RESUMO
Rats with transplantable spontaneously metastasizing mammary tumors have elevated levels of both serum sialoglycoconjugate and serum sialytransferase activity compared with normal female rats or rats with various nonmetastasizing mammary tumors. A direct relationship was observed between the amount of serum protein-bound sialic acid and serum sialyltransferase activity in all rats studied. Serum sialyltransferase activity in rats with a representative metastasizing mammary tumor, SMT-2A, was also correlated with tumor age. Microsomes prepared from the SMT-2A tumor have a sixfold higher sialyltransferase activity than do microsomes prepared from the nonmetastasizing mammary tumor MT-W9B. Normal rat liver microsomes have the same level of activity as microsomes prepared from livers of animals with either SMT-2A or MT-W9B tumors. The data indicate that spontaneously metastasizing mammary tumor cells have an increased production and release, perhaps through cell surface shedding, of a sialyltransferase. It is suggested that this sialyltransferase may increase the serum half-life of certain tumor-specific circulating glycoconjugates by increasing the content of protein-bound sialic acid and may thereby play a role in the immune escape mechanism of metastasizing tumor cells.
Assuntos
Neoplasias Mamárias Experimentais/sangue , Metástase Neoplásica , Ácidos Siálicos/sangue , Sialiltransferases , Transferases , Animais , Feminino , Fígado/enzimologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/enzimologia , Metilcolantreno , Microssomos/enzimologia , Ratos , Fatores de TempoRESUMO
BACKGROUND: The taxane paclitaxel (Taxol) is often of limited efficacy in chemotherapeutic regimens because some cancer cells express high levels of the efflux pump, P-glycoprotein (Pgp), which removes the drug from the cells. The orally active paclitaxel analog IDN-5109 has been reported to overcome Pgp-mediated drug resistance. We tested whether IDN-5109 acts by modulating Pgp activity. METHODS: Human MDA435/LCC6mdr1 and MDA435/LCC6 breast carcinoma cells, which express and do not express Pgp, respectively, were incubated with [3H]IDN-5109 and paclitaxel to determine intracellular drug accumulation. Flow cytometry was used to analyze intracellular retention of two Pgp substrates, rhodamine 123 (Rh-123) and doxorubicin, in both breast carcinoma cell lines and in human colon carcinoma cells (SW-620, DLD1, and HCT-15, whose Pgp levels vary) treated with different taxanes. The effects of IDN-5109 and paclitaxel on tumor growth in vivo were studied with the use of tumors established through xenografts of Pgp-expressing SW-620 and DLD1 cells in severe combined immunodeficiency mice. All statistical tests were two-sided. RESULTS: Pgp-expressing cells treated with IDN-5109 or with the taxane-based drug resistance reversal agent tRA96023, which blocks Pgp activity, retained 8.1- and 9.4-fold more Rh-123 (P =.0001), respectively, and 1.7- and 1.9-fold more doxorubicin (P =.001), respectively, than cells treated with paclitaxel. Non-Pgp-expressing cells treated similarly demonstrated no increased retention of either substrate. MDA435/LCC6mdr1 cells retained 5.3-fold more [3H]IDN-5109 than [3H]paclitaxel after 2 hours (P =.01). IDN-5109 showed statistically significantly higher tumor growth inhibition than paclitaxel against the SW-620 xenograft (P =.003). CONCLUSIONS: IDN-5109 modulates Pgp activity, resulting in superior tumor growth inhibition against Pgp-expressing tumors as compared with paclitaxel. IDN-5109 may broaden the spectrum of taxane use to include colon tumors.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Neoplasias do Colo/tratamento farmacológico , Paclitaxel/análogos & derivados , Paclitaxel/farmacologia , Taxoides , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/efeitos dos fármacos , Administração Oral , Animais , Antibióticos Antineoplásicos , Antineoplásicos Fitogênicos/farmacocinética , Neoplasias da Mama/metabolismo , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Neoplasias do Colo/metabolismo , Doxorrubicina/farmacocinética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Citometria de Fluxo , Fluorescência , Humanos , Masculino , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Distribuição Tecidual , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Tunicamycin, an antibiotic which specifically inhibits the dolichol-mediated synthesis of glycoproteins, significantly decreased the incorporation of tritiated D-mannose and D-glucosamine into L1210 ascites leukemia cell glycoproteins at concentrations which affected the biosynthesis of proteins minimally. Mice receiving inoculations of L1210 cells pretreated with 10 microM tunicamycin in vitro survived nearly twice as long as did mice receiving implants of untreated tumor cells. A nonlethal dose of X-irradiation (350 rads) to mice 24 hr prior to receiving their inoculation of tunicamycin-treated L1210 cells prevented this increase in life span. Thirty-eight % of the long-term surviving mice which received 1 X 10(5) L1210 cells pretreated with 10 microM tunicamycin in vitro were then resistant to a subsequent challenge with 10(6) untreated L1210 ascites cells. Direct i.p. administration of tunicamycin to mice resulted in potent liver toxicity (50% lethal dose, 2.0 mg/kg) which obviated any therapeutic efficacy when administered to L1210 ascites tumor-bearing mice. The administration of nontoxic levels of D-mannose prior to the administration of tunicamycin decreased the toxicity of the antibiotic in vivo and, when combined with D-mannose in vitro, exhibited cytotoxic additivity in terms of the inhibition of L1210 leukemic cell growth. A therapeutic regimen incorporating a 24-hr infusion of the sugar prior to multiple administrations of tunicamycin gave evidence of a small therapeutic response in terms of the survival of tumor-bearing mice. These results suggest that tunicamycin, an inhibitor of glycoprotein biosynthesis, might be able to alter tumor cell growth and immunogenicity provided that host liver toxicity is diminished.
