Role of Yeast Mannoproteins in the Interaction between Salivary Proteins and Flavan-3-ols in a Cell-Based Model of the Oral Epithelium.

Astringency is a highly complex sensation which involves multiple mechanisms occurring simultaneously, such as the interaction between flavan-3-ols and salivary proteins (SP). Moreover, astringency development can be affected by the presence of polysaccharides such as mannoproteins (MP). The aim of this work was to evaluate the molecular mechanisms whereby MP could modulate the astringency elicited by tannins, using a cell-based model of the oral epithelium (TR146 cells), and the effect of salivary proteins on these interactions. The binding of flavan-3-ols to oral cells was evaluated by DMACA assay, while the content of unbound flavan-3-ols after the interactions was assessed by means of HPLC-DAD-MS. Results obtained confirm the existence of cell-tannin interactions, that can be partially inhibited by the presence of SP and/or MP. The most significant decrease was obtained in the system containing MPF (38.16%). Both mannoproteins assayed seem to have modulating effect on flavan-3-ol-SP interactions, acting by two different mechanisms: MPF would lead to the formation of SP/MPF/flavan-3-ols ternary soluble aggregates, while MPL seems to prevent flavan-3-ol-saliva interaction by a competitive mechanism, i.e., MPL would reduce cell-tannin interactions, similar to SP. This study suggests that mannoproteins with different compositional characteristics could exhibit preferential interaction with distinct flavan-3-ol families.

Mechanisms of Resistance to Chemotherapy in Gastric Cancer.

Although surgical resection is the standard curative therapy for gastric cancer, these tumors are often diagnosed at an advanced stage, when surgery is not recommended. Alternative treatments such as radiotherapy and chemotherapy achieve only very modest results. There is therefore an urgent need to advance in this field of oncologic gastroenterology. The poor response of gastric cancer to chemotherapy is usually due to a combination of mechanisms of chemoresistance (MOC), which may include a reduction in drug uptake (MOC-1a), enhanced drug efflux (MOC-1b), a reduced proportion of active agents in tumor cells due to a reduction in pro-drug activation or an enhancement in drug inactivation (MOC-2), changes in the expression/function of the molecular targets of anticancer drugs (MOC-3), an enhanced ability of cancer cells to repair anticancer drug-induced DNA damage (MOC-4), and decreased expression/function of pro-apoptotic factors or up-regulation of anti-apoptotic genes (MOC-5). Two major goals of modern pharmacology aimed at overcoming this situation are the prediction of a lack of response to chemotherapy and the identification of the underlying mechanisms accounting for primary or acquired refractoriness to anticancer drugs. These are important issues if we are to select the best pharmacological regime for each patient and develop novel strategies to overcome chemoresistance. The present review reports updated information regarding the mechanisms of chemoresistance (from MOC-1 to MOC-5) in gastric cancer, the advances made in the prediction of the failure of chemotherapeutic treatment, and novel strategies based on gene therapy currently being developed to treat these tumors.

Effect of maternal cholestasis on TGR5 expression in human and rat placenta at term.

BACKGROUND & AIMS: TGR5 (Gpbar-1) is a plasma membrane-bound bile acid receptor expressed in several tissues, including liver, intestine and brain. High levels of TGR5 mRNA have been detected in human and rodent placenta, however, localization of the TGR5 protein has not been studied in this tissue. We aimed at characterizing TGR5 expression in placental tissue and investigated the effect of bile acids and progesterone metabolites, which accumulate during intrahepatic cholestasis of pregnancy (ICP), on receptor expression and localization. METHODS: TGR5 mRNA levels and cell-specific localization were determined by quantitative PCR and immunofluorescence, respectively. RESULTS: In human term placentas, TGR5 was mainly localized in fetal macrophages and to a lower extent in trophoblasts. In placentas from ICP patients and pregnant rats with obstructive cholestasis a marked down-regulation of TGR5 mRNA expression was observed. However, the cell-specific distribution of the TGR5 protein was unaffected. Besides bile acids, progesterone and its metabolites (5α-pregnan-3α-ol-20-one/5α-pregnan-3ß-ol-20-one), which increase in serum during ICP, were able to dose-dependently activate TGR5. In addition, progesterone metabolites but not their sulfated derivatives nor taurolithocholic acid, significantly down-regulated TGR5 mRNA and protein expression in isolated human macrophages and a macrophage-derived cell line. CONCLUSION: Since fetal macrophages and trophoblast cells are exposed to changes in the flux of compounds across the placental barrier, the expression of TGR5 in these cells together with its sensitivity to bile acids and progesterone metabolites regarding receptor activity and mRNA expression suggest that TGR5 may play a role in the effect of maternal cholestasis on the placenta.

