Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

Combined vitamin C and E deficiency induces motor defects in gulo(-/-)/SVCT2(+/-) mice.

OBJECTIVES: Key antioxidants, vitamins C and E, are necessary for normal brain development and neuronal function. In this study, we depleted both of these vitamins in two mouse models to determine if oxidative stress due to combined vitamin C and E dietary deficiency altered their neurological phenotype. The first model lacked both alleles for the Gulonolactone oxidase gene (Gulo(-/-)) and therefore was unable synthesize vitamin C. To obtain an additional cellular deficiency of vitamin C, the second model also lacked one allele for the cellular vitamin C transporter gene (Gulo(-/-)/SVCT2(+/-)). METHODS: The experimental treatment was 16 weeks of vitamin E deprivation followed by 3 weeks of vitamin C deprivation. Mice were assessed for motor coordination deficits, vitamin levels, and oxidative stress biomarkers. RESULTS: In the first model, defects in motor performance were more apparent in both vitamin C-deficient groups (VE+VC-, VE-VC-) compared to vitamin C-supplemented groups (VE+VC+, VE-VC+) regardless of vitamin E level. Analysis of brain cortex and liver confirmed decreases of at least 80% for each vitamin in mice on deficient diets. Vitamin E deficiency doubled oxidative stress biomarkers (F2-isoprostanes and malondialdehyde). In the second model, Gulo(-/-)/SVCT2(+/-) mice on the doubly deficient diets showed deficits in locomotor activity, Rota-rod performance, and other motor tasks, with no concomitant change in anxiety or spatial memory. DISCUSSION: Vitamin E deficiency alone caused a modest oxidative stress in brain that did not affect motor performance. Adding a cellular deficit in vitamin C to dietary deprivation of both vitamins significantly impaired motor performance.

Syrbactin proteasome inhibitor TIR-199 overcomes bortezomib chemoresistance and inhibits multiple myeloma tumor growth in vivo.

Multiple myeloma (MM) and mantle cell lymphoma (MCL) are blood cancers that respond to proteasome inhibitors. Three FDA-approved drugs that block the proteasome are currently on the market, bortezomib, carfilzomib, and ixazomib. While these proteasome inhibitors have demonstrated clinical efficacy against refractory and relapsed MM and MCL, they are also associated with considerable adverse effects including peripheral neuropathy and cardiotoxicity, and tumor cells often acquire drug resistance. TIR-199 belongs to the syrbactin class, which constitutes a novel family of irreversible proteasome inhibitors. In this study, we compare TIR-199 head-to-head with three FDA-approved proteasome inhibitors. We demonstrate that TIR-199 selectively inhibits to varying degrees the sub-catalytic proteasomal activities (C-L/ß1, T-L/ß2, and CT-L/ß5) in three actively dividing MM cell lines, with Ki50 (CT-L/ß5) values of 14.61 ±â€¯2.68 nM (ARD), 54.59 ±â€¯10.4 nM (U266), and 26.8 ±â€¯5.2 nM (MM.1R). In most instances, this range was comparable with the activity of ixazomib. However, TIR-199 was more effective than bortezomib, carfilzomib, and ixazomib in killing bortezomib-resistant MM and MCL cell lines, as judged by a low resistance index (RI) between 1.7 and 2.2, which implies that TIR-199 indiscriminately inhibits both bortezomib-sensitive and bortezomib-resistant MM and MCL cells at similar concentrations. Importantly, TIR-199 reduced the tumor burden in a MM mouse model (p < 0.01) confirming its potency in vivo. Given the fact that there is still no cure for MM, the further development of TIR-199 or similar molecules that belong to the syrbactin class of proteasome inhibitors is warranted.

Thiasyrbactins Induce Cell Death via Proteasome Inhibition in Multiple Myeloma Cells.

BACKGROUND/AIM: Proteasome inhibition is a validated therapeutic strategy for the treatment of refractory and relapsed multiple myeloma (MM) and mantle cell lymphoma. We previously showed that thiasyrbactins (NAM compounds) are inhibitors with an affinity for the trypsin-like (T-L, ß2) site of the constitutive proteasome, and more profoundly for the T-L site of the immunoproteasome. MATERIALS AND METHODS: In this study, the biological activity of three NAM compounds was evaluated using four MM cell lines (ARD, U266, MM1R, and MM1S). We assessed the effect of (NAM-93, NAM-95, and NAM-105 on cell viability, as well as cell-based proteasomal activities, and determined the EC50 and Ki50 values, respectively. RESULTS: MM cells were most sensitive to NAM-93 with EC50 values <0.75 µM after 48 h of treatment. NAM-105 had a similar profile in most of the MM cells with EC50 values ranging between 0.42 and 3.02 µM. The level of inhibition of the proteasome T-L sub-catalytic activity in actively-growing MM cells was similar for NAM-93 and NAM-105. However, in each cell line, NAM-93 was more effective than NAM-105 at inhibiting overall trypsin-like sub-catalytic activity while NAM-105 was typically more effective at inhibiting overall chymotrypsin-like (CT-L, ß5) sub-catalytic activity. CONCLUSION: These results show for the first time the proteasome-targeted biological activity of thiasyrbactins in MM tumor cells.