Assuntos
Glucosamina/análogos & derivados , Leucemia L1210/tratamento farmacológico , Tunicamicina/uso terapêutico , Animais , Glicoproteínas/biossíntese , Tolerância Imunológica/efeitos da radiação , Cinética , Leucemia L1210/metabolismo , Manose/farmacologia , Camundongos , Camundongos Endogâmicos DBA , Proteínas de Neoplasias/biossíntese , Precursores de Proteínas/biossíntese , Tunicamicina/toxicidade , Irradiação Corporal TotalRESUMO
A murine melanoma variant (B16-F10ir6), resistant to lymphocytic cytolysis, has been shown previously to produce lower numbers of tumor nodules in the lung of C57BL/6J mice following i.v. inoculations. These differences found in tumor implantation and lymphocyte recognition may be due to changes in surface properties of this cell line. Therefore, membrane-bound sialic acid (released by Vibrio cholerae neuraminidase treatment), ectosialyltransferase activity, and total cellular glycosidase levels were measured in this cell line and compared with levels in its parent melanoma tumor cell line, B16-F10, which was selected for its enhanced ability to form tumor nodules. The results of these studies indicate a correlation between the degree of lung implantation and the amount of tumor cell sialic acid accessible to neuraminidase cleavage, tumor cell surface sialyltransferase activity, and several cellular glycosidase activities. These results are consistent with the idea that membrane structural changes in the glycocalyx may account for the ability of a tumor cell to implant and metastasize.
Assuntos
Membrana Celular/enzimologia , Glicosídeo Hidrolases/metabolismo , Melanoma/enzimologia , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Transferases/metabolismo , Fosfatase Ácida/metabolismo , Animais , Linhagem Celular , Neoplasias Pulmonares/enzimologia , Melanoma/análise , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/enzimologiaRESUMO
Following therapeutic administration, cyclophosphamide and Adriamycin are biotransformed to reactive metabolites, some of which are responsible for undesirable systemic toxicities of these chemicals, whereas others are responsible for their chemotherapeutic effectiveness. Microsomal mixed function oxidases activate cyclophosphamide to produce phosphoramide mustard and acrolein, while cytochrome reductase and xanthine oxidase are capable of transforming Adriamycin and forming free radicals. These reactive metabolites produce unwanted toxic side effects; however, their action may be partially ameliorated by the concomitant administration of thiols. In this study we evaluated the therapeutic activity of combinations of mesna (2-mercaptoethanesulfonate) with cyclophosphamide or Adriamycin in mice with a variety of transplantable tumors (L1210 and P-388 leukemia, Lewis lung and colon 26 carcinoma, B16 melanoma, and M5076 sarcoma). In all cases the administration of mesna prior to cyclophosphamide or Adriamycin treatment did not reduce the antitumor effectiveness of these agents and in some instances (C57BL/6 mice with B16 melanoma or M5076 sarcoma) small improvements were observed. Therefore, the addition of thiols, to reduce effectively the buildup of toxic metabolites of cyclophosphamide or Adriamycin may result in the improved therapeutic effectiveness for these agents in the treatment of cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Mercaptoetanol/análogos & derivados , Mesna/uso terapêutico , Neoplasias Experimentais/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Neoplasias do Colo/tratamento farmacológico , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Leucemia Experimental/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma Experimental/tratamento farmacológico , Mesna/administração & dosagem , Camundongos , Camundongos Endogâmicos , Sarcoma Experimental/tratamento farmacológicoRESUMO
Degradation and invasion of basement membrane by tumor cells involves the cooperative hydrolysis of proteoglycans, collagens, and glycoproteins mediated by a number of enzymes including proteases, collagenases, and glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian carcinoma cells. Using this system, a correlation was observed between solubilization of glycoconjugates present in ECM and extracellular levels of beta-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual isoenzymes of beta-N-acetylglucosaminidase (beta-NAG) in ECM degradation, the cellular and secreted forms of the enzyme were fractionated and characterized. Three intracellular isoenzyme forms A, I, and B, were isolated from invasive human ovarian carcinoma cell line A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total beta-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of beta-NAG (As and Bs) were detected in serum-free medium. The separated intracellular and secreted isoenzymes demonstrated similar Km values, ranging from 1 to 5 mM, with p-nitro-B-N-acetylglucosaminide substrate. Treatment of [3H]glucosamine-labeled ECM with the separated isoenzymes of beta-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of 1 greater than B much greater than A. These results suggest that human ovarian carcinoma cell beta-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted enzymes and form I as a membrane enzyme.
