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1.
J Agric Food Chem ; 70(41): 13027-13035, 2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-35640024

RESUMO

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.


Assuntos
Vinho , Humanos , Vinho/análise , Saccharomyces cerevisiae , Proteínas e Peptídeos Salivares , Polifenóis , Flavonoides/química , Adstringentes , Taninos/química , Polissacarídeos/química , Epitélio
2.
Anticancer Agents Med Chem ; 16(3): 318-34, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26234359

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP2A6/metabolismo , Resistencia a Medicamentos Antineoplásicos , Transportadores de Ânions Orgânicos Dependentes de ATP/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carboxilesterase/genética , Carboxilesterase/metabolismo , Citocromo P-450 CYP2A6/genética , Reparo do DNA/efeitos dos fármacos , Terapia Genética , Humanos , MicroRNAs/uso terapêutico , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Transportadores de Ânions Orgânicos Dependentes de ATP/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
3.
Placenta ; 34(9): 810-6, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23849932

RESUMO

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.


Assuntos
Colestase Intra-Hepática/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Macrófagos/metabolismo , Placenta/metabolismo , Complicações na Gravidez/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Trofoblastos/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Células Cultivadas , Colestase Intra-Hepática/imunologia , Colestase Intra-Hepática/patologia , Modelos Animais de Doenças , Feminino , Genes Reporter , Células HEK293 , Humanos , Ativação de Macrófagos , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/patologia , Placenta/imunologia , Placenta/patologia , Gravidez , Complicações na Gravidez/imunologia , Complicações na Gravidez/patologia , Progesterona/análogos & derivados , Progesterona/metabolismo , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trofoblastos/imunologia , Trofoblastos/patologia
4.
Pharmacol Res ; 66(5): 419-27, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22898327

RESUMO

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.


Assuntos
Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Células Tumorais Cultivadas , Regulação para Cima
5.
Mol Pharm ; 9(6): 1693-704, 2012 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-22524153

RESUMO

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.


Assuntos
Neoplasias Hepáticas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/genética , Colangiocarcinoma/metabolismo , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células Hep G2 , Hepatoblastoma/tratamento farmacológico , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Técnicas In Vitro , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Receptores Citoplasmáticos e Nucleares/genética
6.
Curr Cancer Drug Targets ; 12(4): 402-38, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22229248

RESUMO

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.


Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Variação Genética , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas de Transporte/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Humanos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resultado do Tratamento
7.
Placenta ; 28(2-3): 107-17, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16712928

RESUMO

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.


Assuntos
Linhagem Celular/metabolismo , Coriocarcinoma/metabolismo , Trofoblastos/metabolismo , Neoplasias Uterinas/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Fígado/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Placenta/fisiologia , Gravidez , Receptores Citoplasmáticos e Nucleares/metabolismo
8.
Life Sci ; 79(10): 1014-9, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16764892

RESUMO

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.


Assuntos
Ácidos e Sais Biliares/sangue , Colestase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Ácido Ursodesoxicólico/farmacologia , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colestase/sangue , Feminino , Feto/anormalidades , Feto/efeitos dos fármacos , Feto/metabolismo , Fígado/efeitos dos fármacos , Gravidez , Complicações na Gravidez/tratamento farmacológico , Complicações na Gravidez/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Placenta ; 27(1): 34-41, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16310035

RESUMO

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.


Assuntos
Apoptose , Colestase/metabolismo , Estresse Oxidativo , Placenta/efeitos dos fármacos , Complicações na Gravidez/metabolismo , Ácido Ursodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Colestase/tratamento farmacológico , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Placenta/citologia , Placenta/metabolismo , Placenta/patologia , Gravidez , Complicações na Gravidez/tratamento farmacológico , Ratos , Ratos Wistar , Ácido Ursodesoxicólico/uso terapêutico
10.
Placenta ; 24(5): 431-8, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12774824

RESUMO

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.


Assuntos
Sistema Biliar/metabolismo , Fígado/metabolismo , Trofoblastos/metabolismo , Adulto , Animais , Ácidos e Sais Biliares/metabolismo , Pigmentos Biliares/metabolismo , Transporte Biológico , Feminino , Humanos , Gravidez , Ratos
11.
Placenta ; 24(5): 462-72, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12744922

RESUMO

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.


