In the brain, levels of inflammatory cytokines, interleukin-1 beta (IL-1β) and tumor Necrosis factor-alpha (TNF-α), are elevated under traumatic brain injury, neuroinflammatory conditions and glioblastoma multiforme (GBM). In GBM, the levels of small heat shock protein, CRYAB (HspB5) are also reported to be elevated, where it has been shown to exert anti-apoptotic activity. Interestingly, CRYAB is secreted via exosomes by various cells. In order to understand the relation between inflammatory cytokines and CRYAB, U373 glioma cells, were stimulated with proinflammatory cytokines, IL-1β and TNF-α, and their effect on CRYAB levels in cells and secreted exosomes was studied. Our results show that U373 cells produce and secrete CRYAB via exosomes and that stimulation with IL-1β and TNF-α significantly increase the levels of CRYAB in not only the cells but also in the secreted exosomes. In addition, cytokine stimulation of U373 cells brings about changes in the secreted exosomal proteome, many of which are involved in cancer progression.

Hypoxia, a hallmark characteristic of glioblastoma (GBM) induces changes in the transcriptome and the proteome of tumor cells. We discovered that hypoxic stress produces significant qualitative and quantitative changes in the protein content of secreted exosomes from GBM cells. Among the proteins found to be selectively elevated in hypoxic exosomes were protein-lysine 6-oxidase (LOX), thrombospondin-1 (TSP1), vascular derived endothelial factor (VEGF) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), well studied contributors to tumor progression, metastasis and angiogenesis. Our findings demonstrate that hypoxic exosomes induce differential gene expression in recipient glioma cells. Glioma cells stimulated with hypoxic exosomes showed a marked upregulation of small nucleolar RNA, C/D box 116–21 (SNORD116-21) transcript among others while significantly downregulated the potassium voltage-gated channel subfamily J member 3 (KCNJ3) message. This differential expression of certain genes is governed by the protein cargo being transferred via exosomes. Additionally, compared to normoxic exosomes, hypoxic exosomes increased various angiogenic related parameters vis-à-vis, overall tube length, branching intervals and length of isolated branches studied in tube formation assay with endothelial progenitor cells (EPCs). Thus, the intercellular communication facilitated via exosomes secreted from hypoxic GBM cells induce marked changes in the expression of genes in neighboring normoxic tumor cells and possibly in surrounding stromal cells, many of which are involved in cancer progression and treatment resistance mechanisms.

Purpose.-The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. Materials andMethods.-To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lung tumor xenografts in mice.Results.-Inhibition of GLS markedly reduced cell viability, producing ED 50 values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ~30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induce injury.

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