Assuntos
Acetilglucosaminidase/metabolismo , Carcinoma/enzimologia , Matriz Extracelular/metabolismo , Hexosaminidases/metabolismo , Neoplasias Ovarianas/enzimologia , Acetilglucosaminidase/isolamento & purificação , Membrana Celular/enzimologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Frações Subcelulares/enzimologia , Células Tumorais CultivadasRESUMO
Previously, we have shown that galaptin, an endogenous beta-galactoside-binding lectin, is present in extracellular matrix where it may participate in the adhesion of A121 human ovarian carcinoma cells to extracellular matrix via interaction with specific cell surface carbohydrate receptors. We now report that A121 cells adhere to polystyrene plates coated with polymerized human splenic galaptin. The carbohydrate-mediated specificity of this adhesive interaction was demonstrated by inhibition with lactose. Additionally, treatment of A121 cells with neuraminidase increased cellular adherence by 30%, while beta-galactosidase treatment of cells decreased adherence by 65%. These findings prompted us to isolate and identify the cell surface galaptin receptor. In a Western blot of A121 cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 125I-labeled polymerized galaptin bound [corrected] to a unique cellular protein having a molecular mass of 110 kDa. This receptor was enriched by affinity chromatography using polymerized galaptin-Sepharose. Treatment of this material with N-glycanase ablated its galaptin-binding activity. In related studies, A121 cells metabolically labeled with [3H]glucosamine demonstrated a radiolabeled polymerized galaptin-binding protein with an identical molecular mass of 110 kDa. These studies confirmed the glycoprotein nature of this putative endogenous cellular galaptin receptor. Further studies with antibodies directed against two lysosomal associated membrane proteins, lamp-1 and lamp-2, demonstrated specific reactivity in Western blots with the 110-kDa glycoprotein. Additionally, 125I-polymerized galaptin recognized a 110-kDa protein in Western blots of material immunoprecipitated from A121 cell lysates by lamp-1 and lamp-2 antibodies. Finally, indirect immunofluorescence using antibodies directed against lamps detected cell surface antigenicity. Therefore, lamp-1 and/or lamp-2 appear to be the putative cell surface receptors involved in the adhesion of ovarian carcinoma cells to extracellular matrix mediated by galaptin.
Assuntos
Antígenos CD , Adesão Celular/fisiologia , Hemaglutininas/fisiologia , Neoplasias Ovarianas/fisiopatologia , Receptores Mitogênicos/análise , Ligação Competitiva , Adesão Celular/efeitos dos fármacos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Feminino , Galectinas , Hemaglutininas/efeitos dos fármacos , Hemaglutininas/metabolismo , Humanos , Lactose/metabolismo , Proteínas de Membrana Lisossomal , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Neuraminidase/farmacologia , Neoplasias Ovarianas/química , Receptores Mitogênicos/química , Células Tumorais Cultivadas/fisiologia , beta-Galactosidase/farmacologiaRESUMO
The spermine analogues, N1,N12-bis(ethyl)spermine (BESPM), N1,N11-bis(ethyl)norspermine (BENSPM), and N1,N14-bis(ethyl)-homospermine (BEHSPM) behave similarly in down-regulating the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, but differ distinctly in their abilities to induce the polyamine catabolic enzyme, spermidine/spermine-N1-acetyltransferase; BENSPM is 6-fold more effective than BESPM in increasing spermidine/spermine-N1-acetyltransferase activity and BEHSPM is 10-fold less effective. Since MALME-3 human melanoma cells are extremely responsive to spermidine/spermine-N1-acetyltransferase induction (i.e., increases greater than 200-fold) and since this induction correlates with growth inhibition among melanoma cell lines, the ability of these homologues to inhibit the growth of MALME-3 xenografts was examined. Analogues were administered i.p. three times per day (i.e., every 8 h) for 6 days at the following doses per injection: BEHSPM, 1.5, 3, or 6 mg/kg; BESPM, 10, 20, or 40 mg/kg; BENSPM, 20, 40, or 80 mg/kg. At the highest tolerated doses, all of the analogues fully suppressed growth of established (100-200 mm3) MALME-3 tumor during treatment and sustained tumor growth inhibition following treatment as follows: BEHSPM, 14 days; BESPM, 27 days, and BENSPM, 37 days. The tumor delay (to reach 1000 mm3 relative to control) at the highest tolerated doses was as follows: BEHSPM, 20 days; BESPM, 34 days, and BENSPM, 63 days. The rank order of analogue host toxicity as indicated by weight loss was opposite that for antitumor activity, BEHSPM was most toxic, BESPM, intermediate, and BENSPM, least toxic. Thus, the most effective of the three homologues, BENSPM, was best tolerated, and produced an initial tumor regression, full suppression of tumor regrowth during treatment, and sustained inhibition of tumor regrowth for 37 days after treatment stopped. Owing to its potent antitumor activity, mild host toxicity, and novel apparent mechanism of action, BENSPM is being considered for further development toward clinical trial.
