Abstract:
The phenomenon of resistance to established antineoplastic therapies plays a significant role in the survival of patients with cancer. Increases in the expression and activity of enzymes belonging to the glutathione S-transferase (GST) family has been implicated in glutathione-mediated resistance to several antineoplastic agents. The basis of this study is to determine the potential of selected triphenylmethane dyes as in vivo inhibitors of GST activity.
The triphenylmethane dyes, Malachite Green, Crystal Violet, and Ethyl Violet were found to interact with glutathione to form adducts at physiological pH with experimental Kd's of 0.85, 14.36, and 16.19 mM, respectively. The dyes were also found to interact with bovine serum albumin and high molecular weight proteins present in rat liver.
Kinetic investigations at pH 7.4 revealed human GST A 2-2 to be inhibited by the Malachite Green-GSH adduct competitively with respect to GSH (Ki = 2.533 ± 0.756 μM) and noncompetitively with respect to CDNB (Ki = 15.35 ± 1.437 μM).
Investigations revealed a complex kinetic mechanism associated with GST M 2-2 at pH 7.4. This was characterised by a time-dependant increase in activity at GSH concentrations of below 0.5 mM, and resulted in the inability to analyse data at these GSH concentrations. Malachite Green, Crystal Violet, and Ethyl Violet were all found to be partial inhibitors at GSH concentrations ranging from 0.5 to 5 mM. The inhibitory mechanism of these dyes were found to be noncompetitive with respect to CDNB. Data obtained from experiments using GSH as the variable substrate did not discriminate between mixed and noncompetitive inhibitory models.
Human GST P 1-1 was found to be inhibited by Malachite Green at pH 7.4. The inhibition was found to be noncompetitive with respect to both GSH and CDNB with observed Ki's in the order of 10-7 M. Both free Malachite Green, and the Malachite Green-GSH adduct were identified as inhibitory species.
Intravenous injection of [14C]-labelled Malachite Green into rats revealed at least three unidentified metabolites excreted with free dye in the bile, and at least two unidentified metabolites excreted in the urine. The results suggest that Malachite Green is extensively metabolised in a tissue-dependant manner.
The tested triphenylmethane dyes have demonstrated the ability to inhibit human GST activity in vitro, with the inhibition of GST P 1-1 by Malachite Green being particularly effective. However, the application of these dyes as inhibitors of GST activity in vivo is likely to be substantially restricted by interactions with proteins other than GST's and by the metabolism of these dyes. Therefore, it is unlikely that the tested triphenylmethane dyes have clinical potential for the inhibition of GST activity.
