RESUMO
BACKGROUND: 5-Fluorouracil (5-FU) is an antineoplastic drug that targets thymidylate synthase (TS). Tumour cells can develop resistance to anti-TS drugs by a variety of mechanisms including up-regulation of TS protein and alterations in drug uptake and degradation. The possible mechanisms of the observed rapid development of resistance to the pyrimidine analogs 5-FUdR and 5-FU in cultured HCT116 colon cancer cells were investigated. MATERIALS AND METHODS: Cell survival was determined in resistant and control HCT116 cells treated with 5-FUdR and 5-FU for 7 days. The ability of the cells to take up and metabolize these drugs was determined by Western blotting and [3H]thymidine incorporation. RESULTS AND CONCLUSION: Resistant HCT116 cells were 5- and 100-fold more resistant to killing by 5-FU and 5-FUdR, respectively, than the parental cells and exhibited impaired uptake. Although the HCT116R cells were initially Mycoplasma free, a low level of Mycoplasma contamination was found in these cells after several weeks in culture. Sensitivity to 5-FUdR was restored by treatment with an anti-Mycoplasma antibiotic. Our observations emphasize the need for frequent testing for Mycoplasma contamination in any cell line under investigation for resistance to anti-TS drugs.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/microbiologia , Floxuridina/farmacologia , Fluoruracila/farmacologia , Infecções por Mycoplasma/metabolismo , Aminopterina/metabolismo , Aminopterina/farmacologia , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Células HCT116 , Células HT29 , Células HeLa , Humanos , Hipoxantina/metabolismo , Hipoxantina/farmacologia , Infecções por Mycoplasma/tratamento farmacológico , Timidina/metabolismo , Timidina/farmacologia , Timidina Quinase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismo , TrítioRESUMO
In vivo bioconjugation to the free thiol on Cys34 of serum albumin by a strategically placed reactive group on a bioactive peptide is a useful tool to extend plasma half-life. Three maleimido derivates of human GH-releasing factor (hGRF)(1-29) were synthesized and bioconjugated to human serum albumin ex vivo. All three human serum albumin conjugates showed enhanced in vitro stability against dipeptidylpeptidase-IV and were bioactive in a GH secretion assay in cultured rat anterior pituitary cells. When the maleimido derivatives were individually administered sc to normal male Sprague Dawley rats, an acute secretion of GH was measured in plasma. The best compound, CJC-1295, showed a 4-fold increase in GH area under the curve over a 2-h period compared with hGRF(1-29). CJC-1295, a tetrasubstituted form of hGRF(1-29) with an added N epsilon-3-maleimidopropionamide derivative of lysine at the C terminus, was selected for further pharmacokinetic evaluation, where it was found to be present in plasma beyond 72 h. A Western blot analysis of the plasma of a rat injected with CJC-1295 showed the presence of a CJC-1295 immunoreactive species on the band corresponding to serum albumin, appearing after 15 min and remaining in circulation beyond 24 h. These results led to the identification of CJC-1295 as a stable and active hGRF(1-29) analog with an extended plasma half-life.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Fragmentos de Peptídeos/farmacologia , Adeno-Hipófise/metabolismo , Albumina Sérica/farmacologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Dipeptidil Peptidase 4/farmacologia , Estabilidade de Medicamentos , Hormônio Liberador de Hormônio do Crescimento/administração & dosagem , Hormônio Liberador de Hormônio do Crescimento/genética , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/farmacocinética , Meia-Vida , Humanos , Injeções Subcutâneas , Masculino , Dados de Sequência Molecular , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacocinética , Ratos , Ratos Sprague-Dawley , Sermorelina/farmacocinética , Albumina Sérica/metabolismo , Fatores de Transcrição/efeitos dos fármacosRESUMO
