Project description:β-lapachone (b-lap) is an anticancer agent that selectively induces cell death in human cancer cells. A mechanism for b-lap cytotoxicity was shown to be mediated by the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation in several tumour cells. To further characterise the molecular effects of b-lap action, we compared the gene expression profile of yeast cells treated or not with b-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were enriched in the set of differentially expressed genes. Accordingly, b-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, and inhibitor of NADH dehydrogenases (NADH-DH). In addition, a mutant defective in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to b-lap treatment, thus resembling tumour cell responses to b-lap exposure. However, b-lap treatment elicited similar ROS production in WT and nde2 cells, and dicoumarol did not affect cell viability in b-lap treated yeasts. Most interestingly, DNA damage responses triggered by b-lap were abolished in the nde2 mutant. Furthermore, the nde2 mutant was sensitive to DNA damaging agents other than b-lap. These data indicated that ROS production did not mediate b-lap cytotoxicity and highlighted a role of Nde2p in DNA damage checkpoints. Amino acid biosynthesis genes were also differentially expressed in b-lap treated cells, suggesting that b-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Supporting this, b-lap treatment increased phosphorylation of eIF2 alpha in a Gcn2p-dependent manner. eIF2alpha phosphorylation required Gcn1p and Gcn20p and surprisingly Nde2p. Gcn2p was also shown to be required for the G1 checkpoint triggered by b-lap and for cell survival. Remarkably, b-lap treatment also increased phosphorylation of eIF2 alpha in breast tumour cells, which was partially dependent on the Nde2p orthologue AMID. These findings i) suggest that Gcn2p and Nde2p are key determinants of b-lap toxicity in yeast and human cells, ii) uncover a new functional connection between Nde2p, Gcn2p and DNA damage checkpoints conserved throughout evolution and iii) suggest that pharmacological modulation of Gcn2p could provide an effective strategy for antitumour drug discovery.

Project description:β-lapachone (b-lap) is an anticancer agent that selectively induces cell death in human cancer cells. A mechanism for b-lap cytotoxicity was shown to be mediated by the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation in several tumour cells. To further characterise the molecular effects of b-lap action, we compared the gene expression profile of yeast cells treated or not with b-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were enriched in the set of differentially expressed genes. Accordingly, b-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, and inhibitor of NADH dehydrogenases (NADH-DH). In addition, a mutant defective in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to b-lap treatment, thus resembling tumour cell responses to b-lap exposure. However, b-lap treatment elicited similar ROS production in WT and nde2 cells, and dicoumarol did not affect cell viability in b-lap treated yeasts. Most interestingly, DNA damage responses triggered by b-lap were abolished in the nde2 mutant. Furthermore, the nde2 mutant was sensitive to DNA damaging agents other than b-lap. These data indicated that ROS production did not mediate b-lap cytotoxicity and highlighted a role of Nde2p in DNA damage checkpoints. Amino acid biosynthesis genes were also differentially expressed in b-lap treated cells, suggesting that b-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Supporting this, b-lap treatment increased phosphorylation of eIF2 alpha in a Gcn2p-dependent manner. eIF2alpha phosphorylation required Gcn1p and Gcn20p and surprisingly Nde2p. Gcn2p was also shown to be required for the G1 checkpoint triggered by b-lap and for cell survival. Remarkably, b-lap treatment also increased phosphorylation of eIF2 alpha in breast tumour cells, which was partially dependent on the Nde2p orthologue AMID. These findings i) suggest that Gcn2p and Nde2p are key determinants of b-lap toxicity in yeast and human cells, ii) uncover a new functional connection between Nde2p, Gcn2p and DNA damage checkpoints conserved throughout evolution and iii) suggest that pharmacological modulation of Gcn2p could provide an effective strategy for antitumour drug discovery. Direct comparison between wt samples and b-lapachone treated-wt samples. Four biological replicates. Dye was swapped in two of them

Project description:An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 μM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Dataset Information

Characterization and trypanocidal activity of a ?-lapachone-containing drug carrier.

Publications

Characterization and trypanocidal activity of a β-lapachone-containing drug carrier.

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