RESUMO
Cellular drug target identification through affinity chromatography is often hindered by the quantity of nonspecific binders, such as cytoskeletal and heat shock proteins. Thus, we prepared a 2-aminobenzimidazole-tethered depletion resin designed for removal of these proteins, and tested it on human lung carcinoma cell and rat tissue extracts. Column-bound proteins were identified by two-dimensional gel electrophoresis and MS. Among others, tubulins, actins and heat shock proteins were successfully depleted. Due to the reduction of these highly abundant proteins detection of potential drug targets is considerably facilitated in the pre-purified samples.
Assuntos
Benzimidazóis/química , Células/química , Proteínas/isolamento & purificação , Extratos de Tecidos/química , Marcadores de Afinidade , Animais , Química Encefálica , Carbono/análise , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Ecocardiografia , Humanos , Hidrólise , Indicadores e Reagentes , Masculino , Ratos , Ratos Wistar , Propriedades de Superfície , Espectrometria de Massas em Tandem , Tripsina/químicaRESUMO
The new heptapeptide somatostatin analog TT-232 decreases proliferation of HT-29 human colon carcinoma cells in vitro by reducing mitotic and increasing apoptotic activity. We have synthesized and characterized a specifically tritium labeled 3H-Tyr3-TT-232 (30 Ci/mmol) to investigate the effect and the fate of this antitumor peptide on human colon tumor cells. 3H-labeled TT-232 could be detected on the cell surface, on cytoplasmic membranes and also in the nucleus of HT-29 cells, 1-6 h after the administration of 0.5 and 50 microg/mL [3H]TT-232. Binding and internalization of TT-232 to human colon tumor cells at a relatively high dose provide further evidence for the existence of low-affinity somatostatin receptors in such cells, which might mediate the apoptosis-inducing effect. Our data suggest the possible use of TT-232 in the treatment of human colon tumors.
Assuntos
Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/metabolismo , Células HT29/metabolismo , Peptídeos Cíclicos/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Autorradiografia , Divisão Celular/efeitos dos fármacos , Fragmentação do DNA , Citometria de Fluxo , Células HT29/ultraestrutura , Humanos , Marcação por Isótopo , Cinética , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/farmacologia , Somatostatina/análogos & derivados , TrítioRESUMO
Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.