Assuntos
Bilirrubina/metabolismo , Feto/metabolismo , Fígado/metabolismo , Trofoblastos/metabolismo , Animais , Antipirina/farmacocinética , Bilirrubina/farmacocinética , Transporte Biológico , Membrana Celular/metabolismo , Feminino , Troca Materno-Fetal , Perfusão , Gravidez , Ratos , Ratos Wistar
12.
J Pharmacol Exp Ther ; 305(2): 515-24, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12606635

RESUMO

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.


Assuntos
Colestase/fisiopatologia , Fígado/metabolismo , Placenta/metabolismo , Ácido Ursodesoxicólico/farmacologia , Animais , Antipirina/sangue , Antipirina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Primers do DNA , Feminino , Expressão Gênica/efeitos dos fármacos , Ácido Glicocólico/metabolismo , Cinética , Troca Materno-Fetal/efeitos dos fármacos , Gravidez , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Anticancer Res ; 21(4A): 2703-7, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11724343

RESUMO

BACKGROUND AND AIMS: Cisplatin is a well known cytostatic drug, with high efficiency against several solid tumours, among which ovarian cancer diagnosed during pregnancy can be included. The existence of carrier proteins in the plasma membrane of the trophoblast determines vectorial bile acid transfer across the placenta. Thus, the aim of the present work was to elucidate whether the coupling of cisplatin to a bile acid moiety, such as cholylglycinate, could endow the resulting drug, Bamet-R2, with enhanced beneficial properties; namely, the ability of the placenta to prevent the passage of the drug toward the foetal compartment. MATERIALS AND METHODS: On days 15 and 18 of gestation, pregnant rats were anaesthetised with ether and intravenous administration of 1 micromol cisplatin or Bamet-R2 was carried out. Following euthanasia on day 21 of pregnancy, samples from the placenta and maternal and foetal kidney, liver, brain, lung, heart, muscle and blood were collected and digested to measure tissue drug content by flameless atomic absorption spectroscopy of platinum. RESULTS: In addition to the beneficial properties of Bamet-R2 as regards its much lower toxicity than cisplatin, this study revealed the markedly different abilities of cisplatin and Bamet-R2 to cross the placenta, which accounts for higher accumulation of cisplatin in foetal tissues: mainly kidney, lung and heart. Moreover, the amount of drug that was found in the placenta itself was several-folds higher in animals treated with cisplatin than in those receiving Bamet-R2. CONCLUSION: The ability of the placental barrier to more efficiently protect the foetal compartment from cisplatin when the drug was coupled to cholylglycinate suggests the potential usefulness of Bamet-R2 as an alternative cytostatic drug in the treatment of certain tumours during pregnancy.


Assuntos
Antineoplásicos/farmacocinética , Troca Materno-Fetal/fisiologia , Compostos Organoplatínicos/farmacocinética , Placenta/metabolismo , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/farmacocinética , Animais , Feminino , Feto/metabolismo , Gravidez , Ratos , Distribuição Tecidual
14.
J Pharmacol Exp Ther ; 297(3): 1106-12, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11356935

