Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells

Background

Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins.

Methods

These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 μM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis.

Results

All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway’s intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway.

Conclusions

Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

     

Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents

Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA₄ hydrolase and LTC₄ synthase that all may represent potential targets for LT biosynthesis inhibitors.

Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA₄ hydrolase and LTC₄ synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012–2016.

Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA₄ hydrolase and LTC₄ synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.     

Mitochondrial Function in an In Vitro Model of Skeletal Muscle of Patients With Protracted Critical Illness and Intensive Care Unit–Acquired Weakness

Background: Functional mitochondria in skeletal muscle of patients with protracted critical illness and intensive care unit–acquired weakness are depleted, but remaining mitochondria have increased functional capacities of respiratory complexes II and III. This can be an adaptation to relative abundancy of fatty acid over glucose caused by insulin resistance. We hypothesized that the capacity of muscle mitochondria to oxidize fatty acid is increased in protracted critical illness. Methods: We assessed fatty acid oxidation (FAO) and mitochondrial functional indices in vitro by using extracellular flux analysis in cultured myotubes obtained by isolating and culturing satellite cells from vastus lateralis muscle biopsy samples from patients with ICU‐acquired weakness (n = 6) and age‐matched healthy controls (n = 7). Bioenergetic measurements were performed at baseline and after 6 days of exposure to free fatty acids (FFAs). Results: Mitochondrial density in myotubes from ICU patients was 69% of healthy controls (P = .051). After adjustment to mitochondrial content, there were no differences in adenosine triphosphate (ATP) synthesis or the capacity and coupling of the respiratory chain. FAO capacity in ICU patients was 157% of FAO capacity in controls (P = .015). In myotubes of ICU patients, unlike healthy controls, the exposure to FFA significantly (P = .009) increased maximum respiratory chain capacity. Conclusion: In an in vitro model of skeletal muscle of patients with protracted critical illness, we have shown signs of adaptation to increased FAO. Even in the presence of glucose and insulin, elevation of FFAs in the extracellular environment increased maximal capacity of the respiratory chain.     

Markers of individual susceptibility and DNA repair rate in workers exposed to xenobiotics in a tire plant

Workers employed in tire plants are exposed to a variety of xenobiotics, such as 1,3-butadiene (BD), soots containing polycyclic aromatic hydrocarbons, and other organic chemicals (e.g., styrene). In the present study, we investigated markers of genotoxicity [chromosomal aberrations (CAs) and single-strand breaks (SSBs)] in a cohort of 110 tire plant workers engaged in jobs with different levels of xenobiotic exposure in relation to various polymorphisms in genes coding for biotransformation enzymes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, and GSTT1) and in genes involved in DNA repair (XPD exon 23, XPG exon 15, XPC exon 15, XRCC1 exon 10, and XRCC3 exon 7). In addition, the expression of CYP2E1, a gene playing a key role in BD metabolism, was determined by real-time PCR in peripheral blood lymphocytes, and the capacity of lymphocytes to repair gamma-ray-induced SSBs and to convert 8-oxoguanine in HeLa cell DNA into SSBs was assessed using in vitro assays. No positive associations were detected between the CA frequency or SSB induction and levels of workplace exposure; however, a nonsignificant twofold higher irradiation-specific DNA repair rate was found among highly exposed workers. In evaluations conducted with the markers of individual susceptibility, workers with low-EPHX1-activity genotypes exhibited a significantly higher CA frequency as compared to those with medium and high-EPHX1-activity genotypes (P = 0.050). CA frequencies were significantly lower in individuals homozygous for the XPD exon 23 variant allele in comparison to those with the wild-type CC genotype (P = 0.003). Interestingly, CAs were higher in individuals with higher CYP2E1 expression levels, but the association was nonsignificant (P = 0.097). The results from this study suggest the importance of evaluating markers of individual susceptibility, since they may modulate genotoxic effects induced by occupational exposure to xenobiotics.     

Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.     

Top 3 Differentials in Pediatric Radiology: A Case Review – Rebecca Stein-Wexler (ed)

Indian Journal of Pediatrics -