Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. METHODS: FXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. RESULTS: FXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. CONCLUSION: Activation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

New Advances in Polycystic Liver Diseases.

Polycystic liver diseases (PLDs) include a heterogeneous group of congenital disorders inherited as dominant or recessive genetic traits; they are manifested alone or in association with polycystic kidney disease. Ductal plate malformation during embryogenesis and the loss of heterozygosity linked to second-hit mutations may promote the dilatation and/or development of a large number (> 20) of biliary cysts, which are the main cause of morbidity in these patients. Surgical procedures aimed to eliminate symptomatic cysts show short-term beneficial effects, but are not able to block the disease progression. Therefore, liver transplantation is the only curative option. Intense studies on the molecular mechanisms involved in the pathogenesis of PLDs have resulted in different clinical trials, some of them with promising outcomes. Here the authors summarize the key aspects of PLD etiology, pathogenesis, and therapy, highlighting the most recent advances and future research directions.

Loss of the tumor suppressor spinophilin (PPP1R9B) increases the cancer stem cell population in breast tumors.

The spinophilin (Spn, PPP1R9B) gene is located at 17q21.33, a region frequently associated with microsatellite instability and loss of heterozygosity, especially in breast tumors. Spn is a regulatory subunit of phosphatase1a (PP1), which targets the catalytic subunit to distinct subcellular locations. Spn downregulation reduces PPP1CA activity against the retinoblastoma protein, pRb, thereby maintaining higher levels of phosphorylated pRb. This effect contributes to an increase in the tumorigenic properties of cells in certain contexts. Here, we explored the mechanism of how Spn downregulation contributes to the malignant phenotype and poor prognosis in breast tumors and found an increase in the stemness phenotype. Analysis of human breast tumors showed that Spn mRNA and protein are reduced or lost in 15% of carcinomas, correlating with a worse prognosis, a more aggressive tumor phenotype and triple-negative tumors, whereas luminal tumors showed high Spn levels. Downregulation of Spn by shRNA increased the stemness properties along with the expression of stem-related genes (Sox2, KLF4, Nanog and OCT4), whereas ectopic overexpression of Spn cDNA reduced these properties. Breast tumor stem cells appeared to have low levels of Spn mRNA, and Spn loss correlated with increased stem-like cell appearance in breast tumors as indicated by an increase in CD44+/CD24- cells. A reduction of the levels of PPP1CA mimicked the cancer stem-like cell phenotype of Spn downregulation, suggesting that the mechanism of Spn involves PP1a. These increased cancer stem cell-like properties with reduced Spn might account for the malignant phenotype observed in Spn-loss tumors and may contribute to a worse patient prognosis.

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.

p38α limits the contribution of MAP17 to cancer progression in breast tumors.

MAP17 is a small, 17-kDa, non-glycosylated membrane protein that is overexpressed in a percentage of carcinomas. In the present work, we have analyzed the role of MAP17 expression during mammary cancer progression. We have found that MAP17 is expressed in 60% human mammary tumors while it is not expressed in normal or benign neoplasias. MAP17 levels increased with breast tumor stage and were strongly correlated with mammary tumoral progression. A significant increase in the levels of reactive oxygen species (ROS) was observed in MAP17-expressing cells, as compared with parental cells. This increase was further paralleled by an increase in the tumorigenic capacity of carcinoma cells but not in immortal non-tumoral breast epithelial cells, which provides a selective advantage once tumorigenesis has begun. Expression of specific MAP17 shRNA in protein-expressing tumor cells reduced their tumorigenic capabilities, which suggests that this effect is dependent upon MAP17 protein expression. Our data show that ROS functions as a second messenger that enhances tumoral properties, which are inhibited in non-tumoral cells. We have found that p38α activation mediates this response. MAP17 triggers a ROS-dependent, senescence-like response that is abolished in the absence of p38a activation. Furthermore, in human breast tumors, MAP17 activation is correlated with a lack of phosphorylation of p38α. Therefore, MAP17 is overexpressed in late-stage breast tumors, in which oncogenic activity relies on p38 insensitivity to induce intracellular ROS.

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.

Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells.