RESUMO

Cisplatin-bile acid derivatives belonging to the Bamet-family maintain both liver organotropism and cytostatic activity. "In vivo" toxicity and usefulness as chemotherapeutic agent versus liver tumors of a novel drug, Bamet-UD2 [cis-diamminechlorocholylglycinate platinum (II)], with enhanced "in vitro" cytostatic activity was investigated. Using orthotopically implanted mouse Hepa 1-6 hepatoma in the liver of Nude mice, the antitumor effect of Bamet-UD2 was compared with that of a previously characterized compound of this family, Bamet-R2 [cis-diamminebis-ursodeoxycholate platinum(II)], and cisplatin. Life span was significantly prolonged in mice treated with both Bamets (Bamet-UD2 > Bamet-R2), compared with animals receiving saline or cisplatin. All these drugs inhibit tumor growth (Bamet-UD2 = cisplatin > Bamet-R2). However, toxicity-related deaths only occurred under cisplatin treatment. Using rats maintained in metabolic cages, organ-specific toxicity and drug accumulation in tissues were investigated. The amount of both Bamets in the liver was severalfold higher than that of cisplatin. By contrast, a significantly higher amount of cisplatin in kidney and nerve was found. In lung, heart, muscle, brain, and bone marrow the amount of drug was small and also significantly lower in animals receiving Bamets. Signs of neurotoxicity (altered nerve conduction velocity), nephrotoxicity (increased serum urea and creatinine concentrations and decreased creatinine clearance), and bone marrow toxicity (decreased platelet and white blood counts) in animals treated with cisplatin but not with the Bamets were found. These results indicate that, owing to strong antitumor activity together with absence of side effects, Bamet-UD2 may be useful in the treatment of liver tumors.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Compostos Organoplatínicos/administração & dosagem , Ácido Ursodesoxicólico/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Medula Óssea/química , Medula Óssea/efeitos dos fármacos , Carcinoma Hepatocelular/química , Cisplatino/administração & dosagem , Cisplatino/farmacocinética , Cisplatino/toxicidade , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Injeções Intraperitoneais , Rim/química , Rim/efeitos dos fármacos , Testes de Função Renal , Fígado/química , Fígado/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/química , Masculino , Camundongos , Camundongos Nus , Miocárdio/química , Transplante de Neoplasias , Condução Nervosa/efeitos dos fármacos , Compostos Organoplatínicos/farmacocinética , Compostos Organoplatínicos/toxicidade , Platina/análise , Ratos , Ratos Wistar , Nervo Isquiático/química , Nervo Isquiático/efeitos dos fármacos , Taxa de Sobrevida , Distribuição Tecidual , Células Tumorais Cultivadas , Ácido Ursodesoxicólico/análogos & derivados , Ácido Ursodesoxicólico/farmacocinética , Ácido Ursodesoxicólico/toxicidade
15.
J Inorg Biochem ; 84(3-4): 287-92, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11374592

RESUMO

Based on the ability of bile acids for vectorializing the cytostatic activity of other agents, we have designed and synthesized a new bile acid cholylglycinato Au(III) complex, named Bamet-A1. It has been characterized by means of EA (elemental analysis), FT-IR, NMR, FAB-MS (fast atom bombardment-mass spectrometry) and Vis-UV techniques. This characterization allowed us to propose a structure of the type [Au CG(O) CG(N,O) Cl] for the neutral complex, which has the composition C522H84N2O12AuCl and is very soluble in water, methanol, ethanol and DMSO (dimethylsulfoxide). The study in aqueous solution suggested a redox process for its transformation, which is accompanied by the appearance of colloidal gold phase. The behavior in 4 mM NaCl water (in order to mimic the cytoplasmatic fluid) was similar to that observed in water, while in a 150 mM NaCl (similar to extracellular fluid and serum), the apparition of a dark blue precipitate was observed. This complex displays fluorescence, which does not change when incubated with DNA obtained from E. coli. Bamet-A1 was found to inhibit the growth of a variety of cell lines. The cytostatic effect was mild against human hepatoma HepG2, mouse hepatoma Hepa 1-6, rat hepatoma McA RH-7777 and human colon adenocarcinoma LS-174T, and stronger against mouse sarcoma S180-II and mouse leukemia L-1210 cells. The appearance of colloidal Au during the process of hydrolysis under physiological conditions may explains the low cytostatic activity.


Assuntos
Antineoplásicos/química , Ácidos e Sais Biliares/química , Ouro , Compostos Organometálicos/farmacologia , Animais , Antineoplásicos/farmacologia , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/farmacologia , Divisão Celular/efeitos dos fármacos , Coloides , DNA Bacteriano/metabolismo , Escherichia coli , Fluorescência , Humanos , Camundongos , Estrutura Molecular , Compostos Organoáuricos , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Ratos , Cloreto de Sódio/farmacologia , Análise Espectral , Células Tumorais Cultivadas
16.
Anticancer Res ; 20(5A): 3315-21, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11062759

