Vivek K. Vishnudas

Intrinsic Properties of Brown and White Adipocytes Have Differential Effects on Macrophage Inflammatory Responses

Obesity is marked by chronic, low-grade inflammation. Here, we examined whether intrinsic differences between white and brown adipocytes influence the inflammatory status of macrophages. White and brown adipocytes were characterized by transcriptional regulation of UCP-1, PGC1α, PGC1β, and CIDEA and their level of IL-6 secretion. The inflammatory profile of PMA-differentiated U937 and THP-1 macrophages, in resting state and after stimulation with LPS/IFN-gamma and IL-4, was assessed by measuring IL-6 secretion and transcriptional regulation of a panel of inflammatory genes after mono- or indirect coculture with white and brown adipocytes. White adipocyte monocultures show increased IL-6 secretion compared to brown adipocytes. White adipocytes cocultured with U937 and THP-1 macrophages induced a greater increase in IL-6 secretion compared to brown adipocytes cocultured with both macrophages. White adipocytes cocultured with macrophages increased inflammatory gene expression in both types. In contrast, macrophages cocultured with brown adipocytes induced downregulation or no alterations in inflammatory gene expression. The effects of adipocytes on macrophages appear to be independent of stimulation state. Brown adipocytes exhibit an intrinsic ability to dampen inflammatory profile of macrophages, while white adipocytes enhance it. These data suggest that brown adipocytes may be less prone to adipose tissue inflammation that is associated with obesity.

Identification of Filamin-A and -B as potential biomarkers for prostate cancer

Aim: A novel strategy for prostate cancer (PrCa) biomarker discovery is described. Materials & methods: In vitro perturbation biology, proteomics and Bayesian causal analysis identified biomarkers that were validated in in vitro models and clinical specimens. Results: Filamin-B (FLNB) and Keratin-19 were identified as biomarkers. Filamin-A (FLNA) was found to be causally linked to FLNB. Characterization of the biomarkers in a panel of cells revealed differential mRNA expression and regulation. Moreover, FLNA and FLNB were detected in the conditioned media of cells. Last, in patients without PrCa, FLNA and FLNB blood levels were positively correlated, while in patients with adenocarcinoma the relationship is dysregulated. Conclusion: These data support the strategy and the potential use of the biomarkers for PrCa.

Reduced expression of collagen VI alpha 3 (COL6A3) confers resistance to inflammation-induced MCP1 expression in adipocytes

Collagen VI alpha 3 (COL6A3) is associated with insulin resistance and adipose tissue inflammation. In this study, the role of COL6A3 in human adipocyte function was characterized.

Integrated Metabolomics Assessment of Human Dried Blood Spots and Urine Strips

(1) Background: Interest in the application of metabolomics toward clinical diagnostics development and population health monitoring has grown significantly in recent years. In spite of several advances in analytical and computational tools, obtaining a sufficient number of samples from patients remains an obstacle. The dried blood spot (DBS) and dried urine strip (DUS) methodologies are a minimally invasive sample collection method allowing for the relative simplicity of sample collection and minimal cost. (2) Methods: In the current report, we compared results of targeted metabolomics analyses of four types of human blood sample collection methods (with and without DBS) and two types of urine sample collection (DUS and urine) across several parameters including the metabolite coverage of each matrix and the sample stability for DBS/DUS using commercially available Whatman 903TM paper. The DBS/DUS metabolomics protocols were further applied to examine the temporal metabolite level fluctuations within hours and days of sample collection. (3) Results: Several hundred polar metabolites were monitored using DBS/DUS. Temporal analysis of the polar metabolites at various times of the day and across days identified several species that fluctuate as a function of day and time. In addition, a subset of metabolites were identified to be significantly altered across hours within a day and within successive days of the week. (4) Conclusion: A comprehensive DBS/DUS metabolomics protocol was developed for human blood and urine analyses. The described methodology demonstrates the potential for enabling patients to contribute to the expanding bioanalytical demands of precision medicine and population health studies.

