RESUMEN

Native proaerolysin is a channel-forming bacterial protoxin that binds to cell-surface receptors and then is activated by furin or furin-like proteases. We genetically engineered proaerolysin by replacing the furin-cleavage sequence with a prostate-specific antigen-selective sequence. The recombinant modified proaerolysin was expressed and purified from Aeromonas salmonicida in good yields and purity. Recombinant modified proaerolysin had no furin sensitivity and markedly increased prostate-specific antigen sensitivity relative to wild-type proaerolysin. Human prostate cancer cells were significantly more sensitive to recombinant modified proaerolysin in the presence of active prostate-specific antigen when compared with the absence of prostate-specific antigen or the presence of potent prostate-specific antigen inhibitors. Most normal human cells with the exception of prostate and renal epithelial cells showed very low sensitivity to recombinant modified proaerolysin. Our results suggest that recombinant modified proaerolysin is a potent prostate-specific antigen-sensitive protoxin that deserves further development for regional therapy of benign and malignant prostate growths.

Asunto(s)

Antineoplásicos/farmacología , Toxinas Bacterianas/farmacología , Proteínas Citotóxicas Formadoras de Poros/farmacología , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Aeromonas salmonicida/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/química , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Electroforesis en Gel de Poliacrilamida , Ingeniería Genética , Humanos , Masculino , Péptido Hidrolasas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/administración & dosificación , Proteínas Citotóxicas Formadoras de Poros/química , Profármacos

RESUMEN

An automated apparatus is described for measuring the aqueous solubility of a sparingly soluble organic compound at many different temperatures. Water is pumped through a generator column packed with a chromatographic support coated with the organic compound, producing a saturated solution. The solute in a measured volume of this solution is extracted with an extractor column and analyzed by high performance liquid chromatography (HPLC). The temperature of the thermostat bath and the operation of the valves and the HPLC are under the control of a microcomputer. Solubility measurements of ethylbenzene obtained with this apparatus have a standard deviation at any one temperature of about 3% of the mean.