BACKGROUND AND PURPOSE: Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated. EXPERIMENTAL APPROACH: Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide. KEY RESULTS: Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol. CONCLUSIONS AND IMPLICATIONS: The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS.

Strategies for overcoming chemotherapy resistance in enterohepatic tumours.

When considered together, enterohepatic tumours, i.e., those affecting the liver, the biliary tree and gallbladder and the intestine, constitute the first cause of death due to cancer. Although in many cases surgery and radiotherapy are efficacious, these therapeutic strategies cannot always be implemented. Moreover, even when the removal of tumours is possible, pre- and post-operative pharmacological adjuvant regimens are often needed. However, one important limitation to the use of cytostatic drugs to treat enterohepatic tumours is that they generally exhibit marked refractivity to currently available pharmacological approaches. In addition, most of them increase their chemoresistance during treatment. In view of the high refractivity of these tumours to anti-cancer drugs and the existence of undesirable side effects, both of which are drawbacks in the available chemotherapy, several novel therapeutic approaches have been devised. The purpose of the present review is to offer some insight into the different types of strategies that have already been evaluated and incorporated into clinical practice, such as therapies based on the use of molecular targets, as well as into the approaches that are still under experimental development, such as the chemosensitization of cancer cells, genetic manipulation of tumour or host cells, and cell-specific enhancement of intracellular concentrations of the active agent by efficient targeting of pro-drugs or by using inhibitors of efflux pumps.

Molecular bases of liver cancer refractoriness to pharmacological treatment.

Hepatocellular carcinoma and cholangiocarcinoma are the two most important primary malignancies of the liver. These are among the tumours with the lowest response to pharmacological treatment based on currently available drugs. This is due either to the existence of refractoriness of the initial tumour or to the ability of cancer cells to develop chemoresistance during treatment. Liver cancers share some of the mechanisms responsible for drug refractoriness with other types of tumours, such as a reduction in drug uptake; enhanced drug export; intracellular inactivation of the active agent; alteration of the molecular target; an increase in the activity of the target route to be inhibited, or the appearance or stimulation of alternative routes; enhanced repair of drug-induced modifications in the target molecules, and the activation/ inhibition of intracellular signalling pathways, all of which lead to a negative balance between the apoptosis/survival of tumour cells. The aim of the present article is to review how these mechanisms of chemoresistance affect the different families of drugs that are being or have been used to treat hepatocellular carcinoma and cholangiocarcinoma. A better understanding of the molecular bases of drug refractoriness is needed in order to develop novel drugs or pharmacological strategies aimed at overcoming resistance to anticancer agents.

Overview of the molecular bases of resistance to chemotherapy in liver and gastrointestinal tumours.

Primary malignancies of the liver and the gastrointestinal tract constitute one of the main health problems worldwide. Together, these types of tumour are the first cause of death due to cancer, followed by lung and breast cancer respectively. One important limitation in the treatment of these tumours is that, with a few exceptions, they exhibit marked resistance to currently available drugs. Moreover, most of them develop chemoresistance during treatment. The mechanisms responsible for drug refractoriness in gastrointestinal tumours include a reduction in drug uptake; enhanced drug export; intracellular inactivation of the effective agent; alteration of the molecular target; an increase in the activity of the target route to be inhibited or the appearance or stimulation of alternative routes; enhanced repair of drug-induced modifications in the target molecules, and the activation/inhibition of intracellular signalling pathways, which leads to a negative balance between the apoptosis/survival of tumour cells. A better understanding of these mechanisms is needed in order to develop accurate tests to predict the lack of response to chemotherapy and novel approaches aimed at overcoming resistance to anticancer agents. The purpose of the present review is to offer an updated overview of the molecular mechanisms of resistance to cytostatic drugs in the most frequent types of primary malignant tumour affecting the liver and gastrointestinal tract.

Foetal 'flat' bile acids reappear during human liver regeneration after surgery.

