Inhibiting Lactate Dehydrogenase A Enhances the Cytotoxicity of the Mitochondria Accumulating Antioxidant, Mitoquinone, in Melanoma Cells

Limited options exist for inhibitors targeted against melanoma tumors with mutation subtypes other than BRAF. We investigated the cytotoxic activity of mitoquinone (MitoQ), an antioxidant and ubiquinone derivative, on various human melanoma cell lines, alone or in combination with other agents to perturb cellular bioenergetics. This lipophilic cation crosses the cell membrane, enters and accumulates in the mitochondria where it can disrupt mitochondrial function at micromolar concentrations or act as an antioxidant to preserve membrane integrity at nanomolar concentrations. Consistent with previous studies, cells treated with 12.5 μM MitoQ show significantly reduced viability versus control treatments. Although all melanoma cells were susceptible to cytotoxicity induced by MitoQ, cells with wild-type BRAF were responsive to lower doses, compared to cells with activating mutations in BRAF. Mechanistically, the positively charged lipophilic moiety of the MitoQ induced a dose-dependent collapse of the mitochondrial membrane potential (Δψm) and significantly reduced the mitochondrial ATP production and reduced oxygen consumption rate, suggesting mitochondrial dysfunction. We also combined MitoQ with a glycolytic lactate dehydrogenase A inhibitor (FX-11) and observed an enhanced reduction in viability, but not other therapies examined. To summarize, the data suggest that FX-11 enhances the cytotoxic effects of MitoQ in cells with wild-type BRAF

Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy

Tissue mass-normalized values of cytochrome C reduced in vitro by succinate dehydrogenase from homogenized TAs of VEH- or RAPA-treated LC and KO mice. Two-way ANOVA. (PDF 291 kb

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber–type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation

The alpha(2)delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Ca(v)2.2) calcium channels

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on- rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential ( - 120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K] MVIIA and [O10K] GVIA had greater recovery from the block, whereas [K10R] CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states

Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression—whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reduces in vitro BrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using an in vivo mouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression

Inhibiting EGFR dimerization using triazolyl-bridged dimerization arm mimics.

The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop. In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge. We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability. This is the first example of a biologically active triazolyl-bridged peptide targeting the EGFR dimerization interface that effectively downregulates EGFR activation

Model of auto-regulatory feedback loop between ATF3 and miR-30c-2-3p.

<p>(1) Receptor signaling mediated by lysophosphatidic acid (LPA) (2) induces ATF3 expression. (3) The expression of pre-miR-30c-2 also increases, along with ATF3. (4) Processing leads to miR-30c-2-3p transcripts and subsequent binding to ATF3, causing silencing of further ATF3 expression. (Note: model does not accurately depict proper localization of molecules.)</p

Anti-miR-30c-2-3p augments ATF3 expression.

<p>(A) SKOV-3 cells were transfected with either non-targeting-miRNA control or anti-miR-30c-2-3p, prior to treatment with LPA (5 μM) for the times indicated and visualization of ATF3 protein bands. (B) SKOV-3 cells were transfected with anti-miR-30c-2-3p for 48 h and then treated with lysophosphatidic acid for the times indicated prior to measuring ATF3 mRNA expression using qRT-PCR, in comparison to non-targeting-miRNA control. ***p<0.001 (C) ATF3 protein was measured among unstimulated SKOV-3 cells transfected for 48 h with either miR-30c-2-3p, anti-miR-30c-2-3p or a negative, non-targeting-miRNA control.</p

MiR-30c-2-3p inhibits ATF3 expression.

<p>(A) SKOV-3 or (B) OVCAR-3 cells were treated with lysophosphatidic acid (LPA, 5 μM) for 0.5, 1 or 2 h prior to the assessment of ATF3 and miR-30c-2-3p expression. The data is presented as a line graph of the relative ratio. (C) Cells were transfected with miRNA scramble or miR-30c-2-3p for 24 h prior to treatment with lysophosphatidic acid (LPA, 5 μM) for the times indicated and assessment of ATF3 expression. The bar graph shows the average relative ratio normalized to a housekeeping control. ***p<0.001, *p<0.05 (D) SKOV-3 cells were transfected with miR-30c-2-3p or a scramble miRNA control for 48 h prior to the quantification of mRNA transcripts. (E) SKOV-3 or (F) OVCAR-3 cells stably expressing an ATF3 3´UTR encoded luciferase vector (wild type) or a vector with mutations in the 3´UTR sequence (mutated) were transfected with either miR-30c-2-3p, anti-miR-30c-2-3p (anti-miR), miR-30c-2-3p + anti-miR-30c-2-3p or a control (negative-target miRNA) for 48 h prior to luciferase signal intensity measurement. ***p<0.001.</p