Assuntos
Acetiltransferases/biossíntese , Melanoma/tratamento farmacológico , Espermina/análogos & derivados , Animais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Ensaios de Seleção de Medicamentos Antitumorais , Indução Enzimática/efeitos dos fármacos , Feminino , Humanos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Camundongos Nus , Espermina/farmacologia , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Lysosomal enzymes were elevated about two-fold in primary s.c. Lewis lung carcinoma as compared with metastatic nodules in the lung. In a time course experiment, a general two-fold elevation of acid phosphatase and several glycosidases was observed in the primary tumor between the 14th and 17th postimplant day following s.c. inoculation of Lewis lung carcinoma. This increase in hydrolytic enzyme activity was not due to necrosis in the primary tumor since a comparison of enzyme activities in the nonnecrotic and necrotic areas demonstrated much higher activities in the nonnecrotic areas. No increases in lysosomal enzyme activity were observed with time in Sarcoma 180, a tumor which does not metastasize. There was no change with time in primary Lewis lung tumor lactate dehydrogenase activity while a 7-fold increase in serum lactate dehydrogenase activity was observed in tumor-bearing mice. Mitochondrial succinate-2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium reductase levels fell in the primary Lewis lung tumor as the tumor size increased. A positive correlation was observed between the time of the elevations of tumor lysosomal enzymes in Lewis lung carcinoma and the appearance of micro- and macrometastatic lesions in the lungs. The mechanisms accounting for the increased intratumoral lysosomal enzymes are unknown, but they may be related to macrophage infiltration or other tumor-host interactions which may facilitate the dissemination of tumor cells.
Assuntos
Carcinoma/enzimologia , Neoplasias Pulmonares/enzimologia , Lisossomos/enzimologia , Fosfatase Ácida/análise , Animais , Carcinoma/sangue , Carcinoma/patologia , Feminino , Glicosídeo Hidrolases/análise , L-Lactato Desidrogenase/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Oxirredutases/análise , Sarcoma Experimental/enzimologia , Fatores de TempoRESUMO
In in vitro systems, the spermine analogue, N1,N11-bis(ethyl)norspermine (BENSPM), suppresses the polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ornithine decarboxylase and S-adenosylmethionine decarboxylase, respectively), greatly induces the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), depletes polyamine pools, and inhibits cell growth. Against MALME-3 M human melanoma xenografts, BENSPM and related homologues demonstrate potent antitumor activity that has been found to correlate positively with their ability to induce SSAT activity in vitro. Herein, we further evaluate the antitumor activity of BENSPM and at the same time characterize the biochemical effects of BENSPM treatment on polyamine metabolism of selected normal and tumor tissues. At 40 mg/kg 3 times/day for 6 days i.p., BENSPM suppressed growth of MALME-3 M human melanoma xenografts during treatment and for 65 days afterwards. Similar antitumor activity was obtained with 120 mg/kg once daily for 6 days and 40 mg/kg once daily for 6 days, indicating that against this tumor model, the dosing schedule can be relaxed up to sixfold without compromising antitumor activity. When MALME-3 M tumor-bearing mice were retreated with BENSPM 2 weeks after the first treatment at 40 mg/kg 3 times/day for 6 days, initial tumor volumes of 85 mm3 were reduced to < 10 mm3. Analysis of melanoma, liver, and kidney tissues from mice treated with 40 mg/kg 3 times/day for 6 days revealed relatively similar accumulations of BENSPM in all tissues at levels greater than the original total content of polyamine pools. By 2 weeks following treatment, BENSPM pools in normal tissues were almost gone, whereas in tumor tissues significant amounts (40%) were still retained. The biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, gave no indication of enzyme suppression (or increase) by the analogue as typically occurs in vitro. By contrast, SSAT was induced from an average of < 50 pmol/min/mg in control tissues to 320 pmol/min/mg in liver, 1255 pmol/min/mg in kidney, and 13,710 pmol/min/mg in MALME-3M tumor. Two weeks later, SSAT activity was still 12 times higher in tumor than in kidney. Polyamine pools (putrescine, spermidine, and spermine) were reduced after treatment in all tissues and approached near-total depletion in the tumor. Good antitumor activity and even more potent induction of SSAT (i.e., 26,680 pmol/min/mg) was also observed in PANUT-3 human melanoma xenografts. Overall, the findings reveal meaningful antitumor activity by BENSPM against 2 human melanoma xenografts and provide in vivo evidence consistent with SSAT-induced polyamine depletion playing a determining role in at least the initial phase of the antitumor response.
Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Espermina/análogos & derivados , Acetiltransferases/efeitos dos fármacos , Acetiltransferases/metabolismo , Adenosilmetionina Descarboxilase/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Melanoma/enzimologia , Melanoma/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Inibidores da Ornitina Descarboxilase , Poliaminas/metabolismo , Espermina/farmacologia , Transplante HeterólogoRESUMO
Two newly synthesized nitrosoureido sugars have been evaluated for their antitumor activity and diabetogenic potential in a number of in vitro and in vivo preclinical tumor model systems. 2-Amino-2-deoxy-N'-methyl-N'-nitrosoureido-1,3,4,6-tetra-O-acetyl-alpha- D- mannopyranose (MAZ), a lipophilic mannosamine derivative, and ethyl-6-deoxy-3,5-di-O-methyl-6-(3-methyl-3-nitrosoureido)-alph a- D-glucofuranoside (EDOMEN or CGP 6'809), were both found to inhibit L1210 leukemia cell growth in vitro by 50% at approximately 5.0 X 10(-5) M. At these concentrations, little effect was noted immediately on L1210 cell radiolabeled precursor incorporation; however, at higher concentrations, EDOMEN inhibited [3H]leucine and [3H]mannose incorporation, while MAZ specifically decreased L1210 cell [3H]thymidine and [3H]leucine incorporation. Inhibition of Lewis lung carcinoma and B16 melanoma cell growth by 50% in vitro was achieved at higher concentrations of these agents (10(-4) to 10(-3) M). Since the currently available nitrosoureido sugars, streptozotocin and chlorozotocin, have been observed by us to be diabetogenic, EDOMEN and MAZ were evaluated for their specific toxicity to rat pancreatic beta-cells in vitro. Cytotoxicity in beta-cell cultures was monitored both by phase-contrast microscopy and the release of insulin into the culture medium. beta-Cells were found to be 10-fold more sensitive to the toxic effects of MAZ than were pancreatic fibroblasts. EDOMEN, on the other hand, did not damage beta-cells preferentially and therefore was not considered diabetogenic. Both MAZ and EDOMEN had moderate activity as antileukemic agents in mice. At 50 mg/kg/day i.p. for 5 days, MAZ increased the life span of female DBA/2J mice with L1210 leukemia by over 50%. Similarly, doses of EDOMEN at 125 to 250 mg/kg/day i.p. for 5 days increased L1210 leukemic life span by nearly 60%. At these doses, no effect of MAZ was observed on primary Lewis lung carcinoma growth or life span of tumor-bearing C57BL/6 mice. EDOMEN, however, increased life span in Lewis lung carcinoma mice by up to 33% and caused an apparent antimetastatic effect. These studies indicate that EDOMEN may have enhanced value as a cancer chemotherapeutic agent due to its therapeutic effectiveness, lack of diabetogenic potential, and other favorable formulation properties (water solubility) as compared with other clinically available nitrosoureas.
Assuntos
Antineoplásicos/uso terapêutico , Ilhotas Pancreáticas/efeitos dos fármacos , Leucemia L1210/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Compostos de Nitrosoureia/uso terapêutico , Animais , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Feminino , Insulina/metabolismo , Masculino , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos DBARESUMO
Penetration of the extracellular matrix (ECM) by tumor cells, an event which occurs at various stages of the metastatic process, involves tumor cell glycosidase mediated hydrolysis of proteoglycans (PG). Recently, we observed that human ovarian carcinoma cell lines (HOCC) derived from primary tumors, peritoneal effusions, and distant metastases possess a varying ability to degrade radiolabeled PG of the ECM, while normal cells (human mesothelial cells or ovarian fibroblasts) fail to do so. To determine whether a quantitative relationship exists between glycosidase activity and degradation of ECM, both intracellular and extracellular glycosidase activities were measured for HOCC and normal cell lines. No relationship was found between intracellular glycosidase activities and the ability of cells to degrade ECM. However, a correlation was observed between extracellular or secretory glycosidase activities and HOCC mediated ECM degradation. In particular, a 5-8-fold increase, as compared to normal cells, was observed for HOCC extracellular beta-N-acetylglucosaminidase (EC 3.2.2.30) activity. The accumulation or secretion of this enzyme from HOCC into culture medium was found to be time dependent and not related to intracellular levels. Purified hexosaminidase derived from invasive HOCC was able to hydrolyze [3H]-glucosamine radiolabeled ECM (up to 30% radiolabel) and resulted in the cumulative release of free [3H]-N-acetylglucosamine. This enzyme mediated hydrolysis could be completely prevented with 2-acetamido-2-deoxy-1,5-D-gluconolactone, a competitive inhibitor (Ki 10(-6) M). Finally, HOCC mediated degradation of radiolabeled ECM was discerned to be dependent upon active hexosaminidase action, since tumor cell mediated degradation of ECM could be inhibited by up to 60% in the presence of this synthetic competitive inhibitor. In summary, these studies indicate a strong association between HOCC solubilization of glycoconjugates present in the ECM and extracellular levels of hexosaminidase.
