Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 191
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
3.
Eur J Cancer ; 40(12): 1812-24, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15288282

RESUMO

Isolated hepatic perfusion (IHP) involves a method of complete vascular isolation of the liver to allow treatment of liver tumours with toxic systemic doses. The recent clinical studies mainly employed IHP with melphalan with or without tumour necrosis factor-alpha (TNF-alpha) and mild hyperthermia. The results of these studies show that high response rates and high survival rates can be achieved by IHP. In this article, the current status, recent developments and future perspectives of IHP are discussed.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia do Câncer por Perfusão Regional/métodos , Neoplasias Colorretais , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Humanos , Hipertermia Induzida/métodos , Melfalan/administração & dosagem , Melfalan/farmacocinética , Resultado do Tratamento , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacocinética
4.
Eur J Surg Oncol ; 29(9): 757-63, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14602496

RESUMO

AIM: Isolated hepatic perfusion (IHP) is an invasive, technically difficult, non-repeatable and demanding operation. In this study we report the development of a less invasive alternative for the surgical IHP in a pig model. METHODS: Our technique was tested in 8 Yorkshire pigs (60 kg). The liver was isolated from the systemic circuit using minimally invasive techniques: an occlusion stent-graft and balloon catheters, with reversal of the blood flow through the liver during IHP. RESULTS: Tests with varying pressures applied at the PV revealed a clear relation between the suction pressure at the outflow site (PV), intrahepatic pressure and systemic leakage of 99mTc. A leakage-free IHP could be obtained in seven separate experiments. CONCLUSION: Isolated hepatic perfusion using minimally invasive techniques is feasible in pigs when the intrahepatic pressure is controlled. This technique has yet to be tested in patients.


Assuntos
Quimioterapia do Câncer por Perfusão Regional , Neoplasias Hepáticas/tratamento farmacológico , Animais , Modelos Animais de Doenças , Neoplasias Hepáticas/patologia , Procedimentos Cirúrgicos Minimamente Invasivos , Metástase Neoplásica , Suínos , Resultado do Tratamento
5.
Cell Prolif ; 36(1): 35-44, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12558659

RESUMO

Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl-2 gene (CCbcl2 cells) or with bcl-2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CCDeltaBH1 cells), BH3 (CCDeltaBH3 cells), BH4 (CCDeltaBH4 cells) or the transmembrane region (CCDeltaTM cells). We measured GSH levels in exponentially and confluent growing bcl-2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CCDeltaBH1, CCDeltaBH3, and CCDeltaTM cells compared with parental CC531, neo-transfected CC531 and CCDeltaBH4 cells. GSH levels in these bcl-2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CCDeltaBH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation.


Assuntos
Divisão Celular/genética , Regulação da Expressão Gênica , Glutationa/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Sequência de Aminoácidos , Animais , Apoptose/genética , Núcleo Celular/metabolismo , Glutationa/análise , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteína X Associada a bcl-2
6.
Biochem Pharmacol ; 62(8): 1087-97, 2001 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-11597577

RESUMO

Caspase activation is a central event in the execution phase of apoptosis and is associated with phosphatidylserine (PS) externalization and DNA fragmentation. We investigated the role of caspase activity in anticancer drug-induced PS externalization and DNA fragmentation in MTLn3 cells. Caspase activation (DEVD-AMC cleavage) occurred in a time- and concentration-dependent manner after exposure to doxorubicin, in association with cleavage of poly(ADP) ribose polymerase and protein kinase C delta, two caspase-3 substrates. Caspase activation was closely followed by oligonucleosomal DNA fragmentation and PS externalization as determined by flow cytometric analysis. Similar observations were made for etoposide and cisplatin. Inhibition of caspases with zVAD-fmk inhibited almost completely doxorubicin-induced DNA fragmentation as well as proteolysis of protein kinase C delta. In contrast, PS externalization induced by doxorubicin was only partly affected by caspase inhibition. Flow cytometric cell sorting demonstrated that DNA fragmentation in the remaining PS positive cells after doxorubicin treatment in the presence of zVAD-fmk was fully blocked. In conclusion, these data indicate that while DNA fragmentation in anticancer drug-induced apoptosis of MTLn3 cells is fully dependent on caspase activity, PS externalization is controlled by both caspase-dependent and caspase-independent pathways.


