High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

Extracellular vesicles (EVs), including exosomes and microvesicles (MVs), are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs). We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a) The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b) The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c) exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types

Residual Complex I activity and amphidirectional Complex II operation support glutamate catabolism through mtSLP in anoxia

Anoxia halts oxidative phosphorylation (OXPHOS) causing an accumulation of reduced compounds in the mitochondrial matrix which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone reduction extent in isolated mitochondria in real-time, we demonstrate that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. 13C metabolic tracing or untargeted analysis of metabolites extracted during anoxia in the presence or absence of site-specific inhibitors of the electron transfer system showed that NAD+ regenerated by Complex I is reduced by the 2-oxoglutarate dehydrogenase Complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Complex II operated amphidirectionally during the anoxic event, providing quinones to Complex I and reducing fumarate to succinate. Our results highlight the importance of quinone provision to Complex I oxidizing NADH maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.</p

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

The cold-induced lipokine 12,13-diHOME promotes fatty acid transport into brown adipose tissue

Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia1,2. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism3–5. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as ‘lipokines’6–8. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin sensitivity. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders

Abstract 990: Metabolic shift in cancer by re-programming of mitochondrial machinery: Novel insight into Warburg-dependent mechanism

Abstract Otto Warburg described his seminal observation that cancer cells negotiated a switch towards anaerobic respiration from oxidative phosphorylation in return for sustained growth and evasion of normal cellular processes such as cell cycle control and apoptosis. The underlying mechanism that governs this phenomenon has yet to be firmly established. Given the centrality of mitochondria to apoptosis and bioenergetics, we proposed experiments that employed ubidecarenone as an intracellular energy ambassador wherein high-throughput genomic, metabolomic, and proteomic analyses were employed to capture the energetic and molecular signature of cancer cells. Analyses were performed on oncogenic breast, prostate, liver, pancreatic, skin (melanoma, squamous cell carcinoma) and were compared to normal fibroblasts, keratinocytes, hepatocytes, kidney, adipocytes, and human aortic and endothelial cells. The results suggested a hallmark discovery that clearly delineated a differential effect of cancer vs. normal cells under hyperglycemic, hypoxic, and lactate-stressed conditions. A decreased activation of the Pentose Phosphate Shunt was significantly downregulated in cancer cells suggestive of a shift towards utilization of oxygen and glycolysis while Bcl-2 dependent cell death mechanisms were restored. Most notably, cross-talk mechanisms related to p53 and Vegf in the cancer cells and tumor markers and pathology in animal models of melanoma and pancreatic cancer demonstrated efficacy in a topical and intravenous form of Cytotech Labs API 31510, a ubidecarenone based technology that targets the Bcl-2 protein family downstream of a cellular metabolic shift. Conversely, normal cells did not exhibit the aforementioned characteristic indicative of a cancer-specific mechanism. We propose a novel underlying mechanism for the Warburg Hypothesis and demonstrate that pre-clinical work on API 31510 in cancer suggest that it may be a viable agent for control of cancer metabolism and serve as safe, effective anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2011-990</jats:p

Abstract 3568: Treatment of Organ-Specific Chloroleukemia by Cytotech Labs API 31510 and Chemotherapy

Abstract The development of chloroleukemia in the rat after transplantation leads to 100% mortality. Recently, two novel clones have been developed. Following intraperitoneal injection, one clone preferentially migrates and forms tumors in the lungs (CHL-L), while another clone forms tumors in the liver (CHL-Li). In vitro, proliferation of both clones were inhibited in a dose-dependent manner by a novel IV solution of API 31510. Accordingly, in the present study we examined the hypothesis that Cytotech Labs API 31510 alone or in combination with cyclophosphamide (CTX) could be effective in the treatment of both clones in vivo. In the first experiments, 24 rats were randomized into 4 groups of six rats each and injected intraperitoneally with CHL-L. In the control group, 100% developed CHL-L. In the group receiving CTX, 5 out of 6 developed CHL-L, while 1 animal remained disease-free. In the group receiving API 31510, 3 out of 6 rats remained disease-free and 3 out of 6 had a partial therapeutic response. In the group which received API 31510 and CTX, 100% of the animals remained disease-free. In a separate experiment, the same protocol was followed using CHL-Li. As observed with the other clone, animals injected with all control animals developed CHL-Li tumors. Similarly, in the group receiving CTX alone, 5 out of 6 developed CHL-Li and 1 remained disease-free. In the group receiving API 31510, 3 out of 6 animals remained disease-free and 3 out of 6 had a partial response. When combining 31510 and CTX, 5 out of 6 animals remained disease-free and 1 had a partial response. No side effects were observed in the animals receiving API 31510 as evidenced by weight gain and behavior. Taken together, these results indicate that API 31510 as a single agent was effective against both chloroma clones used and as an adjuvant to chemotherapy, API 31510 exhibited treatment efficacy in over 90% of animals treated. These data provide a strong foundation for further development of Cytotech Labs API 31510 intravenous formulation as a viable agent for cancer. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3568.</jats:p

Current Status of Metabolomic Biomarker Discovery: Impact of Study Design and Demographic Characteristics