Abstract 2458: Targeting membrane fluidity as a therapeutic strategy in cancer using BPM 31510

Cancer cells membranes are relatively more fluid compared to healthy cells. Higher fluidity in cancer cells closely relate to their invasive potential, proliferation, and metastatic ability. Pharmacological modulation of membrane fluidity as a novel therapeutic strategy for potential treatment of cancer is investigated in this study. BPM 31510, a proprietary CoQ10 based liposomal formulation currently in clinical trials affects cell membrane fluidity to influence cancer cell survival. To study the effect of BPM 31510 on biophysical parameters of membrane structure in cancer cells, the CoQ10 concentration in the liposomes was systematically increased and the membrane rigidity (Fluorescence Anisotropy) as function of temperature was measured. A progressive (significant, p Citation Format: Sumit Garg, Sirisha Dhavala, Katerina Krumova, Vivek K. Vishnudas, Joaquin J. Jimenez, Michael Kiebish, Rangaprasad Sarangarajan, Niven R. Narain. Targeting membrane fluidity as a therapeutic strategy in cancer using BPM 31510. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2458. doi:10.1158/1538-7445.AM2015-2458

Hsp90β knockdown in DIO mice reverses insulin resistance and improves glucose tolerance

BackgroundInhibition of Hsp90 has been shown to improve glucose tolerance and insulin sensitivity in mouse models of diabetes. In the present report, the specific isoform Hsp90ab1, was identified as playing a major role in regulating insulin signaling and glucose metabolism.MethodsIn a diet-induced obese (DIO) mouse model of diabetes, expression of various Hsp90 isoforms in skeletal tissue was examined. Subsequent experiments characterized the role of Hsp90ab1 isoform in glucose metabolism and insulin signaling in primary human skeletal muscle myoblasts (HSMM) and a DIO mouse model.ResultsIn DIO mice Hsp90ab1 mRNA was upregulated in skeletal muscle compared to lean mice and knockdown using anti-sense oligonucleotide (ASO) resulted in reduced expression in skeletal muscle that was associated with improved glucose tolerance, reduced fed glucose and fed insulin levels compared to DIO mice that were treated with a negative control oligonucleotide. In addition, knockdown of HSP90ab1 in DIO mice was associated with reduced pyruvate dehydrogenase kinase-4 mRNA and phosphorylation of the muscle pyruvate dehydrogenase complex (at serine 232, 293 and 300), but increased phosphofructokinase 1, glycogen synthase 1 and long-chain specific acyl-CoA dehydrogenase mRNA. In HSMM, siRNA knockdown of Hsp90ab1 induced an increase in substrate metabolism, mitochondrial respiration capacity, and insulin sensitivity, providing further evidence for the role of Hsp90ab1 in metabolism.ConclusionsThe data support a novel role for Hsp90ab1 in arbitrating skeletal muscle plasticity via modulation of substrate utilization including glucose and fatty acids in normal and disease conditions. Hsp90ab1 represents a novel target for potential treatment of metabolic disease including diabetes.

Human CCDC47 sandwich immunoassay development with electrochemiluminescence technology

Coiled-Coil Domain Containing 47 (CCDC47) is an endoplasmic reticulum (ER) transmembrane protein involved in calcium signaling through utilization of its calcium binding-acidic luminal domain. CCDC47 also interacts with ERAD (endoplasmic reticulum-associated degradation) complex and is involved in ER stress relief. In this report, we developed human CCDC47 monoclonal antibodies and a sandwich immunoassay for CCDC47 measurement in biological matrices. Specificity of developed antibodies were confirmed by immunoblot and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated cell lysates. To achieve high analytical sensitivity, traditional colorimetric enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence (ECL) technology were compared, and 3 logs of increased sensitivity was observed with the use of ECL. A CCDC47 sandwich ECL assay was subsequently developed and performances evaluated for calibration curves, precision and accuracy, as well as selectivity and interferences for sample measurement. Sample stability was also characterized for freeze/thaw cycles and short/long term storage conditions.

Dysregulation of the calcium handling protein, CCDC47, is associated with diabetic cardiomyopathy

BackgroundDiabetes mellitus is associated with an increased risk in diabetic cardiomyopathy (DCM) that is distinctly not attributed to co-morbidities with other vasculature diseases. To date, while dysregulation of calcium handling is a key hallmark in cardiomyopathy, studies have been inconsistent in the types of alterations involved. In this study human cardiomyocytes were exposed to an environmental nutritional perturbation of high glucose, fatty acids, and l-carnitine to model DCM and iTRAQ-coupled LC–MS/MS proteomic analysis was used to capture proteins affected by the perturbation. The proteins captured were then compared to proteins currently annotated in the cardiovascular disease (CVD) gene ontology (GO) database to identify proteins not previously described as being related to CVD. Subsequently, GO analysis for calcium regulating proteins and endoplasmic/sarcoplasmic reticulum (ER/SR) associated proteins was carried out.ResultsHere, we identified CCDC47 (calumin) as a unique calcium regulating protein altered in our in vitro nutritional perturbation model. The cellular and functional role of CCDC47 was then assessed in rat cardiomyocytes. In rat H9C2 myocytes, overexpression of CCDC47 resulted in increase in ionomycin-induced calcium release and reuptake. Of interest, in a diet-induced obese (DIO) rat model of DCM, CCDC47 mRNA expression was increased in the atrium and ventricle of the heart, but CCDC47 protein expression was significantly increased only in the atrium of DIO rats compared to lean control rats. Notably, no changes in ANP, BNP, or β-MHC were observed between DIO rats and lean control rats.ConclusionsTogether, our in vitro and in vivo studies demonstrate that CCDC47 is a unique calcium regulating protein that is associated with early onset hypertrophic cardiomyopathy.

