RESUMEN
Repair of DNA-targeted anticancer agents is an active area of investigation of both fundamental and clinical interest. However, most studies have focused on a small number of compounds limiting our understanding of both DNA repair and the DNA damage response. S23906 is an acronycine derivative that shows strong activity toward solid tumors in experimental models. S23906 forms bulky monofunctional DNA adducts in the minor groove which leads to destabilization of the double-stranded helix. We now report that S23906 induces formation of DNA double strand breaks that are processed through homologous recombination (HR) but not Non-Homologous End-Joining (NHEJ) repair. Interestingly, S23906 exposure was accompanied by a higher sensitivity of BRCA2-deficient cells compared to other HR deficient cell lines and by an S-phase accumulation in wild-type (wt), but not in BRCA2-deficient cells. Recently, we have shown that S23906-induced S phase arrest was mediated by the checkpoint kinase Chk1. However, its activated phosphorylated form is equally induced by S23906 in wt and BRCA2-deficient cells, likely indicating a role for BRCA2 downstream of Chk1. Accordingly, override of the S phase arrest by either 7-hydroxystaurosporine (UCN-01) or AZD7762 potentiates the cytotoxic activity of S23906 in wt, but not in BRCA2-deficient cells. Together, our findings suggest that the pronounced sensitivity of BRCA2-deficient cells to S23906 is due to both a defective S-phase arrest and the absence of HR repair. Tumors with deficiencies for proteins involved in HR, and BRCA2 in particular, may thus show increased sensitivity to S23906, thereby providing a rationale for patient selection in clinical trials.
Asunto(s)
Acronina/análogos & derivados , Antineoplásicos Alquilantes/metabolismo , Proteína BRCA2/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Reparación del ADN/fisiología , Acronina/metabolismo , Acronina/farmacología , Animales , Antineoplásicos Alquilantes/farmacología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Cricetinae , Reparación del ADN por Unión de Extremidades/efectos de los fármacos , Reparación del ADN por Unión de Extremidades/fisiología , Reparación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , HumanosRESUMEN
S23906 belongs to a novel class of alkylating anticancer agents forming bulky monofunctional DNA adducts. A unique feature of S23906 is its "helicase-like" activity leading to the destabilization of the surrounding duplex DNA. We here characterize the recognition and repair of S23906 adducts by the nucleotide excision repair (NER) machinery. All NER-deficient human cell lines tested showed increased sensitivity to S23906, which was particularly pronounced for cells deficient in XPC, CSB and XPA. In comparison, deficiencies in ERCC1 or XPF had lesser impact on the sensitivity to S23906. The sensitivity was, at least in part, linked to the conversion of unrepaired adducts into toxic DNA strand breaks as shown by single cell electrophoresis and gamma-H2AX formation. The pharmacological relevance of these findings was confirmed by the characterization of KB carcinoma cells with acquired S23906 resistance. These cells showed increased NER activity in vivo as well as toward damaged plasmid DNA in vitro. In particular, both global genome NER, as shown by unscheduled DNA synthesis, and transcription-coupled NER, as shown by transcriptional recovery, were up-regulated in the S23906-resistant cells. The increased NER activity was accompanied by up to 5-fold up-regulation of XPC, CSB and XPA proteins without detectable alterations of ERCC1 on the DNA, RNA or protein levels. Our results suggest that S23906 adducts are recognized and repaired by both NER sub-pathways in contrast to other members of this class, that are only recognized by transcription-coupled NER. We further show that NER activity can be up-regulated without changes in ERCC1 expression.
Asunto(s)
Acronina/farmacología , Antineoplásicos Alquilantes/metabolismo , ADN Helicasas/fisiología , Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/fisiología , ADN/metabolismo , Endonucleasas/fisiología , Acronina/metabolismo , Alquilantes/química , Alquilantes/metabolismo , Antineoplásicos Alquilantes/química , Sitios de Unión/fisiología , Línea Celular , Línea Celular Tumoral , Aductos de ADN/metabolismo , Humanos , Receptores X del Hígado , Receptores Nucleares Huérfanos/deficiencia , Receptores Nucleares Huérfanos/fisiología , Proteínas de Unión a Poli-ADP-Ribosa , Xerodermia Pigmentosa/metabolismoRESUMEN
The benzo[b]acronycine derivative S23906-1 has been recently identified as a promising antitumor agent, showing remarkable in vivo activities against a panel of solid tumors. The anticancer activity is attributed to the capacity of the drug to alkylate DNA, selectively at the exocyclic 2-amino group of guanine residues. Hydrolysis of the C-1 and C-2 acetate groups of S23906-1 provides the diol compound S28907-1 which is inactive whereas the intermediate C-2 monoacetate derivative S28687-1 is both highly reactive toward DNA and cytotoxic. The reactivity of this later compound S28687-1 toward two bionucleophiles, DNA and the tripeptide glutathion, has been investigated by mass spectrometry to identify the nature of the (type II) covalent adducts characterized by the loss of the acetate group at position 2. On the basis of NMR and molecular modeling analyses, the reaction mechanism is explained by a transesterification process where the acetate leaving group is transferred from position C-2 to C-1. Altogether, the study validates the reaction scheme of benzo[b]acronycine derivative with its target.