Assuntos
Antineoplásicos/farmacologia , Apoptose/fisiologia , Caspases/fisiologia , Fragmentação do DNA/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Adenocarcinoma/patologia , Animais , Caspase 3 , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Exocitose/efeitos dos fármacos , Neoplasias Mamárias Animais/patologia , Necrose , Ratos , Células Tumorais Cultivadas
7.
Cell Motil Cytoskeleton ; 49(2): 78-92, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11443738

RESUMO

Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 microM) partly protected against the rearrangement of the actin filaments in the hepatocytes.


Assuntos
Actinas , Apoptose , Citoesqueleto/ultraestrutura , Hepatócitos/citologia , Células Matadoras Ativadas por Linfocina/citologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Cocultura , Dendritos/ultraestrutura , Humanos , Interleucina-2 , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Ativadas por Linfocina/ultraestrutura , Masculino , Ratos , Ratos Wistar , Células Tumorais Cultivadas
8.
Biochem Pharmacol ; 61(4): 417-25, 2001 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11226375

RESUMO

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 microM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2'-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5'-amino-5'-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.


Assuntos
Adenosina/farmacologia , Apoptose , Neuroblastoma/patologia , Receptores Purinérgicos P1/fisiologia , Adenosina/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/fisiologia , Desoxiadenosinas/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/fisiologia , Camundongos , Neuroblastoma/metabolismo , Proteínas de Transporte de Nucleosídeos , Agonistas do Receptor Purinérgico P1 , Receptores Purinérgicos P1/isolamento & purificação , Células Tumorais Cultivadas
9.
Hepatology ; 32(4 Pt 1): 701-10, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11003613

RESUMO

Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21(waf1/cip1) protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21(waf1/cip1), and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic cells in vivo. This may support their clonal expansion and their further malignant transformation because protection against genetic damage is diminished.


Assuntos
Dano ao DNA , Neoplasias Hepáticas/química , Fígado/química , Proteínas Nucleares , Lesões Pré-Cancerosas/química , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas/análise , Proteína Supressora de Tumor p53/análise , Animais , Divisão Celular , Aberrações Cromossômicas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , DNA/biossíntese , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Neoplasias Hepáticas/genética , Masculino , Lesões Pré-Cancerosas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Ratos , Ratos Wistar
10.
Toxicol Appl Pharmacol ; 167(1): 55-62, 2000 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10936079

RESUMO

The cytotoxic monoterpene perillyl alcohol (POH) has anticancer properties. We investigated its cytotoxicity in PC12 cells in relation to its biotransformation. POH is oxidized by alcohol dehydrogenase and aldehyde dehydrogenase to perillaldehyde (PCO) and perillic acid (PCOOH), respectively. Apoptosis was determined by cell cycle (subG(0)G(1)) analysis and AnnexinV staining followed by flow cytometry. PCO caused apoptosis at 200 microM, POH caused apoptosis from 500 microM on, while PCOOH had no effect. The caspase inhibitor zVAD prevented apoptosis. Inhibition of POH oxidation by 4-methylpyrazol did not prevent the apoptotic effect of POH indicating that POH itself is also apoptotic. To find out to what extent POH is metabolized to PCO, the metabolism of POH, PCO, and PCOOH was determined after intravenous injection in the rat and in isolated hepatocytes. Although PCO can form a glutathione conjugate(s), no indication of the formation of GSH conjugates was found either in vivo or in hepatocytes. About 70% of the dose was recovered as glucuronides in bile and urine. PCOOH generated only the acyl glucuronide, while POH and PCO formed both acyl and ether glucuronides. These results indicate that PCO is a major intermediary metabolite of POH in the rat in vivo and suggest that PCO may contribute to the anticancer effect of POH.