Widespread application of omic technologies is evolving our understanding of population health and holds promise in providing precise guidance for selection of therapeutic interventions based on patient biology. The opportunity to use hundreds of analytes for diagnostic assessment of human health compared to the current use of 10&ndash;20 analytes will provide greater accuracy in deconstructing the complexity of human biology in disease states. Conventional biochemical measurements like cholesterol, creatinine, and urea nitrogen are currently used to assess health status; however, metabolomics captures a comprehensive set of analytes characterizing the human phenotype and its complex metabolic processes in real-time. Unlike conventional clinical analytes, metabolomic profiles are dramatically influenced by demographic and environmental factors that affect the range of normal values and increase the risk of false biomarker discovery. This review addresses the challenges and opportunities created by the evolving field of clinical metabolomics and highlights features of study design and bioinformatics necessary to maximize the utility of metabolomics data across demographic groups

Abstract 2865: Protection from chemotherapy-induced alopecia by cytotech lbs API 31543 in a multichemotherapy course model of chloroleukemia

Abstract Alopecia remains the one side-effect of chemotherapy for which there is no therapeutic intervention. We have recently developed a multi-course chemotherapy rat model of transplantable chloroleukemia (MIAC51) in which to study Chemotherapy-[[Unsupported Character - Codename &amp;shy;]]Induced Alopecia (CIA). This model provides the opportunity to study CIA and the cancer cells throughout multiple courses of chemotherapy. In the present study, we investigated the effect of calcitriol in a proprietary delivery system, Cytotech Labs API 31543 to protect from CIA using the following chemotherapy regimens: cyclophosphamide; cyclophosphamide and doxorubicin; cyclophosphamide, doxorubicin and cytarabine; cyclophosphamide, paclitaxel and etoposide; doxorubicin, paclitaxel and etoposide. Animals were injected intraperitoneally with 1×105 MIAC51 on day 5. Thereafter, 0.2µg of API 31543 or vehicle control was applied topically over the head area daily starting on day 6 for 6 consecutive days for the first course. Rats were isolated for 6 hours. Subsequently, the treated area was cleaned with soap and water and rats were returned to their litters. For the first cycle, chemotherapy regimens were administered on day 13, intraperitoneally in a total volume of 0.1 mL. Animals positive for leukemic cells in smears were euthanized on day 23. On day 31, a second anagen cycle was induced by clipping the hair in the neck area on leukemia survivors. Animals were then treated with 0.2 µg API 31543 on day 40 for 6 days in the shaven area. Chemotherapy regimens were then administered on day 46. In both cycles, alopecia was recorded 10 days after the last dose of chemotherapy. Neonatal rats treated with 0.2µg of API 31543 in the head area exhibited localized protection. Similarly, in the adult rats 0.2µg of API 31543 exerted a localized protective effect at the site of application. In both groups, rats that received chemotherapy alone became totally alopecic. Pretreatment with API 31543 protected the hair follicles against chemotherapy without having an effect on the efficacy of chemotherapy treatment of leukemia. In summary, the results herein set a solid foundation for clinical investigation of API 31543. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2865.</jats:p

Parenteral CoQ10 Formulation (BPM31510) Significantly Improves Survival In Animal Model Of Leukemia Including The Resolution Of Paraplegia Due To Brain Metastasis

Abstract Leukemia cells exhibit alterations in intermediary metabolism similar to other cancers, wherein ATP sourcing is shunted from mitochondrial oxphos towards glycolytic preponderance (Warburg Effect) to meet oncogenic proliferative demands. A consequence of this metabolic switch is the simultaneous short-circuit of the programmed death pathways, leading to a immortalization program in cancer cells including leukemia. Delivery of high levels of CoQ10 in a lipid nanodispersion mixture (BPM31510) has been demonstrated to preferentially shift metabolic networks from glycolysis towards mitochondrial-centric oxphos and recapitulation of apoptotic pathways in various cancers in vitro and in vivo models. Given the centrality of the Bcl-2 involvement in the etiology of leukemia, this study focused on investigation of the effectiveness of BPM31510 in animal models of erythroid and myeloid leukemia. Human acute erythro-leukemia (K562) and acute myeloid leukemia (KG1) models were developed in immune-compromised mice. The mice (total n=120 for each model respectively) were randomized into four (n=30/group) treatment groups: Untreated (control); BPM31510 (75 mg/kg, once/day); chemotherapy (Adriamycin [5mg/kg; once/wk]+AraC [25mg/kg; 5 days] and BPM31510+chemotherapy. All dosing were intravenous and followed a protocol of 3wk treatment followed by 1wk rest. In both leukemia models, combination of BPM31510 with chemotherapy was associated with significant increase in survival compared to other groups. BPM31510 alone improved survival compared to chemotherapy in myeloid leukemia, not in erythroleukemia model. In a separate study, a rat (Fisher 344) chloroleukemia (MIA C51) model of CNS leukemia was developed that demonstrated paraplegia and urinary retention as a result of brain metastasis. Administration of BPM31510 (50 mg/kg/day, IP) was associated with complete resolution of limb paralysis demonstrating the ability of BPM31510 in penetrating into the CNS. Moreover, BPM31510 (50 mg/kg/day, IP) administration was associated with significant increase in survival in animals with metastasis to the lungs and liver. The data provides encouraging evidence of the potential translational use of CoQ10 containing BPM31510 in the treatment of leukemia. A Phase 1 study in relapse acute leukemia is to be started shortly. Disclosures: Narain: Berg: Employment. Akmaev:Berg: Employment. Benaim:Berg: Employment. Sarangarajan:Berg: Employment. Jimenez:Berg: Membership on an entity’s Board of Directors or advisory committees