RESUMO

BACKGROUND AND AIMS: Several platinum(II)-bile acid derivatives, named Bamets, have been previously synthesized. Their ability to interact with DNA, their cytostatic activity and their liver organotropic properties have been characterized. Two new compounds of this family, with particular structural properties, have been developed. Bamet-UD2 was formed by two ursodeoxycholic acid moieties bound by the carboxylate groups to cisplatin. In contrast, in Bamet-D3, glycine and a polyamine were used as tandem spacer elements to separate a cholic acid moiety from the platinum(II) atom. The aim of this work was to evaluate how these changes affect the ability of these compounds to interact with DNA and reduce tumour cell growth. MATERIALS AND METHODS: Drug reactivity with DNA was determined by changes in the electrophoretic mobility of the pUC18 plasmid test and by the ethidium bromide (EthBr) displacement assay. Cytostatic activity was measured against two mouse-derived cell lines from lymphocytic leukemia (L1210) and sarcoma (S-180-II). RESULTS: Bamet-UD2, and more markedly Bamet-D3, induced changes in the electrophoretic mobility of pUC18, suggesting the formation of DNA-drug interactions. Bamet-UD2 displaced EthBr from its binding to DNA. This effect was stronger in the case of Bamet-D3. Scatchard plots revealed that pre-incubation with both Bamet-UD2 and Bamet-D3 decreased the number of DNA sites available and their ability to bind EthBr. In spite of the enhanced DNA-reactivity of Bamet-D3, its ability to reduce tumour cell growth was much weaker than that of Bamet-UD2, which was seen to exert a very strong cytostatic effect. CONCLUSION: Although the distance between the platinum atom and the bile acid moiety affects the in vitro Bamet reactivity with DNA, other factors determine the overall cytostatic activity of these compounds.


Assuntos
Antineoplásicos/farmacologia , DNA/efeitos dos fármacos , Ácido Glicocólico/farmacologia , Compostos Organoplatínicos/farmacologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Divisão Celular/efeitos dos fármacos , Cisplatino/farmacologia , DNA/metabolismo , Etídio , Corantes Fluorescentes , Camundongos , Estrutura Molecular , Células Tumorais Cultivadas
17.
Am J Physiol Regul Integr Comp Physiol ; 279(4): R1495-503, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11004020

RESUMO

The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-beta-glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Ligação a DNA/genética , Resistência a Múltiplos Medicamentos , Proteínas de Membrana Transportadoras , Placenta/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/análise , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/análise , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Clonagem Molecular , Proteínas de Ligação a DNA/análise , Endotélio Vascular/metabolismo , Feminino , Glutationa/análogos & derivados , Glutationa/farmacocinética , Ácido Glicocólico/metabolismo , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteína 3 Homóloga a MutS , Placenta/citologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos/metabolismo
18.
Int J Cancer ; 88(2): 287-92, 2000 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11004682

RESUMO

Low water solubility and development of resistance are important drawbacks in the use of cisplatin as a cytostatic agent. A novel bile acid-cisplatin complex, Bamet-R2 [cis-diamminechlorocholylglycinateplatinum (II)], with liver vectoriality, has been synthesized. Our aim was to investigate the usefulness of this compound to overcome cisplatin resistance and to determine whether its encapsulation into liposomes increases its water solubility, uptake by liver tumor cells and cytostatic activity. Highly efficient incorporation of Bamet-R2 into liposomes permitted an increase in the concentration of the drug compared with that in the initial free solution by more than 6 x 10(6)-fold, which is 10(3)-fold higher than the encapsulation obtained for cisplatin. A partially cisplatin-resistant (87-fold) monoclonal cell line (Hepa 1-6/10R) was obtained by 2 subcloning steps of a population of mouse hepatoma Hepa 1-6 cells grown in step-wise increasing cisplatin concentrations up to 10 microM. Decreased sensitivity to cisplatin was accompanied by a 3.2-fold lower drug accumulation compared to wild-type cells. Uptake was markedly increased by the binding of cisplatin to glycocholic acid in both Hepa 1-6 and Hepa 1-6/10R cells. This probably accounts for the partial overcoming (-82%) of resistance when used on Hepa 1-6/10R cells. Inclusion of Bamet-R2 into liposomes further increased the amount of the drug accumulated in both cell types and, hence, enhanced its cytostatic activity. Since both plain liposomes and Bamet-R2 have little toxicity, the formulation of this compound in liposomes may offer a substantial advantage over cisplatin in the treatment of tumors resistant to this anti-neoplastic agent.