BACKGROUND: Changes in bile acid (BA) pool, such as the reappearance of typically foetal-type molecular species with a 'flat' structure at the steroid ring, occur during hepatocarcinogenesis, both in humans and rodents. Moreover flat-BAs also appear during rat liver regeneration. These changes can be detected in urine. The aim of the present study was to investigate whether flat-BAs also reappear during human liver regeneration, and whether this change correlates with the magnitude of liver resection. MATERIALS AND METHODS: Patients undergoing partial hepatectomy were divided in two groups: major hepatectomy group (> 50% of hepatic tissue resection, n = 17) and minor hepatectomy group (< 50%, n = 13). BAs were extracted from serum and urine (collected over 24 h) and analysed by gas chromatography-mass spectrometry. Samples were obtained before surgery (day 0) and on the third and seventh days after hepatectomy. RESULTS: In serum, total BAs significantly increased on day seven after hepatectomy, but only a moderate increase in flat-BA concentrations was observed. By contrast, urinary excretion of total as well as flat-BAs significantly increased at day three and day seven after hepatectomy. Moreover, the amount of flat-BAs excreted in urine during the first week after partial hepatectomy correlated with the magnitude of the resection. CONCLUSIONS: Urinary BA output increases and flat-BAs reappear in urine during human liver regeneration. These results suggest that determination of BAs in urine may be an interesting parameter obtained by non-invasive techniques whose actual clinical value during human liver regeneration warrants further evaluation.

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.

Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat.

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.

Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast.

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.

Bile acids reach the nuclei of hepatocytes, where they may play an important role in controlling gene expression by binding to nuclear receptors. In previous studies, changes in the amounts of the different molecular species of bile acids in the hepatocyte nucleus during rat liver regeneration have been reported. The aim of the present work was to investigate whether this also occurs during rat hepatocarcinogenesis. Liver cell nuclei were isolated after homogenization of livers from healthy adult rats (controls) and from rats at different time points during chemically induced hepatocarcinogenesis, corresponding to the stages of foci (12 weeks), hepatoma (20 weeks) and carcinoma (32 weeks). Bile samples from the cannulated common bile duct were collected for 1h from different sets of animals undergoing hepatocarcinogenesis. Bile acids in bile, liver homogenates and isolated nuclei were measured by GC-MS. Because the yield of nuclei isolated changed during the course of hepatocarcinogenesis (control, 20.1%; 12 weeks, 23.6%; 20 weeks, 7.8%; 32 weeks, 5.1%), amounts of bile acids in nuclei were corrected for the amount of DNA in each preparation. During hepatocarcinogenesis, bile acid concentrations in liver homogenates were reduced to approximately half the values obtained in control livers, while the levels of bile acids in both isolated nuclei and bile were not decreased. Hepatocarcinogenesis induced changes in the composition of bile acid pools. These were manifest as an increase in the proportion of cholic acid and a decrease in that of ursodeoxycholic acid in both bile and liver. These modifications differed from the changes seen in the nuclear bile acid pool, where a decrease in the proportion of cholic acid together with an increase in that of ursodeoxycholic acid were the major changes observed during hepatocarcinogenesis. With regard to the 'flat' bile acids (allo-cholic acid plus Delta(5)- or Delta(4)-unsaturated bile acids), a marked hepatocarcinogenesis-induced increase in the output of these species in bile was found. However, these bile acids were only found in liver homogenates at the hepatoma stage, whereas they were not detected in isolated nuclei at any stage of hepatocarcinogenesis. In summary, these results support the existence of a bile acid pool in hepatocyte nuclei whose composition differs from that of the extranuclear bile acid pool. Moreover, they indicate that, during hepatocarcinogenesis, the composition of the nuclear pool undergoes important alterations.

Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells.

