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The cystine/glutamate exchanger (systemxc-) mediates the transport of cystine into the cell in exchange for glutamate. By releasing glutamate, systemxc-can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and may protect cells against oxidative stress. We tested two different compounds that deplete primary cortical cultures containing both neurons and astrocytes of intracellular GSH,L-buthionine-sulfoximine (L-BSO), and diethyl maleate (DEM). Both compounds caused significant concentration and time dependent decreases in intracellular GSH levels. However; DEM caused an increase in radiolabeled cystine uptake through systemxc-, while unexpectedly BSO caused a decrease in uptake. The compounds caused similar low levels of neurotoxicity, while only BSO caused an increase in oxidative stress. The mechanism of GSH depletion by these two compounds is different, DEM directly conjugates to GSH, while BSO inhibitsγ-glutamylcysteine synthetase, a key enzyme in GSH synthesis. As would be expected from these mechanisms of action, DEM caused a decrease in intracellular cysteine, while BSO increased cysteine levels. The results suggest that negative feedback by intracellular cysteine is an important regulator of systemxc-in this culture system.

Gaps in our understanding of glutamatergic signaling may be key obstacles in accurately modeling complex CNS diseases. System xc - is an example of a poorly understood component of glutamate homeostasis that has the potential to contribute to CNS diseases. This study aims to determine whether system xc - contributes to behaviors used to model features of CNS disease states. In situ hybridization was used to map mRNA expression of xCT throughout the brain. Microdialysis in the prefrontal cortex was used to sample extracellular glutamate levels; HPLC was used to measure extracellular glutamate and tissue glutathione concentrations. Acute administration of sulfasalazine (8–16 mg/kg, IP) was used to decrease system xc - activity. Behavior was measured using attentional set shifting, elevated plus maze, open-field maze, Porsolt swim test, and social interaction paradigm. The expression of xCT mRNA was detected throughout the brain, with high expression in several structures including the basolateral amygdala and prefrontal cortex. Doses of sulfasalazine that produced a reduction in extracellular glutamate levels were identified and subsequently used in the behavioral experiments. Sulfasalazine impaired performance in attentional set shifting and reduced the amount of time spent in an open arm of an elevated plus maze and the center of an open-field maze without altering behavior in a Porsolt swim test, total distance moved in an open-field maze, or social interaction. The widespread distribution of system xc - and involvement in a growing list of behaviors suggests that this form of nonvesicular glutamate release is a key component of excitatory signaling.

Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity.

The V600E mutation of B-Raf kinase results in constitutive activation of the MAPK signaling pathway and is present in approximately 7% of all cancers. Using structure-based design, a novel series of pyrazolopyridine inhibitors of B-Raf(V600E) was developed. Optimization led to the identification of 3-methoxy pyrazolopyridines 17 and 19, potent, selective, and orally bioavailable agents that inhibited tumor growth in a mouse xenograft model driven by B-Raf(V600E) with no effect on body weight. On the basis of their in vivo efficacy and preliminary safety profiles, 17 and 19 were selected for further preclinical evaluation.

As a consequence of a variety of genetic lesions, the PI3K/Akt pathway is constitutively activated in a large proportion of human cancers. The mTOR kinase plays an important role in this pathway as the key component of two independent signaling complexes (TORC1 and TORC2) that are involved at two distinct levels in this signaling cascade. Accordingly, inhibition of mTOR kinase will abrogate signaling from both mTOR complexes and serve as an effective means of targeting this pathway. In addition, the activity of the TORC1 complex is often aberrantly activated in a PI3K-independent manner to allow tumor cells to survive and proliferate despite the many negative influences of the tumor microenvironment such as hypoxia and limited nutrient availability. Therefore, an inhibitor of both TORC1 and TORC2 should effectively block signaling from the PI3K pathway as well as abrogate the cancer cells’ ability to survive in the harsh environment of the tumor thereby providing an effective means of treating cancer. We report here the profile of our small molecule mTOR kinase inhibitor AR-mTOR-26. On enzyme, this compound inhibits mTOR kinase with an IC50 of 1 nM while exhibiting substantial selectivity against PI3Kα as well as a panel of lipid and protein kinases. In cells, AR-mTOR-26 inhibits the TORC1-dependent readouts pS6 (Ser235/6) and p4E-BP1 (Ser37/46) as well as the TORC2 phosphorylation site on Akt, Ser473, with IC50 values of 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 4484.