The rapid degradation of native glucagon-like peptide 1 (GLP-1) by dipeptidyl peptidase-IV (DPP-IV) has fostered new approaches for generation of degradation-resistant GLP-1 analogues. We examined the biological activity of CJC-1131, a DPP-IV-resistant drug affinity complex (DAC) GLP-1 compound that conjugates to albumin in vivo. The CJC-1131 albumin conjugate bound to the GLP-1 receptor (GLP-1R) and activated cAMP formation in heterologous fibroblasts expressing a GLP-1R. CJC-1131 lowered glucose in wild-type mice, but not in GLP-1R-/- mice. Basal glucose and glycemic excursion following glucose challenge remained significantly reduced 10-12 h following a single injection of CJC-1131. Twice daily administration of CJC-1131 to db/db mice significantly reduced glycemic excursion following oral and IP glucose challenge (P < 0.01 to 0.05) but did not significantly lower body weight during the 4-week study period. Levels of random fed glucose were significantly lower in CJC-1131-treated +/+ and db/db mice and remained significantly lower even 1 week following discontinuation of CJC-1131 administration. CJC-1131 increased levels of pancreatic proinsulin mRNA transcripts, percent islet area, and the number of bromodeoxyuridine-positive islet cells. These findings demonstrate that an albumin-conjugated DAC:GLP-1 mimics the action of native GLP-1 and represents a new approach for prolonged activation of GLP-1R signaling.
Assuntos
Glucagon/química , Hipoglicemiantes/farmacologia , Maleimidas/farmacologia , Fragmentos de Peptídeos/química , Peptídeos/farmacologia , Precursores de Proteínas/química , Receptores de Glucagon/metabolismo , Albumina Sérica/química , Animais , Glicemia/análise , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Diabetes Mellitus/sangue , Diabetes Mellitus/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Fibroblastos/metabolismo , Glucagon/agonistas , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Receptor do Peptídeo Semelhante ao Glucagon 1 , Teste de Tolerância a Glucose , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Maleimidas/química , Maleimidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Proinsulina/genética , Precursores de Proteínas/agonistas , Precursores de Proteínas/metabolismo , RNA Mensageiro/análise , Receptores de Glucagon/deficiência , Receptores de Glucagon/genética , Proteínas Recombinantes/metabolismo , Albumina Sérica/metabolismo , TransfecçãoAssuntos
Fusão Celular , Regulação Enzimológica da Expressão Gênica , Células Híbridas/enzimologia , Telomerase/biossíntese , Elementos Alu , Animais , Avidina , Biotinilação , Células Cultivadas/enzimologia , Senescência Celular , Coloração Cromossômica/métodos , Cromossomos Humanos/química , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Melanoma Experimental/enzimologia , Melanoma Experimental/patologia , Camundongos , Microscopia de Fluorescência/métodos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neoplasias/enzimologia , Reação em Cadeia da Polimerase/métodos , Proteínas Repressoras/fisiologia , Especificidade da Espécie , Telomerase/genéticaRESUMO
The plant fenugreek has been used for centuries as a treatment for diabetes. This article presents evidence that the major isomer of 4-hydroxyisoleucine, an atypical branched-chain amino acid derived from fenugreek, is responsible for the effects of this plant on glucose and lipid metabolism. 4-Hydroxyisoleucine was demonstrated to stimulate glucose-dependent insulin secretion by a direct effect on pancreatic islets. In addition to stimulating insulin secretion, 4-hydroxyisoleucine reduced insulin resistance in muscle and/or liver by activating insulin receptor substrate-associated phosphoinositide 3 (PI3) kinase activity. 4-Hydroxyisoleucine also reduced body weight in diet-induced obese mice. The decrease in body weight was associated with a marked decrease in both plasma insulin and glucose levels, both of which are elevated in this animal model. Finally, 4-hydroxyisoleucine decreased elevated plasma triglyceride and total cholesterol levels in a hamster model of diabetes. Based on the beneficial metabolic properties that have been demonstrated, 4-hydroxyisoleucine, a simple, plant-derived amino acid, may represent an attractive new candidate for the treatment of type 2 diabetes, obesity and dyslipidemia, all key components of metabolic syndrome.