Assuntos
Cisplatino/toxicidade , Resistencia a Medicamentos Antineoplásicos , Lipossomos , Compostos Organoplatínicos/administração & dosagem , Compostos Organoplatínicos/toxicidade , Ácido Ursodesoxicólico/análogos & derivados , Animais , Colesterol , Cisplatino/farmacocinética , Portadores de Fármacos , Cinética , Lipossomos/química , Neoplasias Hepáticas Experimentais , Camundongos , Compostos Organoplatínicos/química , Fosfatidilcolinas , Fosfolipídeos , Células Tumorais Cultivadas , Ácido Ursodesoxicólico/administração & dosagem , Ácido Ursodesoxicólico/química , Ácido Ursodesoxicólico/toxicidade
19.
Bioconjug Chem ; 11(2): 167-74, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-10725093

RESUMO

The complexes cis-diamminebis-cholylglycinate (O,O') [Pt(II) C(52)H(90)N(4)O(12)Pt, for convenience referred to as Bamet-R1] and cis-diamminebis-ursodeoxycholate (O,O') Pt(II) (C(48)H(84)N(2)O(8)Pt, Bamet-UD2) were prepared. The structural integrity of the compounds was confirmed by elemental analysis, FT-IR, NMR, FAB-MS, and UV spectroscopies. The kinetic study of both compounds was accomplished by combining the conductivity measurement and those of the analysis of the electronic spectra in aqueous solution for NaCl concentrations of 4 mM (similar to cytoplasmatic concentration), 150 mM (similar to plasmatic concentration), and 500 mM. In water, the compound Bamet-R1 showed a half-life, t(1/2), of 3.0 h. This compound forms the chelate species through loss of a ligand, and the other one acts as a bidentate ligand. Ring opening in the presence of chloride ion was produced with a k(Cl)()-of 0.25 M(-)(1) h(-)(1). The half-life of Bamet-UD2 in aqueous solution was 3.2 h. However, since this species is not able to chelate and has a lower degree of solubility in the presence of chloride ion, its kinetic behavior was very different from that of the other compound. We consider this to be of great interest with regards to its cytostatic activity. All kinetic measurements were performed under pseudo-first-order conditions, and a pseudo-first-order behavior was found. The antitumoral effect of Bamet-UD2 on several cell lines derived from rat hepatoma, human hepatoma, mouse leukemia, and human colon carcinoma was found to be, in general, similar to that of cisplatin, but higher than that observed for Bamet-R1.


Assuntos
Cisplatino/análogos & derivados , Ácido Glicocólico/análogos & derivados , Compostos Organoplatínicos/química , Ácido Ursodesoxicólico/análogos & derivados , Animais , Sobrevivência Celular/efeitos dos fármacos , Ácido Glicocólico/química , Humanos , Concentração Inibidora 50 , Cinética , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Estrutura Molecular , Ratos , Espectrofotometria Infravermelho , Células Tumorais Cultivadas/efeitos dos fármacos , Ácido Ursodesoxicólico/química
20.
Hepatology ; 31(4): 975-83, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10733555

RESUMO

Cholestasis of pregnancy induces alterations in bile acid transport by human trophoblast plasma membrane (TPM) vesicles. We investigated whether maternal cholestasis affects the overall ability of the rat placenta to carry out vectorial bile acid transfer from the fetus to the mother. Complete obstructive cholestasis (OCP) was maintained during the last week of pregnancy and released at term (day 21), before experiments were performed. In situ single-pass perfusion of one placenta per rat with 250 nmol [(14)C]glycocholic acid (GC) revealed an impaired uptake in OCP rats (2.28 vs. 5.53 nmol in control rats). Approximately 100% of GC taken up by control placentas was secreted in maternal bile over 120 minutes (5.38 nmol), whereas this was only 61% (1.40 nmol) of the GC taken up by OCP placentas. When 5 nmol GC was administered through the jugular vein no significant difference between both groups in total GC bile output was found. The efficiency (V(max)/K(M)) of adenosine triphosphate (ATP)-dependent GC transport by vesicles from the maternal side of TPM was decreased (-41%) in OCP. Moreover, histological examination of the placentas suggested a reduction in the amount of functional trophoblast in the OCP group. This was consistent with a lower antipyrine diffusion across the placenta in these animals. In sum, our results indicate that maternal cholestasis affects the ability of the placenta to efficiently carry out bile acid transfer from fetal to maternal blood. Changes in both the structure and the functionality of the chorionic tissue may account for this impairment.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colestase Extra-Hepática/metabolismo , Fígado/metabolismo , Troca Materno-Fetal , Placenta/metabolismo , Complicações na Gravidez , Animais , Membrana Celular/metabolismo , Feminino , Ácido Glicocólico/metabolismo , Cinética , Gravidez , Ratos , Ratos Wistar
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