Assuntos
Antineoplásicos/toxicidade , Monoterpenos , Terpenos/toxicidade , Animais , Bile/metabolismo , Biotransformação , Cicloexenos , Glutationa/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Células PC12 , Ratos , Ratos Wistar , Terpenos/farmacocinética
11.
Br J Cancer ; 82(9): 1539-46, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10789721

RESUMO

A phase I dose-escalation study was performed to determine whether isolated hepatic perfusion (IHP) with melphalan (L-PAM) allows exposure of the liver to much higher drug concentrations than clinically achievable after systemic administration and leads to higher tumour concentrations of L-PAM. Twenty-four patients with colorectal cancer confined to the liver were treated with L-PAM dosages escalating from 0.5 to 4.0 mg kg(-1). During all IHP procedures, leakage of perfusate was monitored. Duration of IHP was aimed at 60 min, but was shortened in eight cases as a result of leakage from the isolated circuit. From these, three patients developed WHO grade 3-4 leukopenia and two patients died due to sepsis. A reversible elevation of liver enzymes and bilirubin was seen in the majority of patients. Only one patient was treated with 4.0 mg kg(-1) L-PAM, who died 8 days after IHP as a result of multiple-organ failure. A statistically significant correlation was found between the dose of L-PAM, peak L-PAM concentrations in perfusate (R = 0.86, P< or =0.001), perfusate area under the concentration-time curve (AUC; R = 0.82, P<0.001), tumour tissue concentrations of L-PAM (R = 0.83, P = 0.011) and patient survival (R = 0.52, P = 0.02). The peak L-PAM concentration and AUC of L-PAM in perfusate at dose level 3.0 mg kg(-1) (n = 5) were respectively 35- and 13-fold higher than in the systemic circulation, and respectively 30- and 5-fold higher than reported for high dose oral L-PAM (80-157 mg m(-2)) and autologous bone marrow transplantation. Median survival after IHP (n = 21) was 19 months and the overall response rate was 29% (17 assessable patients; one complete and four partial remissions). Thus, the maximally tolerated dose of L-PAM delivered via IHP is approximately 3.0 mg kg(-1), leading to high L-PAM concentrations at the target side. Because of the complexity of this treatment modality, IHP has at present no place in routine clinical practice.


Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Quimioterapia do Câncer por Perfusão Regional , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Melfalan/administração & dosagem , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/farmacocinética , Área Sob a Curva , Neoplasias Colorretais/patologia , Humanos , Neoplasias Hepáticas/secundário , Melfalan/efeitos adversos , Melfalan/farmacocinética , Análise de Sobrevida , Resultado do Tratamento
12.
Biochem Pharmacol ; 59(12): 1557-62, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10799652

RESUMO

The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.


Assuntos
Apoptose , Butionina Sulfoximina/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Glutationa/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Antimetabólitos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Glutationa/deficiência , Humanos , Ratos , Células Tumorais Cultivadas
13.
J Biol Chem ; 275(33): 25805-13, 2000 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-10823823

RESUMO

Decreased phosphorylation of focal adhesion kinase and paxillin is associated with loss of focal adhesions and stress fibers and precedes the onset of apoptosis (van de Water, B., Nagelkerke, J. F., and Stevens, J. L. (1999) J. Biol. Chem. 274, 13328-13337). The cortical actin cytoskeletal network is also lost during apoptosis, yet little is known about the temporal relationship between altered phosphorylation of proteins that are critical in the regulation of this network and their potential cleavage by caspases during apoptosis. Adducins are central in the cortical actin network organization. Cisplatin caused apoptosis of renal proximal tubular epithelial cells, which was associated with the cleavage of alpha-adducin into a 74-kDa fragment; this was blocked by a general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk). Hemagglutinin-tagged human alpha-adducin was cleaved into a similar 74-kDa fragment by caspase-3 in vitro but not by caspase-6 or -7. Asp-Arg-Val-Asp(29)-Glu, Asp-Ile-Val-Asp(208)-Arg, and Asp-Asp-Ser-Asp(633)-Ala were identified as the principal caspase-3 cleavage sites; Asp-Asp-Ser-Asp(633)-Ala was key in the formation of the 74-kDa fragment. Cisplatin also caused an increased phosphorylation of alpha-adducin and gamma-adducin in the MARCKS domain that preceded alpha-adducin cleavage and was associated with loss of adducins from adherens junctions; this was not affected by z-VAD-fmk. In conclusion, the data support a model in which increased phosphorylation of alpha-adducin due to cisplatin leads to dissociation from the cytoskeleton, a situation rendered irreversible by caspase-3-mediated cleavage of alpha-adducin at Asp-Asp-Ser-Asp(633)-Ala.


