Sonia Gascón

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.

Rosa canina extracts have antiproliferative and antioxidant effects on caco-2 human colon cancer

The in vitro antiproliferative and antioxidant effects of different fractions of Rosa canina hips on human colon cancer cell lines (Caco-2) was studied. The compounds tested were total extract (fraction 1), vitamin C (fraction 2), neutral polyphenols (fraction 3) and acidic polyphenols (fraction 4). All the extracts showed high cytotoxicity after 72 h, both low and high concentrations. The flow cytometric analysis revealed that all the fractions produce disturbances in the cell cycle resulting in a concomitant cell death by an apoptotic pathway. Changes in the redox status of Caco-2 cells in response to Rosa canina hips were determined. Cells were exposed to hydrogen peroxide in presence of plant fractions and the production of Reactive Oxygen Species (ROS) was significantly decreased. Therefore, our data demonstrate that rosehip extracts are a powerful antioxidant that produces an antiproliferative effect in Caco-2 cells. Therefore, these results predict a promising future for Rosa canina as a therapeutic agent. Thus, this natural plant could be an effective component of functional foods addressed towards colorectal carcinoma.

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.

Cystathionine β-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites

Hyperhomocysteinemia has been reported in human reproduction as a risk factor for early pregnancy loss, preeclampsia, and congenital birth defects like spina bifida. Female infertility was also observed in cystathionine beta synthase-deficient mice (Cbs-KO) as an animal model for severe hyperhomocysteinemia. The aim for the present research was to elucidate the time-point of pregnancy loss and to pinpoint gene and cellular changes involved in the underlying pathological mechanism. By mating 90-day-old wild-type and Cbs-KO female mice with their homologous male partners, we found that pregnancy loss in Cbs-KO occurred between the 8th and 12th gestation day during placenta formation. DNA microarrays were carried out on uterus from implantation and interimplantation samples obtained on day 8. The results allowed us to select genes potentially involved in embryo death; these were individually confirmed by RT-qPCR, and their expressions were also followed throughout pregnancy. We found that changes in expression of Calb1, Ttr, Expi, Inmt, Spink3, Rpgrip1, Krt15, Mt-4, Gzmc, Gzmb, Tdo2, and Afp were important for pregnancy success, since a different regulation in Cbs-KO mice was found. Also, differences in relationships among selected genes were observed, indicating a dysregulation of these genes in Cbs-KO females. In conclusion, our data provide more information on the gene expression cascade and its timely regulated process required for a successful pregnancy. In addition, we unveil new potential avenues to explore further investigations in pregnancy loss.

Cysteinemia, rather than homocysteinemia, is associated with plasma apolipoprotein A-I levels in hyperhomocysteinemia: lipid metabolism in cystathionine β-synthase deficiency.

OBJECTIVE Genetic and dietary hyperhomocysteinemia has been found to decrease high density lipoproteins (HDL) and their apolipoprotein A1 (APOA1). To test the hypothesis that the presence of cysteine could normalize HDL levels in hyperhomocysteinemic cystathionine beta-synthase (Cbs)-deficient mice and that the inclusion of glycine would block this effect. METHODS Lipids and HDL cholesterol were studied in Cbs-deficient mice and wild-type animals fed a low-methionine diet supplemented with cysteine and glycine and in Cbs-deficient mice on the same diet supplemented only with cysteine. RESULTS Triglyceride and homocysteine levels were significantly decreased and increased, respectively in Cbs-deficient mice irrespective of treatment. However, plasma cholesterol, glucose and APOA1 were significantly decreased in homozygous Cbs-deficient mice when they received the cysteine and glycine-enriched beverage. This group of mice also showed decreased mRNA levels and increased hepatic content of APOA1 protein, the latter increase was observed in endothelial cells. A significant, inverse relationship was observed between plasma and hepatic APOA1 concentrations while a positive one was found between plasma levels of cysteine and APOA1. CONCLUSION These data suggest an altered hepatic management of APOA1 and that cysteine may be involved in the control of this apolipoprotein at this level. Overall these findings represent a new aspect of dietary regulation of HDL at the hepatic transendothelial transport.

The anticancer effect related to disturbances in redox balance on Caco-2 cells caused by an alkynyl gold(I) complex

The alkynyl gold(I) derivative [Au(C≡CPh)(PTA)] (PTA=1,3,5-triaza-7-phosphaadamantane) induces apoptosis in colorectal carcinoma tumour cells (Caco-2) without affecting to normal enterocytes. [Au(C≡CPh)(PTA)] is a slight lipophilic drug, stable in PBS (Phosphate Buffered Saline) and able to bind BSA (Bovin Serum Albumin) by hydrophobic interactions. Once inside the cell, [Au(C≡CPh)(PTA)] targets seleno proteins such as Thioredoxin Reductase 1, increasing ROS (Reactive Oxygen Species) levels, reducing cell viability and proliferation and inducing mitochondrial apoptotic pathway, pro-apoptotic and anti-apoptotic protein imbalance, loss of mitochondrial membrane potential, cytochrome c release and activation of caspases 9 and 3. Moreover, unlike other metal-based drugs such as cisplatin, [Au(C≡CPh)(PTA)] does not target nucleic acid, reducing the risk of side mutation in the DNA. In consequence, our results predict a promising future for [Au(C≡CPh)(PTA)] as a chemotherapeutic agent for colorectal carcinoma.

