title: Role of glutathione transferases in the mechanism of brostallicin activation.
abstract: Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.
authors: Department of Chemical Sciences and Technologies, University of Tor Vergata, Rome, Italy.
authors: Silvia
authors: S
authors: Pezzola
authors: Giovanni
authors: G
authors: Antonini
authors: Cristina
authors: C
authors: Geroni
authors: Italo
authors: I
authors: Beria
authors: Maristella
authors: M
authors: Colombo
authors: Massimo
authors: M
authors: Broggini
authors: Sergio
authors: S
authors: Marchini
authors: Nicola
authors: N
authors: Mongelli
authors: Loris
authors: L
authors: Leboffe
authors: Robert
authors: R
authors: MacArthur
authors: Alessia Francesca
authors: AF
authors: Mozzi
authors: Giorgio
authors: G
authors: Federici
authors: Anna Maria
authors: AM
authors: Caccuri
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D005260:Female; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D011758:Pyrroles / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D006146:Guanidines; D011758:Pyrroles; D004247:DNA; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase; C412602:glutathione S-transferase Mu 2; C455279:brostallicin