Gold(I) and Silver(I) Complexes with 2-Anilinopyridine-Based Heterocycles as Multitarget Drugs against Colon Cancer.

A series of gold(I) and silver(I) derivatives with N- or S-donor ligands derived from 2-anilinopyridine has been synthesized and characterized. The mononuclear structure of [Au(L1)(PPh3)](TfO) (1a) and [Au(L2)(PPh3)](TfO) (1b) was confirmed by X-ray diffraction studies, as well as the dinuclear structure in the case of [Ag(TfO)(L1)]2 (4a). Most of the complexes are cytotoxic against a model of colorectal adenocarcinoma (Caco-2 cell line) and breast adenocarcinoma cancer cell lines (MCF-7). [Au(L1)(PPh3)](TfO) (1a) was able to induce caspases 8 and 3 activation, loss of mitochondrial membrane potential, and reactive oxygen species (ROS)-dependent cell death on Caco-2 cells upon 24 h incubation. In addition, the gold complex 1a produced a significant inhibition of the redox enzyme thioredoxin reductase as well as 20S proteasome. However, the silver(I) analogue, [Ag(TfO)(L1)(PPh3)] (2a), induced cell death independent of inhibition of thioredoxin reductase and 20S proteasome, triggered ROS-independent apoptosis mediated by caspase 8 and 3 activation, and loss of mitochondrial membrane potential, which points to a different mechanism of action for both derivatives, dependent on the metal center.

Alkynyl Gold(I) complexes derived from 3-hydroxyflavones as multi-targeted drugs against colon cancer.

The design of multi-targeted drugs has gained considerable interest in the last decade thanks to their advantages in the treatment of different diseases, including cancer. The simultaneous inhibition of selected targets from cancerous cells to induce their death represents an attractive objective for the medicinal chemist in order to enhance the efficiency of chemotherapy. In the present work, several alkynyl gold(I) phosphane complexes derived from 3-hydroxyflavones active against three human cancer cell lines, colorectal adenocarcinoma Caco-2/TC7, breast adenocarcinoma MCF-7 and hepatocellular carcinoma HepG2, have been synthesized and characterized. Moreover, these compounds display high selective index values towards differentiated Caco-2 cells, which are considered as a model of non-cancerous cells. The antiproliferative effect of the most active complexes [Au(L2b)PPh3] (3b) and [Au(L2c)PTA] (4c) on Caco-2 cells, seems to be mediated by the inhibition of the enzyme cyclooxygenase-1/2 and alteration of the activities of the redox enzymes thioredoxin reductase and glutathione reductase. Both complexes triggered cell death by apoptosis, alterations in cell cycle progression and increased of ROS production. These results provide support for the suggestion that multi-targeting approach involving the interaction with cyclooxygenase-1/2 and the redox enzymes that increases ROS production, enhances cell death in vitro. All these results indicate that complexes [Au(L2b)PPh3] and [Au(L2c)PTA] are promising antiproliferative agents for further anticancer drug development.

Hepatic subcellular distribution of squalene changes according to the experimental setting

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies

Gold(I) complexes with alkylated PTA (1,3,5-triaza-7-phosphaadamantane) phosphanes as anticancer metallodrugs.

New stable thiolate gold(I) derivatives containing the alkylated phosphanes [PTA-CH2Ph]Br and [PTA-CH2COOMe]Br derived from 1,3,5-triaza-7-phosphaadamantane (PTA) have been prepared by different routes of synthesis. By the use of basic media to deprotonate the corresponding thiol in the former and by transmetallation reactions from tin (IV) complexes, in the later, thus avoiding side reactions on the phosphane. Strong antiproliferative effects are observed for most of the compounds, including the chloro- and bromo precursors with the series of phosphanes derived from PTA, in human colon cancer cell lines (Caco-2, PD7 and TC7 clones). Apoptosis-induced cell death is found for all compounds, being the thiolate derivatives with [PTA-CH2Ph]Br the most effective, as shown by an annexin-V/propidium iodide double-staining assay.

TNFα regulates sugar transporters in the human intestinal epithelial cell line Caco-2.