Clinical Validation of a Serum Protein Panel (FLNA, FLNB and KRT19) for Diagnosis of Prostate Cancer

This study reports on the development of a novel serum protein panel of three prostate cancer biomarkers, Filamin A, Filamin B and Keratin-19 (FLNA, FLNB and KRT19) using multivariate models for disease screening and prognosis. ELISA and IPMRM (LC-MS/MS) based assays were developed and analytically validated by quantitative measurements of the biomarkers in serum. Retrospectively collected and clinically annotated serum samples with PSA values and Gleason scores were analyzed from subjects who underwent prostate biopsy, and showed no evidence of cancer with or without indication of prostatic hyperplasia, or had a definitive pathology diagnosis of prostatic adenocarcinoma. Probit linear regression models were used to combine the analytes into score functions to address the following clinical questions: does the biomarker test augment PSA for population screening? Can aggressive disease be differentiated from lower risk disease, and can the panel discriminate between prostate cancer and benign prostate hyperplasia? Modelling of the data showed that the new prostate biomarkers and PSA in combination were better than PSA alone in identifying prostate cancer, improved the prediction of high and low risk disease, and improved prediction of cancer versus benign prostate hyperplasia.

Abstract 1497: BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers

Dysregulated mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. Perturbations in mitochondrial fluxes (i.e inhibition of electron chain complexes activity, impaired electron flow) have substantial effects on cell viability, suggesting that targeting mitochondrial function could be effective for therapeutic response in cancer. BPM31510, containing oxidized coenzyme Q10 elicits an anti-Warburg effect is currently in phase II clinical trials for solid tumors. Previously, we have demonstrated the anti-cancer properties of BPM31510 in breast and pancreatic xenograft models. Here, we examined the mechanism of action of BPM31510 in vitro. Using a multi-cancer cell panel, BPM31510 was shown to be consistently and selectively cytotoxic to cancer cells, compared to normal and non-tumorigenic controls, and sensitivity did not correlate to cell doubling time or mutational status. Treatment with BPM 31510 (EC50) in breast and pancreatic cancer cells resulted in a time- and dose-dependent decrease in mitochondrial membrane potential which preceded an increase in early and late apoptosis cells, suggesting BPM31510 initiates a mitochondrial mediated cell death pathway. Using a fluorescently labeled CoQ10 we were able to trace the subcellular location of the CoQ10, which predominantly accumulates in mitochondria and lipid droplets in a time dependent manner. Additionally, the mitochondrial enrichment of CoQ10 is accompanied by morphological changes that varied amongst the different cancer cell types. As CoQ10 is a redox molecule required for electron transfer activity between complexes, we hypothesized that disruption of Q-pool homeostasis would alter complex activity. To investigate this, Complex driven respiration was measured in cells treated with BPM31510 and compared to untreated. Alterations in mitochondrial respiration characterized by a dose-dependent decrease in succinate (Complex II) and glycerol-3-phosphate (Complex III)-fueled respiration were observed in cells treated with BPM31510, while no changes were seen in pyruvate driven respiration (Complex I), suggesting that BPM31510 specifically impairs respiration responses that are more dependent on Q-pool functionality. As impairment of the electron transport chain increases intracellular oxidative stress, we next investigated if BPM 31510 treatment increases ROS levels. After 24h treatment, BPM31510 significantly increased ROS levels in treated cancer cells compared to untreated. Furthermore, BPM31510 induced death could be in part prevented by co-treatment with antioxidants. Together, these data demonstrates BPM31510 has anti-cancer activity in multiple cancer cell types and define a unique and novel functional link between mitochondrial Q-pool disruption and the mechanism of action of BPM31510. Citation Format: Tulin Dadali, Katerina Krumova, Anne R. Diers, Pallavi Awate, Ryan Ng, Arleide Lee, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2017-1497