Identification of the molecular mechanisms underlying the cytotoxic action of a potent platinum metallointercalator

Abstract

Platinum-based DNA metallointercalators are structurally different from the covalent DNA binders such as cisplatin and its derivatives but have potent in vitro activity in cancer cell lines. However, limited understanding of their molecular mechanisms of cytotoxic action greatly hinders their further development as anticancer agents. In this study, a lead platinum-based metallointercalator, [(5,6-dimethyl-1,10-phenanthroline) (1S,2S-diaminocyclohexane)platinum(II)](2+) (56MESS) was found to be 163-fold more active than cisplatin in a cisplatin-resistant cancer cell line. By using transcriptomics in a eukaryotic model organism, yeast Saccharomyces cerevisiae, we identified 93 genes that changed their expressions significantly upon exposure of 56MESS in comparison to untreated controls (p ≤ 0.05). Bioinformatic analysis of these genes demonstrated that iron and copper metabolism, sulfur-containing amino acids and stress response were involved in the cytotoxicity of 56MESS. Follow-up experiments showed that the iron and copper concentrations were much lower in 56MESS-treated cells compared to controls as measured by inductively coupled plasma optical emission spectrometry. Deletion mutants of the key genes in the iron and copper metabolism pathway and glutathione synthesis were sensitive to 56MESS. Taken together, the study demonstrated that the cytotoxic action of 56MESS is mediated by its ability to disrupt iron and copper metabolism, suppress the biosynthesis of sulfur-containing amino acids and attenuate cellular defence capacity. As these mechanisms are in clear contrast to the DNA binding mechanism for cisplatin and its derivative, 56MESS may be able to overcome cisplatin-resistant cancers. These findings have provided basis to further develop the platinum-based metallointercalators as anticancer agents.

Electronic supplementary material: The online version of this article (doi:10.1007/s12154-011-0070-x) contains supplementary material, which is available to authorized users.

Keywords: 56MESS; Anticancer; Gene; Iron; Metallointercalators; Platinum.

Fig. 1

Growth inhibition of yeast cells by 56MESS. BY4743 wild-type yeast cultures at exponential growth phase (OD₆₀₀ 0.8) were exposed to a range of 56MESS concentrations (0.4 to 3.5 mM) for 180 min at 30 °C with agitation. Growth inhibition was expressed as the percentage of colony count at each of time-points over that at 0 time-point. Diamond, control; triangle, 0.4 mM; multiplication symbol, 0.5 mM; square, 1.0 mM; circle, 3.5 mM. The data are the averages from at least two biological replicates and error bars indicate standard deviations

Fig. 2

The differentially expressed genes (a) and the major pathways (b, c) in response to 56MESS treatment. Genes whose expression changed by 1.5-fold or more at p ≤ 0.05 under 56MESS treatment were identified as differentially expressed genes using ANOVA Partek® Genomic Suite™. Each dot in (a) represents one gene. Out of 5,841 genes, 48 up-regulated genes are shown in the yellow area and 45 down-regulated genes in the green area. The major pathways represented by the up- and down-regulated genes were revealed using GO Slim Mapper and shown in b and c, respectively. Pathways represented by a to n in (b) and I to X in (c) are listed in Table S2 in “Electronic supplementary material”

Fig. 3

The proposed molecular mechanism for the up-regulation of iron and copper under 56MESS treatment. 56MESS interacts with iron and copper membrane transporters (Ctr1p, Ftr1p, Fre1p, Fre2p, Fre3p and Arn1–4p). Such interaction then leads to conformational changes of these transporter proteins and subsequent inhibition of iron and copper uptake, with ultimate depletion of intracellular iron and copper. The iron and copper depletion results in Aft1p translocation from cytoplasm into the nucleus where this transcription factor binds to target genes (such as ARN1 and FTR1) to trigger their expression

Fig. 4

Phenotypic screening of deletion mutants of AFT1 (a) and FTR1 (b) under 56MESS treatment. The exponential phase of yeast cells was exposed to a gradient of 56MESS concentrations in 96-well plates for 22 h and then spotted to agar plates for further 48 h of incubation. The spots were the colonies of the yeast cells

Fig. 5

Glutathione involvement in the cytotoxicity of 56MESS. a The growth inhibition of the gsh1Δ mutant under 56MESS treatment. Square, untreated; multiplication symbol, 2.5 mM 56MESS; circle, 5.0 mM 56MESS. b Effects of supplementing glutathione into L1210 cell culture on the 56MESS’ cytotoxicity. Square, 1.2 mM GSH only; cross, control; circle, 0.045 mM 56MESS with 0.6 mM GSH; multiplication symbol, 0.045 mM 56MESS with 1.2 mM GSH; triangle, 0.045 mM 56MESS only