RESUMEN
The tumor-suppressor protein p53 is considered to be one of the most important transport hubs of cell signal transduction, playing critical roles in the control of cell cycle arrest, apoptosis and many other processes as a nuclear transcription factor. p53 also acts in the cytoplasm to trigger apoptosis. Paclitaxel and other microtubule inhibitors can inhibit the growth of different types of cancer cells and induce apoptosis which is believed to be p53-independent. In the present study, we demonstrated that UTD1, a genetically engineered epothilone analog and a new microtubule inhibitor, activated p53 as a transcription factor at low concentrations demonstrated by its enhanced transcriptional activity and accumulation of p21, which led to cell cycle arrest. However, at high concentrations of UTD1, p53 was accumulated in the cytoplasm which contributed to induction of apoptosis. These observations indicate that the epothilone analog has differential effects on intracellular signaling and implies that p53 plays different roles in cells exposed to different concentrations of the anticancer agent.
Asunto(s)
Neoplasias de la Mama/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Epotilonas/administración & dosificación , Proteína p53 Supresora de Tumor/genética , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Humanos , Células MCF-7 , Paclitaxel/administración & dosificación , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Plásmidos/genética , Sintasas Poliquetidas/genética , Escherichia coli/enzimología , Proteínas de Escherichia coli/genética , Vectores Genéticos , Familia de Multigenes , Sintasas Poliquetidas/metabolismo , Recombinación Genética , Proteína Ribosómica S9 , Proteínas Ribosómicas/genética , Análisis de Secuencia de ADNRESUMEN
Fostriecin (FST, 1) is a natural product with promising antitumor activity produced by Streptomyces pulveraceus. Its antitumor activity is associated with the selective inhibition of protein phosphatase activities. The biosynthetic gene cluster for FST has recently been cloned and sequenced. To better understand the post-polyketide synthase (PKS) modification steps in the biosynthetic pathway of FST, we constructed and characterized three post-PKS modification gene mutants of fosG, fosK, and fosM by knockout inactivation in S. pulveraceus. As a result, we determined that a fosK-encoded cytochrome P450 monooxygenase is responsible for C-18 hydroxylation, formation of an unsaturated lactone is dependent upon FosM, and the fosG gene product is involved in hydroxylation at C-4 after the action of FosM to yield PD 113,271 from FST. The accumulated analogues from the ΔfosK and ΔfosM mutant strains possessed a malonyl ester moiety that suggested that all the post-PKS modification steps in FST biosynthesis occur with the polyketide chain bearing a malonyl ester at the C-3 position, with formation of the unsaturated six-membered lactone as the last step in FST biosynthesis.
Asunto(s)
Antineoplásicos/aislamiento & purificación , Polienos/aislamiento & purificación , Sintasas Poliquetidas/metabolismo , Pironas/aislamiento & purificación , Streptomyces , Antineoplásicos/química , Antineoplásicos/farmacología , Estructura Molecular , Familia de Multigenes , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Polienos/química , Polienos/farmacología , Pironas/química , Pironas/farmacología , Streptomyces/química , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
The epothilones represent a new class of bacterial natural products with broad spectrum of antiproliferative activity against various types of human tumors and tumor cell lines. The attractive preclinical profile of epothilones has made them promising lead compounds for novel anticancer agents and has spurred a strong interest in obtaining different derivatives to fully evaluate their therapeutic potentials. We have generated a number of novel epothilone D and 10,11-dehydroepothilone D (Epo490) analogs via biotransformation using Amycolata autotrophica to alter the oxidation state of the parental compounds. The bioconverted compounds displayed different degrees of potency in cytotoxicity assays against a panel of human tumor cell lines, with 11-hydroxyepothilone D, 14-hydroxyepothilone D, and 21-hydroxyepothilone D showing comparable activity to that of epothilone D, and 21-hydroxy Epo490 being comparable to Epo490. The addition of hydroxyl group(s) seems to cause a decrease in cytotoxic activity against multiple drug resistant cell lines (with overexpressed P-glycoprotein). The compounds generated by biotransformation exert differential effects on tubulin polymerization, which correlate with their biological activities.
Asunto(s)
Actinomycetales/metabolismo , Epotilonas/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Biotransformación , División Celular/efectos de los fármacos , Epotilonas/química , Epotilonas/farmacología , Humanos , Macrólidos/química , Macrólidos/farmacología , Conformación Molecular , Tiazoles/química , Tiazoles/farmacología , Células Tumorales CultivadasRESUMEN
A new epothilone, 10,11-didehydroepothilone D (5), was isolated from a strain of the heterologous host Myxococcus xanthus genetically engineered to produce epothilone D (4). The structure of 5 was determined from NMR and MS data. The epothilone polyketide synthase was further modified in a recombinant M. xanthus strain to produce 5 as the major epothilone-related metabolite. The cytotoxicity of 5 against a panel of tumor cell lines, including several with multidrug resistance, and its effect on tubulin polymerization were comparable to epothilone D (4).