PURPOSE: During intestinal inflammation TNFα levels are increased and as a consequence malabsorption of nutrients may occur. We have previously demonstrated that TNFα inhibits galactose, fructose and leucine intestinal absorption in animal models. In continuation with our work, the purpose of the present study was to investigate in the human intestinal epithelial cell line Caco-2, the effect of TNFα on sugar transport and to identify the intracellular mechanisms involved. METHODS: Caco-2 cells were grown on culture plates and pre-incubated during different periods with various TNFα concentrations before measuring the apical uptake of galactose, α-methyl-glucoside (MG) or fructose for 15 min. To elucidate the signaling pathway implicated, cells were pre-incubated for 30min with the PKA inhibitor H-89 or the PKC inhibitor chelerythrine, before measuring the sugar uptake. The expression in the apical membrane of the transporters implicated in the sugars uptake process (SGLT1 and GLUT5) was determined by Western blot. RESULTS: TNFα inhibited 0.1mM MG uptake after pre-incubation of the cells for 6-48h with the cytokine and in the absence of cytokine pre-incubation. In contrast, 5mM fructose uptake was stimulated by TNFα only after long pre-incubation times (24 and 48 h). These effects were mediated by the binding of the cytokine to its specific receptor TNFR1, present in the apical membrane of the Caco-2 cells. Analysis of the expression of the MG and fructose transporters at the brush border membrane of the cells, after 24h pre-incubation with the cytokine, revealed decrease on the amount of SGLT1 and increase on the amount of GLUT5 proteins. Short-term inhibition of MG transport by TNFα was not modified by H-89 but was blocked by chelerythrine. CONCLUSIONS: SGLT1 and GLUT5 expression in the plasma membrane is regulated by TNFα in the human epithelial cell line Caco-2 cells, leading to alteration on sugars transport, suggesting that TNFα could be considered as a physiological local regulator of nutrients absorption in response to an intestinal inflammatory status.

Changes in plasma hormone levels following lipopolysaccharide injection in rabbits.

Lipopolysaccharide (LPS) is an endotoxin comprising part of the cell wall of Gram-negative bacteria. It is able to induce a septic state and the release of several pro-inflammatory cytokines that are known to be responsible for hormonal changes in humans and animals. The aim of this study was to evaluate changes in plasma adrenocorticotrophic hormone (ACTH), corticosterone and cortisol levels in a rabbit model in which sepsis was induced by the intravenous administration of LPS. The possible involvement of several protein kinases, namely protein kinase A (PKA), C (PKC) and mitogen-activated protein kinases (MAPKs), and proteasome was also assessed. The results indicated that LPS induced significant increases in plasma ACTH, corticosterone and cortisol concentrations in rabbits. Moreover, protein kinases and proteasome seemed to mediate the hormone response to LPS as treatment with specific inhibitors prior to LPS administration was able to reduce, delay, or, in some cases, inhibit the hormone increases. The findings may help to construct strategies to protect and treat animals with endotoxaemia.

Lipopolysaccharide induces inhibition of galactose intestinal transport in rabbits in vitro.

BACKGROUND/AIMS: Previous studies from our laboratory have revealed impaired intestinal absorption of D-galactose in lipopolysaccharide-treated rabbits. The aim of the present work was to examine the effect of LPS on D-galactose intestinal absorption in vitro. METHODS: D-galactose intestinal transport was assessed employing three techniques: sugar uptake in rings of everted jejunum, transepithelial flux in Ussing-type chambers and transport assays in brush border membrane vesicles. The level of expression of the Na(+)/D-galactose cotransporter (SGLT1) was analyzed by Western blot. RESULTS: LPS decreased the mucosal D-galactose transport in rabbit jejunum but a preexposition to the endotoxin was required. LPS affected the Na(+)-dependent transport system by increasing the apparent Km value without affecting the Vmax. It also decreased the Na(+), K(+)-ATPase activity. However, it did not inhibit neither the uptake of D-galactose by brush border membrane vesicles nor modified the SGLT1 protein levels in the brush border, suggesting an indirect endotoxin effect. This inhibitory effect, was reduced by selective inhibitors of Ca(2+)-calmodulin (W13), protein kinase C (GF 109203X), p38 mitogen-activated protein kinase (SB 203580), c-Jun N-terminal kinase (SP 600125) and mitogen extracellular kinase (U 0126). CONCLUSION: LPS inhibits the mucosal Na(+)-dependent D-galactose intestinal absorption and the Na(+), K(+)-ATPase activity when it is added to the tissue. Intracellular processes related to protein kinases seem to be implicated in the endotoxin effect.

Protein kinases, TNF-{alpha}, and proteasome contribute in the inhibition of fructose intestinal transport by sepsis in vivo.

Lipopolysaccharide (LPS) endotoxin is a causative agent of sepsis. The aim of this study was to examine LPS effects on intestinal fructose absorption and to decipher mechanisms. Sepsis was induced by intravenous injection of LPS in rabbits. The ultrastructural study and DNA fragmentation patterns were identical in the intestine of LPS and sham animals. LPS treatment reduced fructose absorption altering both mucosal-to-serosal transepithelial fluxes and uptake into brush border membrane vesicles (BBMVs). Cytochalasin B was ineffective on fructose uptake, indicating that GLUT5, but not GLUT2, transport activity was targeted. GLUT5 protein levels in BBMvs were lower in LPS than in sham-injected rabbits. Thus lower fructose transport resulted from lower levels of GLUT5 protein. LPS treatment decreased GLUT5 levels by proteasome-dependent degradation. Specific inhibitors of PKC, PKA, and MAP kinases (p38MAPK, JNK, MEK1/2) protected fructose uptake from adverse LPS effect. Moreover, a TNF-alpha antagonist blocked LPS action on fructose uptake. We conclude that intestinal fructose transport inhibition by LPS is associated with diminished GLUT5 numbers in the brush border membrane of enterocytes triggered by activation of several interrelated signaling cascades and proteasome degradation.