Chloramidine/Bisindolylmaleimide-I-Mediated Inhibition of Exosome and Microvesicle Release and Enhanced Efficacy of Cancer Chemotherapy.

Microvesicle (MV) release from tumour cells influences drug retention, contributing to cancer drug resistance. Strategically regulating MV release may increase drug retention within cancer cells and allow for lower doses of chemotherapeutic drugs. The contribution of exosomes to drug retention still remains unknown. Potential exosome and MV (EMV) biogenesis inhibitors, tested on human prostate cancer (PC3) cells for their capacity to inhibit EMV release, were also tested on PC3 and MCF-7 (breast cancer) cells for improving chemotherapy. Agents inhibiting EMV release most significantly, whilst maintaining cell viability, were chloramidine (Cl-amidine; 50 µM) and bisindolylmaleimide-I (10 µM). Apoptosis mediated by the chemotherapy drug 5-fluorouracil (5-FU) was significantly enhanced in PC3 cells in the presence of both these EMV inhibitors, resulting in a 62% (Cl-amidine + 5-FU) and 59% (bisindolylmaleimide-I + 5-FU) decrease in numbers of viable PC3 cells compared to 5-FU alone after 24 h. For MCF-7 cells, there were similar increased reductions of viable cells compared to 5-FU treatment alone ranging from 67% (Cl-amidine + 5-FU) to 58% (bisindolylmaleimide-I + 5-FU). Using combinatory treatment, the two EMV inhibitors further reduced the number of viable cancer cells tested. Neither inhibitor affected cell viability. Combining selected EMV inhibitors may pose as a novel strategy to enhance the efficacy of chemotherapeutic drug-mediated apoptosis.

Signaling regulates activity of DHCR24, the final enzyme in cholesterol synthesis.

The role of signaling in regulating cholesterol homeostasis is gradually becoming more widely recognized. Here, we explored how kinases and phosphorylation sites regulate the activity of the enzyme involved in the final step of cholesterol synthesis, 3ß-hydroxysterol Δ24-reductase (DHCR24). Many factors are known to regulate DHCR24 transcriptionally, but little is known about its posttranslational regulation. We developed a system to specifically test human ectopic DHCR24 activity in a model cell-line (Chinese hamster ovary-7) using siRNA targeted only to hamster DHCR24, thus ensuring that all activity could be attributed to the human enzyme. We determined the effect of known phosphorylation sites and found that mutating certain residues (T110, Y299, and Y507) inhibited DHCR24 activity. In addition, inhibitors of protein kinase C ablated DHCR24 activity, although not through a known phosphorylation site. Our data indicate a novel mechanism whereby DHCR24 activity is regulated by signaling.

Neuropeptide Y promotes hepatic apolipoprotein A1 synthesis and secretion through neuropeptide Y Y5 receptor.

OBJECTIVES: Apolipoprotein A1 (ApoA1), a major component of high-density lipoprotein (HDL), is a protective factor against cardiovascular disease (CVD). A recent epidemiological study found an association between neuropeptide Y (NPY) gene polymorphism and serum HDL levels. However, the direct effect of NPY on ApoA1 expression remains unknown. This study was designed to investigate the molecular mechanisms underlying the NPY-mediated regulation of hepatic ApoA1. METHODS: Serum ApoA1, total cholesterol, and HDL-c and hepatic ApoA1 levels were measured after intraperitoneal administration of NPY or an NPY Y5 receptor (NPY5R) agonist in vivo. HepG2 and BRL-3A hepatocytes were treated in vitro with NPY in the presence or absence of NPY receptor antagonists, agonists, or signal transduction pathway inhibitors. Subsequently, the protein and mRNA expression of cellular and secreted ApoA1 were determined. RESULTS: NPY considerably upregulated hepatic ApoA1 expression and stimulated ApoA1 secretion, both in vivo and in vitro. NPY5R inhibition blocked NPY-induced upregulation of ApoA1 expression, and NPY5R activation stimulated ApoA1 expression and secretion in hepatocytes. Moreover, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and protein kinase A (PKA) inhibition almost completely blocked the upregulation of ApoA1 expression and secretion induced by NPY5R. CONCLUSIONS: For the first time, we demonstrated that NPY5R activation promotes hepatic ApoA1 synthesis and secretion through the ERK1/2 and PKA signal transduction pathways. Thus, NPY5R may be a potential therapeutic target for treating CVD by promoting cholesterol reverse transport.

