Fatty acid composition in matrix vesicles and in microvilli from femurs of chicken embryos revealed selective recruitment of fatty acids.

Hypertrophic chondrocytes participate in matrix mineralization by releasing matrix vesicles (MVs). These MVs, by accumulating Ca(2+) and phosphate initiate the formation of hydroxyapatite. To determine the types of lipids essential for mineralization, we analyzed fatty acids (FAs) in MVs, microvilli and in membrane fractions of chondrocytes isolated from femurs of chicken embryos. The FA composition in the MVs was almost identical to that in microvilli, indicating that the MVs originated from microvilli. These fractions contained more monounsaturated FAs especially oleic acid than in membrane homogenates of chondrocytes. They were enriched in 5,8,11-eicosatrienoic acid (20:3n-9), in eicosadienoic acid (20:2n-6), and in arachidonic acid (20:4n-6). In contrast, membrane homogenates from chondrocytes were enriched in 20:1n-9, 18:3n-3, 22:5n-3 and 22:5n-6. Due to their relatively high content in MVs and to their selective recruitment within microvilli from where MV originate, we concluded that 20:2n-6 and 20:3n-9 (pooled values), 18:1n-9 and 20:4n-6 are essential for the biogenesis of MVs and for bone mineralization.

Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species.

BACKGROUND: Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action. METHODS: Multiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN-metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo. RESULTS: We show that metal chelation using TPEN (5µM) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts. CONCLUSION: Our data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells.

Study of microRNA expression profiling as biomarkers for colorectal cancer patients in Lebanon.

The high incidence and mortality rates of colorectal cancer (CRC) reveal its hazardous effect globally. Thus, it is important to diagnose CRC at an early stage to decrease its burden and improve survival rates. Previous studies have investigated the role of short non-coding microRNAs (miRNAs or miRs) in numerous types of cancer, including CRC. Previous studies have been performed to investigate the role of miRNAs as biomarkers in diagnosis, prognosis and prediction of CRC development. The aim of the present retrospective study was to identify the expression levels of miR-31, miR-145, miR-146b and miR-186 to highlight their role in CRC diagnosis and progression at different stages of the disease (precancerous polyp, adenoma and adenocarcinoma) in a Lebanese population. The expression levels of miRNAs was revealed using TaqMan reverse transcription-quantitative PCR on formalin-fixed paraffin-embedded tissues from Lebanese patients at different stages; their diagnostic value was determined using a receiver operating characteristics curve. Compared with healthy controls, miR-31 was upregulated (P<0.0001) at all stages. By contrast, miR-145, miR-186, and miR-146b were significantly downregulated at all stages (P<0.0001, P=0.0009 and P=0.0241, respectively). Of the four miRNAs studied, miR-31 and miR-145 were identified as potentially useful diagnostic factors, with an area under the curve of 0.7771 and 0.8269 and diagnostic accuracy of 71.3 and 78.5%, respectively. These data suggested that miR-31 and miR-145, upon further clinical validation, may be used as potential diagnostic biomarkers for the early detection of CRC at the polyp stage.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Thiazole derivatives as inhibitors of cyclooxygenases in vitro and in vivo.

Cyclooxygenases (COXs) are important membrane-bound heme containing enzymes important in platelet activation and inflammation. COX-1 is constitutively expressed in most cells whereas COX-2 is an inducible isoform highly expressed in inflammatory conditions. Studies have been carried out to evaluate thiazole derivatives as anti-inflammatory molecules. In this study, we investigated the in vitro and in vivo effects of two novel thiazole derivatives compound 1 (N-[4-(4-hydroxy-3-methoxyphenyl)-1,3-thiazol-2-yl] acetamide) and compound 2 (4-(2-amino-1,3-thiazol-4-yl)-2-methoxyphenol) on prostaglandin E2 (PGE2) production and COX activity in inflammatory settings. Our results reveal a potent inhibition of both compound 1 (IC50 9.01±0.01µM) and 2 (IC50 11.65±6.20µM) (Mean±S.E.M.) on COX-2-dependent PGE2 production. We also determined whether COX-1 activity was inhibited. Using cells stably over-expressing COX-1 and human blood platelets, we showed that compound 1 is a specific inhibitor of COX-1 with IC50 (5.56×10(-8)±2.26×10(-8)µM), whereas compound 2 did not affect COX-1. Both compounds exhibit anti-inflammatory effect in the dorsal air pouch model of inflammation as shows by inhibition of PGE2 secretion. Modeling analysis of docking in the catalytic site of COX-1 or COX-2 further confirmed the difference in the effect of these two compounds. In conclusion, this study contributes to the design of new anti-inflammatory agents and to the understanding of cyclooxygenase inhibition by thiazole.

New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity.

BACKGROUND: Thromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors. RESULTS AND DISCUSSION: We synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 µM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 µM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1. CONCLUSIONS: In conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1.

Human T-cell lymphotropic virus type 1-transformed cells induce angiogenesis and establish functional gap junctions with endothelial cells.

The role of angiogenesis in the growth and metastasis of solid tumors is well established. However, the role of angiogenesis in hematologic malignancies was only recently appreciated. We show that HTLV-I-transformed T cells, but not HTLV-I-negative CD4(+) T cells, secrete biologically active forms of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and, accordingly, induce angiogenesis in vitro. Furthermore, fresh ATL leukemic cells derived from patients with acute ATL produce VEGF and bFGF transcripts and proteins. The viral transactivator Tax activates the VEGF promoter, linking the induction of angiogenesis to viral gene expression. Angiogenesis is associated with the adhesion of HTLV-I-transformed cells to endothelial cells and gap junction-mediated heterocellular communication between the 2 cell types. Angiogenesis, cell adhesion, and communication likely contribute to the development of adult T-cell leukemia-lymphoma and represent potential therapeutic targets.