Asunto(s)
Acronina/metabolismo , Antineoplásicos Fitogénicos/metabolismo , ADN/metabolismo , Glutatión/metabolismo , Acronina/análisis , Acronina/química , Antineoplásicos Fitogénicos/análisis , Antineoplásicos Fitogénicos/química , Sitios de Unión , ADN/análisis , Glutatión/análisis , Unión Proteica/fisiología , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
The benzoacronycine derivative, S23906-1, was characterized recently as a novel potent antitumor agent through alkylation of the N2 position of guanines in DNA. We show here that its reactivity towards DNA can be modulated by glutathione (GSH). The formation of covalent adducts between GSH and S23906-1 was evidenced by EI-MS, and the use of different GSH derivatives, amino acids and dipeptides revealed that the cysteine thiol group is absolutely required for complex formation because glutathione disulfide (GSSG) and other S-blocked derivatives failed to react covalently with S23906-1. Gel shift assays and fluorescence measurements indicated that the binding of S23906-1 to DNA and to GSH are mutually exclusive. Binding of S23906-1 to an excess of GSH prevents DNA alkylation. Additional EI-MS measurements performed with the mixed diester, S28053-1, showed that the acetate leaving group at the C1 position is the main reactive site in the drug: a reaction scheme common to GSH and guanines is presented. At the cellular level, the presence of GSH slightly reduces the cytotoxic potential of S23906-1 towards KB-3-1 epidermoid carcinoma cells. The GSH-induced threefold reduction of the cytotoxicity of S23906-1 is attributed to the reduced formation of lethal drug-DNA covalent complexes in cells. Treatment of the cells with buthionine sulfoximine, an inhibitor of GSH biosynthesis, facilitates the formation of drug-DNA adducts and promotes the cytotoxic activity. This study identifies GSH as a reactant for the antitumor drug, S23906-1, and illustrates a pathway by which GSH may modulate the cellular sensitivity to this DNA alkylating agent. The results presented here, using GSH as a biological nucleophile, fully support our initial hypothesis that DNA alkylation is the major mechanism of action of the promising anticancer drug S23906-1.
Asunto(s)
Acronina/análogos & derivados , Acronina/metabolismo , Antineoplásicos Alquilantes/metabolismo , ADN/metabolismo , Glutatión/metabolismo , Acronina/química , Antineoplásicos Alquilantes/química , Butionina Sulfoximina/metabolismo , Dicroismo Circular , ADN/química , Inhibidores Enzimáticos/metabolismo , Humanos , Sustancias Macromoleculares , Estructura Molecular , Espectrometría de Masa por Ionización de Electrospray , Células Tumorales CultivadasRESUMEN
Seven 1,2-dihydroxy-1,2-dihydroacronycine and 1,2-dihydroxy-1,2-dihydro-6-demethoxyacronycine esters and diesters were synthesized via osmic oxidation of acronycine or 6-demethoxyacronycine followed by acylation. The 6-demethoxyacronycine derivatives were found to be inactive, whereas in contrast, all of the acronycine derivatives were more potent than acronycine itself when tested against L1210 cells in vitro. Four selected acronycine derivatives (17,19, 21, and 22) were evaluated in vivo against murine P388 leukemia and colon 38 adenocarcinoma implanted in mice. All compounds were markedly active against P388 at doses 4-16-fold lower than acronycine itself. Against the colon 38 adenocarcinoma, the three compounds 17, 21, and 22 were highly efficient. 1,2-Diacetoxy-1,2-dihydroacronycine (17) was the most active, all the treated mice being tumor-free on day 23.
Asunto(s)
Acridinas/síntesis química , Acronina/análogos & derivados , Antineoplásicos/síntesis química , Ésteres/farmacología , Acridinas/farmacología , Acridinas/toxicidad , Acronina/síntesis química , Acronina/metabolismo , Acronina/farmacología , Adenocarcinoma/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Ciclo Celular/efectos de los fármacos , Ésteres/toxicidad , Leucemia Experimental/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Estructura Molecular , Células Tumorales CultivadasAsunto(s)
Acronina/metabolismo , Alcaloides/metabolismo , Animales , Femenino , Absorción Intestinal , Masculino , Ratas , Distribución TisularRESUMEN
A solid basis for the M4-approach has been developed over the past 10 years. Recent examples of the production of difficult-to-synthesize mammalian metabolites through microbial transformations attest to the utility of the methodology. There is, however, much more to be done. Model studies should be conducted to test parallels between microbial and mammalian S- and N-oxidations, O-glucuronidations, and ester and amide hydrolyses. Subsequently, even greater applications of M4- work can be envisioned. We have been pleased to see our colleagues in industry and academia adopt the M4- approach to solve difficult pharmacological and toxicological problems. In large measure, this has been our greatest reward for efforts initially presented before the membership of the American Society of Pharmacognosy in 1973.