Up-regulation of FXR isoforms is not required for stimulation of the expression of genes involved in the lack of response of colon cancer to chemotherapy.

Several mechanisms are involved in the poor response of colorectal adenocarcinoma (CRAC) to pharmacological treatment. Since preliminary evidences have suggested that the enhanced expression of farnesoid X receptor (FXR) results in the stimulation of chemoresistance, we investigated whether FXR up-regulation is required for the expression of genes that characterize the multidrug resistance (MDR) phenotype of CRAC. Samples of tumours and adjacent healthy tissues were collected from naive patients. Using Taqman Low-Density Arrays, the abundance of mRNA of 87 genes involved in MDR was determined. Relevant changes were re-evaluated by conventional RT-QPCR. In healthy tissue the major FXR isoforms were FXRα2(+/-) (80%). In tumours this predominance persisted (91%) but was accompanied by a consistent reduction (3-fold) in total FXR mRNA. A lower FXR expression was confirmed by immunostaining, in spite of which there was a significant change in the expression of MDR genes. Pharmacological challenge was simulated "in vitro" using human CRAC cells (LS174T cells). Short-term (72h) treatment with cisplatin slightly increased the almost negligible expression of FXR in wild-type LS174T cells, whereas long-term (months) treatment induced a cisplatin-resistant phenotype (LS174T/R cells), which was accompanied by a 350-fold up-regulation of FXR, mainly FXRα1(+/-). However, the changed expression of MDR genes in LS174T/R cells was not markedly affected by incubation with the FXR antagonist Z-guggulsterone. In conclusion, although the enhanced expression of FXR may be involved in the stimulation of chemoresistance that occurs during pharmacological treatment, FXR up-regulation is not required for the presence of the MDR phenotype characteristic of CRAC.

No correlation between the expression of FXR and genes involved in multidrug resistance phenotype of primary liver tumors.

Farnesoid X receptor (FXR) has been recently reported to enhance chemoresistance through bile acid-independent mechanisms. Thus, FXR transfection plus activation with GW4064 resulted in reduced sensitivity to cisplatin-induced toxicity. This is interesting because primary tumors of the liver, an organ where FXR is expressed, exhibit marked refractoriness to pharmacological treatment. Here we have determined whether FXR is upregulated in hepatocellular carcinoma (HCC), cholangiocarcinoma (CGC) and hepatoblastoma (HPB) and whether this is related with the expression of genes involved in mechanisms of chemoresistance. Using RT-QPCR and Taqman low density arrays we have analyzed biopsies from healthy livers or surgically removed tumors from naive patients and cell lines derived from HCC (SK-HEP-1, Alexander and Huh7), CGC (TFK1) and HPB (HepG2), before and after exposure to cisplatin at IC50 for 72 h. In liver tumors FXR expression was not enhanced but significantly decreased (healthy liver > HCC > HPB ≈ CGC). Except for CGC, this was not accompanied by changes in the proportions of FXR isoforms. Changes in 36 genes involved in drug uptake/efflux and metabolism, expression/function of molecular targets, and survival/apoptosis balance were found. Changes affecting SLC22A1, CYP2A1 and BIRC5 were shared by HCC, CGC and HPB. Similarity in gene expression profiles between cell lines and parent tumors was found. Pharmacological challenge with cisplatin induced changes that increased this resemblance. This was not dependent upon FXR expression. Thus, although FXR may play a role in inducing chemoresistance under certain circumstances, its upregulation does not seem to be involved in the multidrug resistance phenotype characteristic of HCC, CGC and HPB.

Genetic variants in genes involved in mechanisms of chemoresistance to anticancer drugs.

Refractoriness to the pharmacological treatment of cancer is dependent on the expression levels of genes involved in mechanisms of chemoresistance and on the existence of genetic variants that may affect their function. Thus, changes in genes encoding solute carriers may account for considerable inter-individual variability in drug uptake and the lack of sensitivity to the substrates of these transporters. Moreover, changes in proteins involved in drug export can affect their subcellular localization and transport ability and hence may also modify the bioavailability of antitumor agents. Regarding pro-drug activation or drug inactivation, genetic variants are responsible for changes in the activity of drug-metabolizing enzymes, which affect drug clearance and may determine the lack of response to anticancer chemotherapy. The presence of genetic variants may also decrease the sensitivity to pharmacological agents acting through molecular targets or signaling pathways. Recent investigations suggest that changes in genes involved in DNA repair may affect the response to platinum-based drugs. Since most anticancer agents activate cell death pathways, the evasion of apoptosis plays an important role in chemoresistance. Several genetic variants affecting death-receptor pathways, the mitochondrial pathway, downstream caspases and their natural modulators, and the p53 pathway, whose elements are mutated in more than half of tumors, and survival pathways, have been reported. The present review summarizes the available data regarding the role of genetic variants in the different mechanisms of chemoresistance and discusses their potential impact in clinical practice and in the development of tools to predict and overcome chemoresistance.