In vitro and in vivo evaluation of organometallic gold(I) derivatives as anticancer agents

Alkyne gold(I) derivatives with the water soluble phosphanes PTA (1,3,5-triaza-7-phosphaadamantane) and DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) were described and their anticancer potential against the colon cancer cell line Caco-2 (PD7 and TC7 clones) was studied. Strong antiproliferative effects are found, for all the new complexes, to be even more pronounced than for the reference drug cisplatin, and similar to auranofin. The interaction of these derivatives with bovine serum albumin (BSA) was studied by fluorescence spectroscopy. The types of quenching and binding constants were determined by a fluorescence quenching method. Moderate values of the binding constants are calculated for the tested derivatives indicating that these complexes can be stored and carried easily by this protein in the body. The study of the thermodynamic parameters in the case of [Au(C[triple bond, length as m-dash]CCH2Spyridine)(PTA)] points out to the presence of van der Waals interactions or hydrogen bonding between the metallic complex and the protein. In addition, the complex [Au(C[triple bond, length as m-dash]CCH2Spyridine)(PTA)] has shown inhibition in colon cancer proliferation of HTC-116-luc2 cell lines via the apoptotic pathway and S-phase arrest of the cell cycle. Intraperitoneal injection of this derivative in athymic nude mice inoculated with HTC-116-luc2 cells prolonged their survival and displayed moderate inhibition of the tumour growth with no subsequent organ (kidney and liver) damage after treatment.

In Vivo Anticancer Activity, Toxicology and Histopathological Studies of the Thiolate Gold(I) Complex [Au(Spyrimidine)(PTA-CH 2 Ph)]Br

A physiologically stable thiolate gold(I) derivative [Au(Spyrimidine)(PTA-CH2Ph)]Br has shown inhibition in colon cancer proliferation of Caco-2/TC7, Caco-2/PD7 and HTC-116-luc2 cell lines via apoptotic pathway and S-phase arrest in the cell cycle. Intraperitoneal injection of [Au(Spyrimidine)(PTA-CH2Ph)]Br in athymic nude mice inoculated with HTC-116-luc2 cells prolonged their survival and greatly inhibited tumour growth, near to disappearance. Low concentration of gold in urine and blood were detected in mice after 48 h of administration of 5 mg/kg body weight (bw) of the gold complex and non-organ (kidney and liver) damage has been detected after gold treatment. The results obtained suggested that the thiolate gold(I) derivative shown here could be considered as a candidate for therapeutic treatment in colon cancer.

Nitric oxide involved in the IL‐1β‐induced inhibition of fructose intestinal transport

Interleukin‐1β (IL‐1β) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL‐1β effect on D‐fructose transport across rabbit jejunum and try to identify the mediators implicated in this process. A sepsis condition was induced for 90 min after intravenous (iv) administration of IL‐1β and body temperature was recorded. Studies on cellular intestinal integrity have not shown modifications of the epithelium and the basement membrane. D‐fructose intestinal transport was studied in rabbit jejunum from control and treated animals and it was reduced in the latter ones. This cytokine decreased both the mucosal to serosal transepithelial flux and the transport across brush‐border membrane vesicles of D‐fructose. The inhibition was reversed by L‐NAME (nitric oxide [NO] synthase inhibitor), but not by indomethacin (cyclooxygenase 1 and 2 inhibitor). Both inhibitors were administered iv 15 min before the IL‐1β. The protein levels of GLUT5 were not changed in all animal groups and those of mRNA were even increased. In summary, these findings indicate that IL‐1β, at the time assayed, induced a significant reduction in the relative intrinsic activity of GLUT5 and in this decrease are involved NO signalling pathways. In this way, blockage of D‐fructose intestinal uptake by IL‐1β may be playing an essential role in the pathophysiology of septic shock. J. Cell. Biochem. 111: 1321–1329, 2010.

Interleukin-1beta reduces galactose transport in intestinal epithelial cells in a NF-kB and protein kinase C-dependent manner.

Interleukins (IL), aside from their role in the regulation of the immune cascade, they have also been shown to modulate intestinal transport function. IL-1β is a potent inflammatory cytokine involved in many important cellular functions. The aim of this work was to study the in vitro effect of IL-1β on d-galactose transport across intestinal epithelia in rabbit jejunum and Caco-2 cells. The results showed that d-galactose intestinal absorption was diminished in IL-1β treated jejunum rabbits without affecting the Na(+), K(+)-ATPase activity. The presence of IL-1 cell-surface receptors was confirmed by addition to tissue of a specific IL-1 receptor antagonist (IL-1ra). The cytokine did not inhibit either the uptake of d-galactose nor modified the sodium-glucose transport (SGLT1) protein levels in the brush border membrane vesicles, suggesting an indirect IL effect. The IL-inhibition was significantly reversed in the presence of inhibitors of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs). The proteasome selective inhibitor completely abolished the IL-effect. Furthermore, the cytokine inhibition on galactose transport related to NF-kB activation was also confirmed in Caco-2 cells. In summary, the direct addition of IL-1β to intestinal epithelia inhibits d-galactose transport by a possible reduction in the SGLT1 activity. This event may be mediated by several transduction pathways activated during the inflammatory processes related to several protein kinases and nuclear factor, NF-kB. The IL-effect is independent of hormonal milieu and nervous stimuli.