Assuntos
Apoptose , Proteínas de Ligação a Calmodulina/metabolismo , Caspases/metabolismo , Rim/metabolismo , Actinas/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Animais , Antineoplásicos/farmacologia , Células COS , Proteínas de Transporte/metabolismo , Caspase 3 , Morte Celular , Cisplatino/farmacologia , Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Imunofluorescência , Humanos , Immunoblotting , L-Lactato Desidrogenase/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Plasmídeos/metabolismo , Isoformas de Proteínas , Proteína Quinase C/química , Proteína Quinase C/metabolismo , Ratos , Serina/metabolismo , Fatores de Tempo , Transfecção
14.
Eur J Cancer ; 36(1): 106-12, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10741303

RESUMO

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.


Assuntos
Neoplasias Hepáticas/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetoxiacetilaminofluoreno , Animais , Carcinógenos , Cisteína Endopeptidases , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Feminino , Imuno-Histoquímica , Neoplasias Hepáticas/etiologia , Complexos Multienzimáticos , Lesões Pré-Cancerosas/etiologia , Complexo de Endopeptidases do Proteassoma , Ratos , Ratos Sprague-Dawley , Raios X
16.
Biochem Pharmacol ; 58(12): 1891-8, 1999 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10591143

RESUMO

The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.


Assuntos
Adenosina/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Cicloeximida/antagonistas & inibidores , Fígado/efeitos dos fármacos , Animais , Cicloeximida/farmacologia , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Fígado/citologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/fisiologia , Substâncias Protetoras/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar
17.
Biochim Biophys Acta ; 1452(1): 12-24, 1999 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-10525156

RESUMO

The presence and localisation of G-actin in various cell lines was studied using the highly G-actin specific, fluorescence-labelled vitamin D-binding protein. In various cell-types, pig kidney-derived cells (LLC-PK1), Chinese hamster ovary (CHO) cells, SV-40 transformed African green monkey kidney (COS) cells and human hepatoma (HepG2) cells, G-actin was only visible in the cytoplasm of interphase cells. However, in mitotic cells, depending on the mitotic phase, intense G-actin staining was found associated with the mitotic spindle (early mitosis) or overlapping the DNA-staining pattern (late mitosis). Also after heat shock (60-180 min at 43 degrees C), an intense nuclear staining of G-actin was observed. In LLC-PK1 cells, the increase of nuclear G-actin staining disappeared again after 24 h at 37 degrees C, but in COS, CHO and HepG2 cells, it was still present in the nucleus after 24 h at 37 degrees C, indicating that the process was not rapidly reversible in these cells; the increased nuclear G-actin was not associated with cell division. Comparison of the amount of G-actin present in the nucleus and in the cytosol before and after heat shock using Western blotting demonstrated that the total amount of G-actin in both nucleus and cytosol was unchanged after heat shock. This indicates that the increased G-actin staining is not a result of import of G-actin into the nucleus. These observations suggest a rearrangement of G-actin in the nucleus during both mitosis and heat shock, which may be due to changes in interaction of G-actin with chromosomes.


Assuntos
Actinas/análise , Protocolos de Quimioterapia Combinada Antineoplásica , Núcleo Celular/metabolismo , Fuso Acromático/metabolismo , Animais , Linhagem Celular , Núcleo Celular/efeitos da radiação , Ciclofosfamida , Citoesqueleto/efeitos da radiação , Citosol/metabolismo , DNA/análise , Doxorrubicina , Corantes Fluorescentes , Haplorrinos , Histocitoquímica , Temperatura Alta , Humanos , Microscopia Confocal , Mitose , Compostos de Quinolínio , Suínos , Tiazóis , Células Tumorais Cultivadas , Vincristina , Proteína de Ligação a Vitamina D
18.
Toxicol Appl Pharmacol ; 159(3): 214-23, 1999 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-10486308

