RESUMEN
A rapid and very sensitive enzyme immunoassay was developed for the measurement of paclitaxel and related taxanes in crude extracts of Taxus sp., in human serum and in culture medium of paclitaxel-producing microorganisms such as Erwinia taxi. For the ELISA, paclitaxel was chemically modified by the introduction of an amine to enable coupling with biotin. The presence of paclitaxel or related taxanes competitively inhibited the binding of paclitaxel-biotin to anti-taxane monoclonal antibody. This method detected paclitaxel in concentrations as low as 33 pM; the affinity of the antibody was higher for paclitaxel than for cephalomanine, baccatin and DAB. The sensitivity of this assay makes it useful for estimating the paclitaxel and taxanes content of Taxus sp. extracts, monitoring the paclitaxel serum level of paclitaxel treated patients and in other biological fluids.
Asunto(s)
Antineoplásicos Fitogénicos/análisis , Paclitaxel/análisis , Anticuerpos Monoclonales/inmunología , Antineoplásicos Fitogénicos/inmunología , Cromatografía Líquida de Alta Presión , Ensayo de Inmunoadsorción Enzimática , Mediciones Luminiscentes , Paclitaxel/análogos & derivados , Paclitaxel/inmunología , Sensibilidad y EspecificidadRESUMEN
Microtubules are a major component of cell cytoskeleton. Microtubules constitute the cellular target of paclitaxel. The interaction of paclitaxel with microtubules causes an increase in tubulin polymerization and microtubules stabilization, leading to a G2/M phase cell cycle arrest and cell death by apoptosis. Three paclitaxel fluorescent analogues were prepared by introducing fluorescein or BODIPY moiety onto the 2' of the 7-carbon. These were then used to study the interaction of paclitaxel with its cellular binding site and the microtubule network was visualized directly by fluorescence microscopy. Free paclitaxel was able to inhibit 7-FITC paclitaxel binding, demonstrating that both products bind to the same site and possess similar biological properties. Using the carbon 7 derivatives, microtubules were labeled as cytoplasmic fibers extending centripedly. A few other cellular components such as nuclear membrane, nucleoli and some other cytoplasmic structures were also labeled. The labeling intensity was reduced by preincubation with free paclitaxel. The interaction of paclitaxel with microtubules was also investigated using flow cytometry. No binding of the 2'C derivative was detected, confirming that a free 2'C is a pre-requisite for paclitaxel microtubules interaction.
RESUMEN
Transferrin is a serum glycoprotein involved in iron transport. Transferrin acts also in cell growth regulation through membrane receptors. The number of transferrin receptors is increased in tumor and other rapidly dividing cells. This renders transferrin suitable for use in cytotoxic drugs targetting tumor cells. Paclitaxel was derivatized on 2' carbon and coupled with trasferrin using glutaraldehyde. The cytotoxicity of the conjugate was evaluated on small cell carcinoma of the lung cell line (H69). As compared to paclitaxel, the conjugate exhibited a slight decrease in cytotoxicity.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Paclitaxel/análogos & derivados , Paclitaxel/toxicidad , Taxoides , Transferrina/toxicidad , Carcinoma de Células Pequeñas , Humanos , Neoplasias Pulmonares , Análisis de Regresión , Células Tumorales CultivadasRESUMEN
We have developed a fluorescence polarization immunoassay (FPIA) for the quantitative determination of paclitaxel in serum, crude Taxus extracts and Erwinia taxi culture medium. To achieve this, we used an antipaclitaxel monoclonal antibody and an FITC-labeled paclitaxel. Paclitaxel was chemically modified by introducing an amino function to enable coupling with fluorescein isothiocyanate. Paclitaxel competitively inhibited the binding of the monoclonal antibody with FITC-paclitaxel causing a decrease in polarization. We were able to detect paclitaxel in a concentration as low as 2 nM. Fluorescence polarization immunoassay requires only the addition of the fluorescent probe to the antibody, followed by an incubation and measurement of polarization. Both the simplicity and the sensitivity of this method make it useful for estimating the paclitaxel content in yew tree crude extracts and culture medium of paclitaxel producing micro-organisms and a possible assay for monitoring paclitaxel level in patients under treatment.
Asunto(s)
Antineoplásicos Fitogénicos/análisis , Inmunoensayo de Polarización Fluorescente/métodos , Paclitaxel/análisis , Anticuerpos Monoclonales , Antineoplásicos Fitogénicos/inmunología , Cromatografía Líquida de Alta Presión/métodos , Paclitaxel/inmunología , Sensibilidad y EspecificidadRESUMEN
Upon incubation with paclitaxel, B16-F1 melanoma cells showed a flattening and darkening of their cytoplasm. By electron microscopy analysis, an increase in the number of melanosomes associated with an increase of their melanin content was observed. The cells also showed prominent morphological changes such as flattening, loss of prolongation and increase in size. Tyrosinase activity was increased 2.2 fold after 16 hours of incubation with 10 microM paclitaxel. The tyrosinase gene transcription was evaluated by semi-quantitative RT-PCR. The mRNA level was not significantly increased indicating that the effect of paclitaxel on melanogenesis was probably due to post-transcriptional events.