Methylglyoxal augments uridine diphosphate-induced contraction via activation of p38 mitogen-activated protein kinase in rat carotid artery.

The methylglyoxal elicits diverse adverse effects on the body. Uridine diphosphate, an extracellular nucleotide, plays an important role as a signaling molecule controlling vascular tone. This study aimed to evaluate the relationship between methylglyoxal and uridine diphosphate-induced carotid arterial contraction in rats. Additionally, we examined whether p38 mitogen-activated protein kinase (MAPK) would involve such responses. Organ baths were conducted to determine vascular reactivity in isolated carotid arterial rings, and western blotting was used for protein analysis. Treatment with methylglyoxal to carotid arterial rings showed concentration-dependent augmentation to uridine diphosphate-induced contraction in the absence and presence of NG-nitro-L-arginine, which is a nitric oxide synthase inhibitor, whereas, methylglyoxal did not affect serotonin- or isotonic high K+-induced contraction in the presence of a nitric oxide synthase inhibitor. Under nitric oxide synthase inhibition, SB203580, which is a selective p38 MAPK inhibitor, suppressed uridine diphosphate-induced contraction in both the control and methylglyoxal-treated groups, and the difference in uridine diphosphate-induced contraction was abolished by SB203580 treatment. The levels of phosphorylated p38 MAPK were increased by methylglyoxal in carotid arteries, not only under the basal condition but also under uridine diphosphate stimulation. The suppression of uridine diphosphate-induced contraction by a highly selective cell-permeable protein kinase C inhibitor bisindolylmaleimide I was observed in the methylglyoxal-treated group but not in the controls. Moreover, methylglyoxal-induced augmentation of uridine diphosphate-induced contraction was prevented by N-acetyl-L-cysteine. These results suggest that methylglyoxal could enhance uridine diphosphate-induced contraction in rat carotid arteries and may be caused by activation of p38 MAPK and protein kinase C and increased oxidative stress.

Formulation of Liver-Specific PLGA-DY-635 Nanoparticles Loaded with the Protein Kinase C Inhibitor Bisindolylmaleimide I.

Bisindolylmaleimide I (BIM-I) is a competitive pan protein kinase C inhibitor with anti-inflammatory and anti-metastatic properties, suggested to treat inflammatory diseases and various cancer entities. However, despite its therapeutic potential, BIM-I has two major drawbacks, i.e., it has a poor water solubility, and it binds the human ether-à-go-go-related gene (hERG) ion channels, potentially causing deadly arrhythmias. In this case, a targeted delivery of BIM-I is imperative to minimize peripheral side effects. To circumvent these drawbacks BIM-I was encapsulated into nanoparticles prepared from poly(lactic-co-glycolic acid) (PLGA) functionalized by the near-infrared dye DY-635. DY-635 served as an active targeting moiety since it selectively binds the OATP1B1 and OATP1B3 transporters that are highly expressed in liver and cancer cells. PLGA-DY-635 (BIM-I) nanoparticles were produced by nanoprecipitation and characterized using dynamic light scattering, analytical ultracentrifugation, and cryogenic transmission electron microscopy. Particle sizes were found to be in the range of 20 to 70 nm, while a difference in sizes between the drug-loaded and unloaded particles was observed by all analytical techniques. In vitro studies demonstrated that PLGA-DY-635 (BIM-I) NPs prevent the PKC activation efficiently, proving the efficacy of the inhibitor after its encapsulation, and suggesting that BIM-I is released from the PLGA-NPs. Ultimately, our results present a feasible formulation strategy that improved the cytotoxicity profile of BIM-I and showed a high cellular uptake in the liver as demonstrated in vivo by intravital microscopy investigations.