Assuntos
Carcinoma/metabolismo , Matriz Extracelular/metabolismo , Glicosídeo Hidrolases/metabolismo , Neoplasias Ovarianas/metabolismo , Acetilglucosamina/metabolismo , Células Cultivadas , Feminino , Glucosamina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Inibidores de Proteases/farmacologia , beta-N-Acetil-Hexosaminidases/farmacologiaRESUMO
1-beta-D-Arabinofuranosylcytosine 5'-diphosphate-rac-1-S-octadecyl-2-O- palmitoyl-1-thioglycerol (ara-CDP-DL-PTBA) is an effective stable 1-beta-D-arabinofuranosylcytosine (ara-C) conjugate of thioether phospholipid against a variety of transplantable tumors in mice. The conjugate was formulated in a micellar solution by sonication, in which the conjugate exists as micellar discs (size, 0.01 to 0.04 micron). Analyses on thin-layer and high-pressure liquid chromatography showed that the conjugate was chemically stable upon storage at 3-4 degrees C for more than a 6-mo period. However, stored at room temperature for 3 mo it began to degrade (3 to 11%) to 1-beta-D-arabinofuranosylcytosine 5'-monophosphate and phosphatidic acid. At 3-4 degrees C, the micellar structure remained generally unchanged for 6 mo (size, less than 0.1 micron). Samples stored for 4 mo at room temperature formed some larger vesicles (size, 0.1 to 0.4 micron). Antitumor activity against i.p. implanted L1210 leukemia in mice remained relatively constant with samples stored for 6 mo at 3-4 degrees C or 3 mo at room temperature. 1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (ara-CTP) levels were elevated (greater than 500 pmol/10(7) cells) in L1210 leukemia cells within 1 h following i.p. administration of 400 mg/kg of ara-CDP-DL-PTBA to mice. More importantly, retention of cellular ara-CTP was prolonged (greater than 24 h) in these tumor cells as compared with ara-C treatments. Administration of ara-CDP-DL-PTBA to mice with colon 26 carcinoma (s.c.) resulted in both significant antitumor activity with an increased life span greater than 100% and decreased tumor size. The conjugate also demonstrated a dose-dependent therapeutic effect in mice with M5076 sarcoma (s.c.) as demonstrated by decreases in tumor size and liver metastases. Overall, ara-CDP-DL-PTBA, a stable lipid conjugate of ara-C in a micellar solution, appears to offer substantial therapeutic benefit to mice with leukemia and solid tumors warranting its further development and clinical investigation.
Assuntos
Antineoplásicos/uso terapêutico , Citarabina/análogos & derivados , Leucemia L1210/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Éteres Fosfolipídicos/uso terapêutico , Animais , Neoplasias do Colo/tratamento farmacológico , Citarabina/síntese química , Citarabina/metabolismo , Citarabina/uso terapêutico , Estabilidade de Medicamentos , Feminino , Técnica de Fratura por Congelamento , Masculino , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Microscopia Eletrônica , Estrutura Molecular , Éteres Fosfolipídicos/síntese química , Sarcoma Experimental/tratamento farmacológicoRESUMO
The goal of this study was to establish an optimal in vitro growth assay system for human urological tumor explants. Bovine corneal endotelial cell extracellular matrix (ECM) coated dishes were evaluated as a growth substrate for tumor cultures. Growth success for different urological carcinomas (prostatic, bladder, kidney, and testicular) was compared after seeding fresh surgical explants onto bovine corneal endothelial cell ECM and plastic culture flasks. Tumor samples were disaggregated enzymatically, and 1 X 10(4) cells were seeded onto the different substrates using RPMI 1640 medium containing 10% fetal calf serum and/or different growth factors, nutrients, and hormones. Cell growth on ECM was quantitated on days 7-15 by [3H]thymidine uptake, cell counting, and total protein. Tumor cells were characterized by flow cytometry and cytology. It was observed that ECM provides superior culture conditions for urological carcinomas. By increasing the initial number of cells plated on ECM and by adding different growth factors or hormones, the growth rate for specific tumor types was increased significantly. Several tumors (11 cases) grown on ECM were examined under the light microscope, and in all cases pre- and post-cytology confirmed malignancy. Tumor cells maintained on ECM and transplanted into nude mide retained their tumorigenic and morphological characteristics. Clinically aggressive tumors were associated with extensive ECM degradation. In addition, the growth of fresh human tumors on ECM provides a biologically relevant model system (for assessing the invasiveness of tumors in vitro) and should also be useful for drug evaluation studies.