Expression in human trophoblast and choriocarcinoma cell lines, BeWo, Jeg-3 and JAr of genes involved in the hepatobiliary-like excretory function of the placenta.

Using cytokeratin-7-positive trophoblast cells (hTr) isolated from human term placentas and the choriocarcinoma cell lines (hCC) BeWo, Jeg-3 and JAr, the expression of genes involved in the hepatobiliary excretion of cholephilic compounds was investigated by RT-PCR/sequencing followed by measurement of the absolute abundance of mRNA by real-time RT-PCR. Although mRNA of BSEP was detectable and its expression confirmed by Western blotting, its very low expression (higher in hTr than in whole placenta and hCC) did not permit its detection by immunohistochemistry. In hTr, the expression was high for OATP-B/2B1, OATP-8/1B3, MRP1, MRP3, BCRP, FIC1, RARalpha, FXR and SHP, low for OSTalpha, MRP2, MRP4, MRP8, MDR1, CAR and SXR, very low for OATP-A/1A2 and MDR3, and not detectable for OATP-C/1B1, HNF1alpha and HNF4. Expression patterns in hCC mimicked those in hTr, although some important cell line-specific differences were found. The functionality of transporters expressed in hCC was confirmed by their ability to take up and export estradiol 17beta-d-glucuronide in a self-inhibitable and temperature-sensitive manner. In conclusion, several transporters, export pumps, and nuclear receptors involved in the liver excretory function may play a similar role in the placenta, whose specific aspects can be studied by selectively using BeWo, Jeg-3 or JAr cells.

Effect of maternal cholestasis and treatment with ursodeoxycholic acid on the expression of genes involved in the secretion of biliary lipids by the neonatal rat liver.

In juvenile rats born from mothers with obstructive cholestasis during pregnancy (OCP), transient latent cholestasis together with alterations in the secretion of biliary lipids have been reported. Here we investigated whether the expression of genes involved in this function is already modified at birth and examined the effect of treating pregnant rats with ursodeoxycholic acid (UDCA; i.g., 60 microg/100 g b.w./day). Cholanemia was markedly higher in mothers with OCP, and was further increased by UDCA. In the Control pups, cholanemia increased after birth, whereas in OCP and OCP+UDCA pups, hypercholanemia decreased after birth. Steady-state mRNA levels in neonatal liver were measured by real-time quantitative RT-PCR. The expression of basolateral bile acid transporters was not affected by OCP and was unchanged (Oatp1/1a1 and Oatp4/1b2) or moderately increased (Ntcp and Oatp2/1a4) by UDCA. In both groups, the expression of ABC proteins was either not modified (Bsep, Bcrp and Mrp2) or enhanced (Mrp1 and Mrp3), that of phospholipid flippase Mdr2 was not changed, whereas that of cholesterol transporter Abcg5/Abcg8 was impaired. The expression of the nuclear receptor FXR was not affected by OCP or UDCA, whereas that of SHP and key enzymes in bile acid synthesis (Cyp7a1, Cyp8b1 and Cyp27) was increased in both groups. In conclusion, OCP affects the expression in the neonatal liver of genes involved in hepatobiliary function, which cannot be prevented, at this stage, by treating pregnant rats with UDCA, even though this treatment has been found to partially restore normal lipid secretion later during post-natal development.

Maternal cholestasis induces placental oxidative stress and apoptosis. Protective effect of ursodeoxycholic acid.

We have investigated whether maternal obstructive cholestasis during pregnancy (OCP) causes oxidative stress and apoptosis in rat placenta and whether treatment with ursodeoxycholic acid (UDCA, i.g., 60 microg/100 g b.wt./day, following complete biliary obstruction on day 14 of pregnancy) has protective effects on this organ. In rats with OCP, increased (15-fold) serum bile acid concentrations (BAs) together with signs of placental oxidative stress (lipid peroxidation and protein carbonylation) were found. The latter were partly prevented by UDCA, even though hypercholanemia was not corrected. Some elements of the antioxidant system (total glutathione content, GSH/GSSG ratio and catalase, glutathione peroxidase, and glutathione-S-transferase--but not glutathione reductase--activities) were impaired in placentas from the OCP group. UDCA treatment partly prevented changes in the antioxidant system. OCP induced an increase in Bax-alpha/Bcl-2 mRNA ratio, as determined by real-time quantitative PCR, suggesting enhanced susceptibility to apoptosis activation through the mitochondria-mediated pathway. Accordingly, the activity of caspase-3, but not caspase-8, was increased in OCP placentas, in which DNA-ladder analysis and TUNEL confirmed the existence of apoptosis. UDCA prevented changes in the Bax-alpha/Bcl-2 mRNA ratio and caspase-3 activity. In conclusion, OCP causes oxidative stress and apoptosis in rat placenta, which can be prevented by treatment with UDCA.