The rat hepatoma-human fibroblast hybrid cell line WIF-B9 stably exhibits the structural and functional characteristics of normal differentiated hepatocytes. The abilities of these cells to synthesize bile acids and amidate them with glycine and taurine were investigated. The release of bile acids into the culture media over 72 h was assessed by gas chromatography-mass spectrometry. WIF-B9 cells were able to synthesize bile acids (1.10+/-0.17 nmol/mg protein) but less efficiently than rat hepatocytes in primary culture (2.19+/-0.19 nmol/mg protein; P<0.01). The patterns of major bile acid species produced by both types of cells were also different. Cholic acid (CA; 72%) and beta-muricholic acid (19%) were the major bile acids produced by rat hepatocytes, while chenodeoxycholic acid (CDCA) accounted for only 4.5% of total bile acids. In contrast, muricholic acids were absent, while CA (62%) and CDCA (34%) were the most abundant bile acids synthesized by WIF-B9 cells. Using reverse transcription-polymerase chain reaction and gene- and species-specific primers for key enzymes involved in bile acid synthesis, the expression of human, but not rat, orthologues of CYP7A1, CYP27, CYP8B and CYP7B1 was found in WIF-B9 cells. Induction of cell stress by serum deprivation did not change the amount of total bile acids synthesized by these cells, but an inversion of the CA-to-CDCA ratio from 1.8 to 0.3 together with a marked increase in the proportion of intermediate metabolites related to the acidic pathway was found. Using 500 microM radiolabeled CA and 2 mM of taurine or glycine, the ability to amidate CA over 48 h was determined by high performance liquid chromatography. Rat hepatocytes conjugated more than 90% CA with either amino acid, whereas this ability was very poor (< 2%) in WIF-B9 cells. Regarding the expression of enzymes and the products of bile acid synthesis, it may be concluded that the human phenotype predominates over that of the rat in WIF-B9 cells. Moreover, these cells are almost completely unable to further conjugate primary bile acids, which facilitates the manipulation of these steroids in analytical procedures. These characteristics make WIF-B9 cells a suitable in vitro model to carry out studies on bile acid synthesis by 'human-like' metabolic pathways.

Liver organotropism and biotransformation of a novel platinum-ursodeoxycholate derivative, Bamet-UD2, with enhanced antitumour activity.

BACKGROUND/AIMS: Several members of a novel family of bile acid derivatives with cytostatic and virostatic activity have been synthesized and characterized. The aim of this work was to investigate the liver organotropism and biotransformation of two novel compounds with enhanced DNA-reactivity: Bamet-D3, in which a glycine-polyamine tandem was used as a spacer to separate the glycocholic acid moiety from the platinum(II) atom, and Bamet-UD2, in which cisplatin was directly bound to the carboxylate group of two ursodeoxycholic acid moieties. METHODS: Drug uptake and "in vitro" toxicity were investigated using rat hepatocytes in primary culture. Following i.v. administration of 0.5 mumol cisplatin, Bamet-D3 or Bamet-UD2, bile output, urinary and fecal excretion, organ distribution and pharmacokinetic parameters were determined in short-term (3 h) and long-term (14 days) experiments carried out on anaesthetized and conscious rats, respectively. Liver biotransformation was investigated by HPLC analysis of bile samples. Total platinum was measured by flameless atomic absorption spectroscopy. Using Nude mice, antitumour activity was investigated in subcutaneously implanted Hepa 1-6 mouse hepatoma cells. RESULTS: Uptake by rat hepatocytes was Bamet-UD2 (11.3 nmol/mg protein) > Bamet-D3 (5.6 nmol/mg protein) > cisplatin (2.1 pmol/mg protein). Bamet-UD2 induced "in vitro" cell toxicity, which was not observed for Bamet-D3 or cisplatin. On the contrary, no toxicity "in vivo" for Bamet-UD2 was found which was observed for cisplatin and Bamet-D3. This may be related with the fact that bile output of Bamet-UD2, which occurs with no major biotransformation, was > 10 fold higher than that of cisplatin and 3-fold higher than that of Bamet-D3, which was previously transformed into at least three different metabolites. Fecal excretion was Bamet-UD2 > Bamet-D3 > cisplatin, whereas urinary output was Bamet-D3 > cisplatin > Bamet-UD2. Accordingly, a marked liver- and a reduced kidney-vectoriality for Bamet-UD2, but not for Bamet-D3, was observed. Bamet-UD2 and cisplatin, but not Bamet-D3, were efficient in inhibiting tumour growth whereas, only Bamet-UD2 significantly prolonged survival time. CONCLUSIONS: There results indicate that Bamet-UD2 is a cisplatin-ursodeoxycholate derivative with strong antitumour activity, marked hepatobiliary organotropism, and reduced toxic side-effects as compared to the parent drug cisplatin.