Assuntos
Matriz Extracelular/fisiologia , Neoplasias Renais/patologia , Neoplasias da Próstata/patologia , Neoplasias Testiculares/patologia , Neoplasias da Bexiga Urinária/patologia , Adesão Celular , Ciclo Celular , Células Cultivadas , Meios de Cultura , Endotélio , Fibroblastos/patologia , Substâncias de Crescimento , Humanos , Masculino , PlásticosRESUMO
On the basis of encouraging preclinical findings, polyamine analogues have emerged as a novel class of experimental antitumor agents. The spermine derivative N1,N11-diethylnorspermine (DE-333, also known as DENSPM) is currently undergoing Phase I clinical trials against solid tumors. A series of systematically modified DE-333 analogues differing in intra-amine carbon distances and in N-alkyl terminal substituents (i.e., methyl, ethyl, and propyl) were evaluated in MALME-3M human melanoma cells, a cell line known to be cytotoxically affected by DE-333 and especially responsive to analogue induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase. Analogues accumulated to comparable intracellular concentrations and similarly affected cell growth with IC50 values in the 0.5-1.0 microM range. During prolonged incubations, diethyl and dipropyl analogues were cytotoxic, whereas two dimethyl analogues were cytostatic. Cell cycle analysis following treatment with the cytotoxic analogues revealed a prominent G1 block apparent as an accumulation of cells in G0/G1 and depletion of S-phase cells as well as a less restrictive G2 block. By contrast, cytostatic analogues incompletely arrested cells in G1, leaving a significant number of S-phase cells. Morphological and immunocytochemical analysis of detached cells revealed a far greater proportion of apoptotic cells with cytotoxic analogues than with cytostatic analogues. Although spermidine/spermine N1-acetyltransferase activity was differentially induced by the analogues, there was no obvious correlation with cell cycle effects. Overall, these data indicate a previously unrecognized combined effect of polyamine analogues on cell cycle progression and apoptosis. On the basis of structure-function relationships, these activities may be manipulated to optimize therapeutic efficacy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Melanoma/tratamento farmacológico , Melanoma/patologia , Espermina/análogos & derivados , Acetiltransferases/biossíntese , Antineoplásicos/farmacocinética , Poliaminas Biogênicas/metabolismo , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Espermina/farmacocinética , Espermina/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The spermine analogue N1,N11-diethylnorspermine (DENSPM) has been shown to induce the polyamine-acetylating enzyme spermidine/spermine N1-acetyltransferase, disrupt polyamine pool homeostasis, and inhibit tumor growth. DENSPM is currently being developed as an anti-neoplastic agent and is about to enter Phase II clinical trials. In this report, the antitumor efficacy of DENSPM was evaluated against a human transitional cell bladder BL13 carcinoma xenograft implanted orthotopically and s.c. in nude athymic mice. DENSPM was administered via continuous s.c. infusion at 93 mg/kg/day for 5 days. Treatment with DENSPM was well tolerated and produced tumor regressions in all mice with a significant proportion (up to 50%) of apparent cures. On the basis of low toxicity and good therapeutic efficacy, there is a strong rationale for evaluation of the therapeutic efficacy of DENSPM against bladder carcinomas in Phase II clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células de Transição/tratamento farmacológico , Espermina/análogos & derivados , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Carcinoma de Células de Transição/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Putrescina/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Espermina/farmacocinética , Espermina/uso terapêutico , Espermina/toxicidade , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismoRESUMO
Certain N-alkylated analogues of the natural polyamine spermine have been found to disrupt polyamine pool homeostasis and inhibit tumor cell growth. The most effective of these analogues, N1, N11-diethylnorspermine (DENSPM), apparently depletes intracellular polyamine pools primarily by inducing the polyamine acetylating enzyme spermidine/spermine N1-acetyltransferase, which contributes to polyamine depletion via increased polyamine excretion and catabolism. In this report, the experimental therapeutic efficacy of DENSPM was further examined with the use of other human solid tumor xenografts, including A121 ovarian carcinoma, A549 lung adenocarcinoma, HT29 colon carcinoma, and SH-1 melanoma, and compared with previously obtained findings with MALME-3M and PANUT-3 human melanomas. In vitro studies indicated that the growth sensitivity of most tumor cell lines to DENSPM was similar, with characteristically flat dose-response curves and IC50s ranging between 0.1 and 1 micrometer the only exception was the HT29 colon carcinoma cell line, which had an IC50 of >100 micrometer. For in vivo studies, DENSPM was administered by i.p. injection to female nude athymic mice at 40 and/or 80 mg/kg 3 times a day (every 8 h) for 6 days or by continuous s.c. infusion with the use of Alzet pumps at 120, 240, or 360 mg/kg/day for 4 days. Treatment began after s.c. tumor xenografts had reached 100-200 mm3. The SH-1 melanoma, A549 lung adenocarcinoma, and A121 ovarian carcinoma xenografts responded well to the i.p. administration of analogue with obvious tumor regressions, long-term tumor growth suppressions, and a significant proportion (up to 40%) of apparent cures (i.e., lack of tumor regrowth). However, in similarity to in vitro findings, HT29 colon carcinoma xenografts responded poorly to DENSPM treatment. Massive induction of N1-acetyltransferase activity and extensive depletion of polyamine pools were consistent findings in most tumor types after in vivo or in vitro treatment with DENSPM. The rapidly growing human LOX melanoma xenograft, however, demonstrated poor induction of N1-acetyltransferase activity and the poorest response to DENSPM treatment. In nude athymic mice with MALME-3M melanoma xenografts, constant infusion delivery of DENSPM resulted in prolonged inhibition of tumor growth and long-term tumor regressions comparable to those produced by multiple i.p. injections. On the basis of the unique structure of DENSPM, novel target and mode of intervention, mild host toxicity, and activity against different human solid tumor xenografts, DENSPM is currently being developed as an antitumor agent in humans.