The hepatobiliary-like excretory function of the placenta. A review.

In the adult, several endogenous compounds, such as bile acids and biliary pigments, as well as many xenobiotics are mainly biotransformed and eliminated by the hepatobiliary system. However, because this function is immature in the foetus, this role is carried out by the placenta during the intrauterine life. This review describes current knowledge of the trophoblastic machinery responsible for this function, which includes transport and metabolic processes, similar in part to those existing in the mature liver. Because many of the studies reviewed here were conducted on human or rat near-term placentae, two aspects should be borne in mind: (i) although both types of placenta are haemochorial, profound species-specific differences at the structural, molecular and functional levels do exist, and (ii) the placenta is an organ undergoing continuous developmental changes, including its hepatobiliary-like excretory function.

Excretion of foetal bilirubin by the rat placenta-maternal liver tandem.

Using plasma membrane vesicles from human trophoblast, carrier-mediated transport of unconjugated bilirubin (UCBR) has been reported. In the present work, using the in situ perfused rat placenta-maternal liver tandem, the relevance of this pathway in vivo was investigated. After single-pass perfusion of rat placenta through the umbilical artery with 0.25 micromol [(3)H]-UCBR, approximately 15 per cent of it was taken up by the placenta, detected in maternal serum (>96 per cent was unconjugated) and subsequently secreted into maternal bile (approximately 15 per cent of administered dose; >88 per cent was glucuronidated bilirubin). Co-administration through the umbilical artery of 0.25 micromol [(3)H]-UCBR and 2.5 micromol unlabelled UCBR, bromosulfophthalein, cholic acid or biliverdin IXalpha, reduced [(3)H]-UCBR placenta uptake, and the amount of radioactivity found in the maternal serum and bile. Co-administration into maternal jugular vein of 0.1 micromol [(3)H]-UCBR-a dose 3-fold higher than that reaching the maternal compartment in placenta perfusion experiments-and 1.0 micromol bromosulfophthalein, cholic acid or biliverdin IXalpha, resulted in no marked inhibition of the amount of radioactivity bile output. When antipyrine and [(3)H]-UCBR were continuously co-infused to the mother, similar antipyrine concentrations in maternal and foetal serum were reached in approximately 15 min, while progressive increase in [(3)H]-bilirubin concentrations in maternal serum above 70 microM was accompanied by a very low transfer of this compound into foetal compartment where [(3)H]-bilirubin concentrations were always <10 microM. These results suggest that the transfer of UCBR across the rat placenta occurs, without biotransformation, via a foetal-to-maternal mainly unidirectional pathway that can be cis-inhibited by UCBR and other cholephilic organic anions.

Effect of ursodeoxycholic acid on the impairment induced by maternal cholestasis in the rat placenta-maternal liver tandem excretory pathway.

We investigated the effects of ursodeoxycholic acid (UDCA; 60 microg/day/100 g b.wt.) on the impairment induced by maternal obstructive cholestasis during pregnancy (OCP) in the rat placenta-maternal liver tandem excretory pathway. A blunted catheter was implanted in the common bile duct on day 14 of pregnancy, and the tip was cut on day 21. [(14)C]Glycocholate (GC) was then administered through the umbilical artery of "in situ" perfused placenta (placental transfer test) or through the maternal jugular vein (biliary secretion test), and GC bile output was measured. OCP impaired both GC placental transfer and maternal biliary secretion. UDCA moderately improved the latter but had a more marked beneficial effect on GC placental transfer. Histological examination revealed trophoblast atrophy and structural alterations, e.g., loss of apical membrane microvilli in OCP placentas. Gene expression level was investigated by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis. OCP reduced both placental lactogen II (a trophoblast-specific gene) mRNA and the functional amount of epithelial tissue, determined by transplacental diffusion of antipyrin. Using a rapid filtration technique, impairment in the ATP-dependent GC transport across trophoblast apical plasma membranes obtained from OCP placentas was found. UDCA partially prevented all these changes. The expression level of organic anion transporters Oatp1, Oatp2, and Oatp4, and multidrug resistance-associated proteins Mrp1, Mrp2, and Mrp3 in whole placenta were not affected or were moderately affected by OCP but greatly enhanced by UDCA. In summary, UDCA partially prevents deleterious effects of OCP on the rat placenta-maternal liver tandem excretory pathway, mainly by preserving trophoblast structure and function.