RESUMO

The formation of glutathione (GSH) conjugates of racemic 4-hydroxy-trans-2,3-nonenal (4-HNE) in the rat in vivo in the perfused rat liver and rat erythrocytes has been studied. An HPLC system was developed for the assay of 4-HNE-glutathione conjugates (HNE-SG). The very sensitive electrochemical detection method (detection limit 5 pmol) can also be used to study endogenously formed HNE-SG. Three diastereomeric HNE-SG conjugates could be separated by this system. Rat liver cytosol catalyzed the formation of 2 of the 3 conjugates. When 17 micromol/kg [(3)H] 4-HNE was injected intravenously in the rat, 21% of the radioactivity was excreted within 90 min in bile and 37% in urine. Most of the 4-HNE in bile was present as 2 of the HNE-SG conjugates (molecular mass 463). In addition, 25% was excreted as a third GSH conjugate (molecular mass of 461), which was identified as the lactone of the 4-hydroxynonenoic acid glutathione conjugate. Erythrocytes in vitro eliminated 4-HNE very rapidly, in part by GSH conjugation, suggesting that they may also play an important role in vivo. To study the role of the liver selectively, we used the recirculating perfused rat liver without erythrocytes in the perfusion medium; the same conjugates were found, but the third conjugate was a minor component. These results present direct evidence for the in vivo formation of 4-HNE glutathione conjugates in which the liver may play an important role.


Assuntos
Aldeídos/metabolismo , Eritrócitos/metabolismo , Glutationa/metabolismo , Fígado/metabolismo , Aldeídos/farmacocinética , Aldeídos/toxicidade , Animais , Reagentes de Ligações Cruzadas , Eritrócitos/efeitos dos fármacos , Peroxidação de Lipídeos , Fígado/efeitos dos fármacos , Masculino , Taxa de Depuração Metabólica , Perfusão , Ratos , Ratos Wistar
19.
Biochem Pharmacol ; 58(4): 655-64, 1999 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10413303

RESUMO

Intracellular levels of glutathione have been shown to affect the sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, SW837, and SW1116) that GSH depletion by L-buthionine-[S,R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cycle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enhance the inhibition of S phase progression as induced by L-PAM; at high concentration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Apoptose , Ciclo Celular/fisiologia , Neoplasias Colorretais/tratamento farmacológico , Glutationa/fisiologia , Melfalan/farmacologia , Antioxidantes/farmacologia , Apoptose/fisiologia , Butionina Sulfoximina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias Colorretais/patologia , Células HT29 , Humanos , Células Tumorais Cultivadas
20.
Cancer Chemother Pharmacol ; 44(2): 111-6, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10412944

RESUMO

PURPOSE: Glutathione (GSH) plays an important role in the resistance of tumors to cytostatics. Therefore, depletion of GSH by the GSH synthesis inhibitor buthionine sulfoximine (BSO) has been proposed to enhance the efficacy of certain anticancer agents. We studied the effect of BSO in rats bearing intrahepatically implanted tumors of the CC531 colorectal cancer cell line on the antitumor activity of melphalan (L-PAM). Since these liver tumors tend to derive most of their blood supply from the hepatic artery, we evaluated whether delivery of BSO into the hepatic artery would more selectively decrease GSH levels in the implanted tumor tissue as compared with normal liver and extrahepatic tissues. METHODS: Tumor-bearing rats were treated with a 24-h continuous infusion of 0.375 mmol/ kg BSO via the jugular vein, immediately followed by a bolus L-PAM (15 micromol/kg; 4.5 mg/kg) infusion via the hepatic artery. Laparotomy was performed on day 14 and 28 after treatment for measurement of the liver tumors. For the evaluation of locoregional administration of BSO, a 24-h continuous infusion of 0.375 mmol/kg BSO was delivered into either the hepatic artery, the portal vein, or the jugular vein in freely moving rats and GSH levels in the tumor, liver, kidney, lung, heart, bone marrow, and blood were measured. RESULTS: BSO infusion via the jugular vein increased the antitumor efficacy of L-PAM injected into the hepatic artery 2-fold as determined at 14 days after treatment. Although infusion of BSO via the hepatic artery depleted GSH more severely in the tumor as compared with jugular vein or portal vein administration, the additional effect was only slight (10%). No difference was observed in any other tissue. CONCLUSION: GSH depletion increased the cytostatic efficacy of L-PAM 2-fold in vivo as determined at 14 days after treatment. Hepatic artery infusion of BSO translated into a statistically significant, but probably not therapeutically relevant, increase in tumor GSH depletion as compared with the other routes of BSO administration.


Assuntos
Butionina Sulfoximina/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Glutationa/análise , Neoplasias Hepáticas Experimentais/secundário , Melfalan/administração & dosagem , Animais , Sinergismo Farmacológico , Infusões Intra-Arteriais , Masculino , Ratos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...