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Espermina/análogos & derivados , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/toxicidade , Divisão Celular/efeitos dos fármacos , Esquema de Medicação , Feminino , Humanos , Camundongos , Camundongos Nus , Espermina/administração & dosagem , Espermina/uso terapêutico , Espermina/toxicidade , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Better in vitro models are needed to elucidate the mechanisms underlying tissue destruction by human tumor cells. To address this matter recently isolated and characterized human ovarian carcinoma cell lines derived from either primary tumors, ascitic effusions or metastatic growths were plated in direct contact with extracellular matrix (ECM) previously deposited on culture dishes by bovine corneal endothelial cells. Light and electron microscopy of four of the five ovarian tumor cell lines demonstrated morphologic digestion with penetration of ECM by tumor cell microvilli, along with associated rarefaction. The ability of these same ovarian tumor cell lines to solubilize specific carbohydrate and protein moieties present in intact ECM was assessed with the use of metabolically prelabeled ECM employing tritiated fucose, galactose, glucosamine and proline. Results from these studies corroborated morphologic observations in which four of the five tumor cell lines tested extensively solubilized radiolabeled ECM. The kinetics of radiolabel release from ECM illustrated that three of the four invasive tumors released [3H]fucose, [3H]glucosamine and [3H]proline at high rates. Normal human ovarian fibroblasts and mesothelial cells were observed to be unable to digest ECM and this was consistent with their inability to release radiolabeled material from prelabeled ECM. The results from these studies suggest that some ovarian carcinomas have the ability to degrade basement membrane components. Knowledge regarding the mechanisms responsible for tissue degradation may eventually lead to the development of new chemotherapeutic modalities designed to restrict tumor cell invasion, growth and metastasis.
Assuntos
Matriz Extracelular/ultraestrutura , Neoplasias Ovarianas/ultraestrutura , Células Cultivadas , Feminino , Humanos , Técnicas In Vitro , Cinética , Microscopia Eletrônica de Varredura , Microscopia de Contraste de Fase , SolubilidadeRESUMO
The compounds N6-allyl-, N6-isopropyl-, N6-propargyl-, and N6-(2-methylallyl)adenosine were prepared by reacting 6-chloropurine riboside with an excess of the corresponding amines in ethanol, in the presence of two acid acceptors resulting in virtually quantitative yields. The compounds showed biological activity in a number of in vitro and in vivo tumor cell systems. Very good increases in life spans of mice bearing mammary carcinoma were obtained by treatment with the N6-allyl, N6-isopropyl, and N6-propargyl analogues, respectively. In rats, the N6-allyl analogue slowed the rate of transplantable mammary tumor growth by one-fourth. The short-chain adenosine analogues are more active in the treatment of animal carcinomas than in the leukemia or sarcoma tumor cell systems.
Assuntos
Adenosina/análogos & derivados , Antineoplásicos/síntese química , Adenosina/síntese química , Adenosina/farmacologia , Animais , Células Cultivadas , Fenômenos Químicos , Química , Feminino , Leucemia L1210/tratamento farmacológico , Camundongos , Neoplasias Experimentais/tratamento farmacológico , RatosRESUMO
Several new analogues of L-fucose modified in the 2 position and the 5-methyl group have been synthesized as potential plasma-membrane glycoconjugate inhibitors or modifiers, and their biological effects have been studied. 2-Chloro-, 2-bromo-, and 2-iodo-2-deoxy-L-fucose (9a, 9b, and 13, respectively) have been prepared by addition of the appropriate halogen to 3,4-di-O-acetyl-L-fucal, followed by hydrolysis of the anomeric halogen and the acetyl groups. A series of four halogenated 5-methyl analogues of L-fucose (4, X = F, Cl, Br, and I) have been obtained starting from 1,2:3,4-di-O-isopropylidene-L-galactose. The synthesis of this latter compound has been improved. A corresponding series of 6-deoxy-6-halo-D-galactose analogues, which are enantiomers of the 5-(halomethyl)-L-fucose analogues, has also been synthesized. Analogues 4b, 4c, and 9b at 1 x 10(-3) M specifically inhibited the incorporation of L-[3H]fucose into macromolecular components of SW613 human mammary tumor cells. Analogue 13 inhibited the growth of L1210 murine leukemic cells with an IC50 of 6 x 10(5) M in culture. 6-Deoxy-6-fluoro-D-galactose and its enantiomer 4a were found to be effective inhibitors of D-[3H]galactose and L-[3H]fucose incorporation, respectively, into macromolecular components of human mammary tumor cells. The effectiveness of inhibition was reduced with an increase in size of the halogen atom. Analogue 4a and its enantiomer have been tritiated at C-1 and both were found to be activated to a nucleotide sugar, which was followed by incorporation into the macromolecular fraction of SW613 human mammary tumor cells in vitro.