated clones. Collectively, these results indicate a novel role for TCEAL7 in the negative regulation of NF-kappaB signaling at the basal level by modulating transcriptional activity of NF-kappaB on its target gene promoters, potentially providing a novel mechanism by which NF-kappaB activity may be deregulated in ovarian cancer cells.

The pathophysiological mechanisms that drive the development and progression of epithelial ovarian cancer remain obscure. Recently, we identified TCEAL7 as a transcriptional regulatory protein often downregulated in epithelial ovarian cancer. However, the biological significance of such downregulation in cancer is not currently known. Here, we show that TCEAL7 is downregulated frequently in many human cancers and that in immortalized human ovarian epithelial cells this event promotes anchorage-independent cell growth. Mechanistic investigations revealed that TCEAL7 associates with cyclin D1 promoter containing Myc E-box sequence and transcriptionally represses cyclin D1 expression. Moreover, downregulation of TCEAL7 promotes DNA-binding activity of Myc-Max, and upregulates the promoter activity of c-Myc-target gene, ornithine decarboxylase (ODC), whereas enhanced expression of TCEAL7 inhibits Myc-induced promoter activity of ODC. Our findings suggest that TCEAL7 may restrict ovarian epithelial cell transformation by limiting Myc activity. These results also suggest a potential, alternative mechanism by which c-Myc activity may be deregulated in cancer by the downregulation of TCEAL7.

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

Globoid cell leukodystrophy (Krabbe disease) is an inherited neurological disorder caused by the pathogenomic accumulation of psychosine (galactosylsphingosine), a substrate for the deficient enzyme galactocerebroside beta-galactosidase. This study underscores the mechanism of action of psychosine in the regulation of oligodendrocyte cell death via the generation of lysophosphatidylcholine (LPC) and arachidonic acid (AA) by the activation of secretory phospholipase A2 (sPLA2). There was a significant increase in the level of LPC, indicating a phospholipase A2 (PLA2)-dependent pathobiology, in the brains of Krabbe disease patients and those of twitcher mice, an animal model of Krabbe disease. In vitro studies of the treatment of primary oligodendrocytes and the oligodendrocyte MO3.13 cell line with psychosine also showed the generation of LPC and the release of AA in a dose- and time-dependent manner, indicating psychosine-induced activation of PLA2. Studies with various pharmacological inhibitors of cytosolic phospholipase A2 and sPLA2 and psychosine-mediated induction of sPLA2 enzymatic activity in media supernatant suggest that psychosine-induced release of AA and generation of LPC is mainly contributed by sPLA2. An inhibitor of sPLA2, 7,7-dimethyl eicosadienoic acid, completely attenuated the psychosine-mediated accumulation of LPC levels, release of AA, and generation of reactive oxygen species, and blocked oligodendroyte cell death, as evident from cell survival, DNA fragmentation, and caspase 3 activity assays. This study documents for the first time that psychosine-induced cell death is mediated via the sPLA2 signaling pathway and that inhibitors of sPLA2 may hold a therapeutic potential for protection against oligodendrocyte cell death and resulting demyelination in Krabbe disease.

This study emphasizes the importance of Rho/ROCK pathway in lovastatin-induced apoptosis as replenishment with exogenous isoprenoid, geranylgeranylpyrophosphate (GGPP), resulted in inhibition of apoptosis in cultured tumor cells. Treatment of C6 glioma cells with Toxin B and exoenzyme C3 resulted in cell death suggesting the role of geranylgeranylated protein(s) in the survival of glioma cells. Relative apoptotic death observed in cells transfected with dominant negative constructs of RhoA, Rac, and cdc42 imply Rho A as playing the major role in cell survival. Furthermore, the inhibition of Rho A kinase (ROCK), a direct downstream effector of Rho A, by Y-27632 or dominant negative of ROCK, induced apoptosis in glioma cells. These findings indicate that RhoA/ROCK pathway is involved negatively in the regulation of glioma cell death pathway. Moreover, in vivo studies of lovastatin treatment in animals implanted with C6 glioma cell tumors also resulted in smaller tumor size and induced apoptosis in the tumor tissue. The implantation of stably transfected C6 glioma cells with expression vector of C3 exoenzyme, dominant negative of RhoA and ROCK, resulted in significant smaller tumor mass, further establishing the importance of geranylgeranylated proteins, specifically RhoA and its downstream effecter ROCK, in cell survival and tumor genesis.

Background Acquisition of metabolic alterations has been shown to be essential for the unremitting growth of cancer, yet the relation of such alterations to chemosensitivity has not been investigated. In the present study our aim was to identify the metabolic alterations that are specifically associated with platinum resistance in ovarian cancer. A global metabolic analysis of the A2780 platinum-sensitive and its platinum-resistant derivative C200 ovarian cancer cell line was performed utilizing ultra-high performance liquid chromatography/mass spectroscopy and gas chromatography/mass spectroscopy. Per-metabolite comparisons were made between cell lines and an interpretive analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic library and the Ingenuity exogenous molecule library. Results We observed 288 identified metabolites, of which 179 were found to be significantly different (t-test p

Globoid cell leukodystrophy (Krabbe disease) is characterized by the accumulation of a toxic metabolite, psychosine (galactosylsphingosine), which is a substrate for the deficient enzyme (galactocerebroside beta-galactosidase). This study underscores the possible role of psychosine in the effect of inducible nitric oxide synthase (iNOS) -derived NO in the pathophysiology of this demyelinating disease. For the first time, we provide evidence of the expression of iNOS in CNS of Krabbe patient and show that the iNOS-expressing cells in the CNS were astrocytes. Psychosine potentiated the LPS-induced production of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in primary rat astrocytes and regulated the cytokine-mediated production of NO in C6 glioma and primary rat astrocyte. Psychosine induced cytokine-mediated nuclear translocation of AP-1 and C/EBP by potentiating the expression of Fra-1 and C/EBP-delta proteins. This suggests that psychosine maintained or sustained the cytokine-primed expression of iNOS by further potentiating the nuclear translocation of AP-1 and C/EBP without modulating the cytokine-mediated transcription activity of NF-kappaB. This study hypothesizes that accumulated psychosine leads to production of cytokines and iNOS expression. The ensuing excessive production of NO and ONOO- may play a role in pathogenesis of Krabbe disease.