DMSO reduces the cytotoxicity of anticancer ruthenium complex KP1019 in yeast.

Low solubility in aqueous solutions is a significant limitation of the otherwise promising anticancer ruthenium complex KP1019. In laboratory studies, this challenge is often overcome by using DMSO to help drive the drug into solution. Since DMSO was previously shown to alter the bioactivity of platinum-based chemotherapeutics, here we examine DMSO's effects on KP1019. Using Saccharomyces cerevisiae as a model organism, we apply multiple measures of growth inhibition to demonstrate that DMSO reduces the drug's toxicity. This reduction in bioactivity correlates with spectrophotometric changes consistent with DMSO-dependent increases in the stability of the KP1019 pro-drug. The impact of DMSO on the biology and chemistry of KP1019 suggests this solvent should not be used in studies of this and similar anticancer ruthenium complexes.

Proteomic analysis of the S. cerevisiae response to the anticancer ruthenium complex KP1019.

Like platinum-based chemotherapeutics, the anticancer ruthenium complex indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(iii)], or KP1019, damages DNA, induces apoptosis, and causes tumor regression in animal models. Unlike platinum-based drugs, KP1019 showed no dose-limiting toxicity in a phase I clinical trial. Despite these advances, the mechanism(s) and target(s) of KP1019 remain unclear. For example, the drug may damage DNA directly or by causing oxidative stress. Likewise, KP1019 binds cytosolic proteins, suggesting DNA is not the sole target. Here we use the budding yeast Saccharomyces cerevisiae as a model in a proteomic study of the cellular response to KP1019. Mapping protein level changes onto metabolic pathways revealed patterns consistent with elevated synthesis and/or cycling of the antioxidant glutathione, suggesting KP1019 induces oxidative stress. This result was supported by increased fluorescence of the redox-sensitive dye DCFH-DA and increased KP1019 sensitivity of yeast lacking Yap1, a master regulator of the oxidative stress response. In addition to oxidative and DNA stress, bioinformatic analysis revealed drug-dependent increases in proteins involved ribosome biogenesis, translation, and protein (re)folding. Consistent with proteotoxic effects, KP1019 increased expression of a heat-shock element (HSE) lacZ reporter. KP1019 pre-treatment also sensitized yeast to oxaliplatin, paralleling prior research showing that cancer cell lines with elevated levels of translation machinery are hypersensitive to oxaliplatin. Combined, these data suggest that one of KP1019's many targets may be protein metabolism, which opens up intriguing possibilities for combination therapy.

DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

The anticancer ruthenium complex KP1019 induces DNA damage, leading to cell cycle delay and cell death in Saccharomyces cerevisiae.

The anticancer ruthenium complex trans-[tetrachlorobis(1H-indazole)ruthenate(III)], otherwise known as KP1019, has previously been shown to inhibit proliferation of ovarian tumor cells, induce DNA damage and apoptosis in colon carcinoma cells, and reduce tumor size in animal models. Notably, no dose-limiting toxicity was observed in a Phase I clinical trial. Despite these successes, KP1019's precise mechanism of action remains poorly understood. To determine whether Saccharomyces cerevisiae might serve as an effective model for characterizing the cellular response to KP1019, we first confirmed that this drug is internalized by yeast and induces mutations, cell cycle delay, and cell death. We next examined KP1019 sensitivity of strains defective in DNA repair, ultimately showing that rad1Δ, rev3Δ, and rad52Δ yeast are hypersensitive to KP1019, suggesting that nucleotide excision repair (NER), translesion synthesis (TLS), and recombination each play a role in drug tolerance. These data are consistent with published work showing that KP1019 causes interstrand cross-links and bulky DNA adducts in mammalian cell lines. Published research also showed that mammalian cell lines resistant to other chemotherapeutic agents exhibit only modest resistance, and sometimes hypersensitivity, to KP1019. Here we report similar findings for S. cerevisiae. Whereas gain-of-function mutations in the transcription activator-encoding gene PDR1 are known to increase expression of drug pumps, causing resistance to structurally diverse toxins, we now demonstrate that KP1019 retains its potency against yeast carrying the hypermorphic alleles PDR1-11 or PDR1-3. Combined, these data suggest that S. cerevisiae could serve as an effective model system for identifying evolutionarily conserved modulators of KP1019 sensitivity.