The Lipid Metabolism as Target and Modulator of BOLD-100 Anticancer Activity: Crosstalk with Histone Acetylation.

The leading first-in-class ruthenium-complex BOLD-100 currently undergoes clinical phase-II anticancer evaluation. Recently, BOLD-100 is identified as anti-Warburg compound. The present study shows that also deregulated lipid metabolism parameters characterize acquired BOLD-100-resistant colon and pancreatic carcinoma cells. Acute BOLD-100 treatment reduces lipid droplet contents of BOLD-100-sensitive but not -resistant cells. Despite enhanced glycolysis fueling lipid accumulation, BOLD-100-resistant cells reveal diminished lactate secretion based on monocarboxylate transporter 1 (MCT1) loss mediated by a frame-shift mutation in the MCT1 chaperone basigin. Glycolysis and lipid catabolism converge in the production of protein/histone acetylation substrate acetyl-coenzymeA (CoA). Mass spectrometric and nuclear magnetic resonance analyses uncover spontaneous cell-free BOLD-100-CoA adduct formation suggesting acetyl-CoA depletion as mechanism bridging BOLD-100-induced lipid metabolism alterations and histone acetylation-mediated gene expression deregulation. Indeed, BOLD-100 treatment decreases histone acetylation selectively in sensitive cells. Pharmacological targeting confirms histone de-acetylation as central mode-of-action of BOLD-100 and metabolic programs stabilizing histone acetylation as relevant Achilles' heel of acquired BOLD-100-resistant cell and xenograft models. Accordingly, histone gene expression changes also predict intrinsic BOLD-100 responsiveness. Summarizing, BOLD-100 is identified as epigenetically active substance acting via targeting several onco-metabolic pathways. Identification of the lipid metabolism as driver of acquired BOLD-100 resistance opens novel strategies to tackle therapy failure.

Human serum albumin as a copper source for anticancer thiosemicarbazones.

Thiosemicarbazones (TSCs) are a class of biologically active compounds with promising anticancer activity. Their typical mechanism, especially of the clinically far developed representative Triapine, is chelation of iron (Fe), with the Fe-containing enzyme ribonucleotide reductase as primary intracellular target. However, for the subclass of terminally disubstituted, nanomolar-active derivatives like Dp44mT and Me2NNMe2, recent findings suggest that the chelation, stability, and reduction properties of the copper(II) (Cu) complexes are essential for their modes of action. Consequently, it is important to elucidate whether blood serum Cu(II) is a potential metal source for these TSCs. To gain more insights, the interaction of Triapine, Dp44mT or Me2NNMe2 with purified human serum albumin (HSA) as the main pool of labile Cu(II) was investigated by UV-vis and electron paramagnetic resonance measurements. Subsequently, a size-exclusion chromatography inductively coupled plasma mass spectrometry method for the differentiation of Cu species in serum was developed, especially separating the non-labile Cu enzyme ceruloplasmin from HSA. The results indicate that the TSCs specifically chelate copper from the N-terminal Cu-binding site of HSA. Furthermore, the Cu(II)-TSC complexes were shown to form ternary HSA conjugates, most likely via histidine. Noteworthy, Fe-chelation from transferrin was not overserved, even not for Triapine. In summary, the labile Cu pool of HSA is a potential source for Cu-TSC complex formation and, consequently, distinctly influences the anticancer activity and pharmacological behavior of TSCs.

Semiquantitative Analysis for High-Speed Mapping Applications of Biological Samples Using LA-ICP-TOFMS.

Laser ablation (LA) in combination with inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS) enables monitoring of elements from the entire mass range for every pixel, regardless of the isotopes of interest for a certain application. This provides nontargeted multi-element (bio-)imaging capabilities and the unique possibility to screen for elements that were initially not expected in the sample. Quantification of a large range of elements is limited as the preparation of highly multiplexed calibration standards for bioimaging applications by LA-ICP-(TOF)MS is challenging. In this study, we have developed a workflow for semiquantitative analysis by LA-ICP-TOFMS based on multi-element gelatin micro-droplet standards. The presented approach is intended for the mapping of biological samples due to the requirement of matrix-matched standards for accurate quantification in LA-ICPMS, a prerequisite that is given by the use of gelatin-based standards. A library of response factors was constructed based on 72 elements for the semiquantitative calculations. The presented method was evaluated in two stages: (i) on gelatin samples with known elemental concentrations and (ii) on real-world samples that included prime examples of bioimaging (mouse spleen and tumor tissue). The developed semiquantification approach was based on 10 elements as calibration standards and provided the determination of 136 nuclides of 63 elements, with errors below 25%, and for half of the nuclides, below 10%. A web application for quantification and semiquantification of LA-ICP(-TOF)MS data was developed, and a detailed description is presented to easily allow others to use the presented method.

Multiparametric Tissue Characterization Utilizing the Cellular Metallome and Immuno-Mass Spectrometry Imaging.

In this study, we present a workflow that enables spatial single-cell metallomics in tissue decoding the cellular heterogeneity. Low-dispersion laser ablation in combination with inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) provides mapping of endogenous elements with cellular resolution at unprecedented speed. Capturing the heterogeneity of the cellular population by metals only is of limited use as the cell type, functionality, and cell state remain elusive. Therefore, we expanded the toolbox of single-cell metallomics by integrating the concepts of imaging mass cytometry (IMC). This multiparametric assay successfully utilizes metal-labeled antibodies for cellular tissue profiling. One important challenge is the need to preserve the original metallome in the sample upon immunostaining. Therefore, we studied the impact of extensive labeling on the obtained endogenous cellular ionome data by quantifying elemental levels in consecutive tissue sections (with and without immunostaining) and correlating elements with structural markers and histological features. Our experiments showed that the elemental tissue distribution remained intact for selected elements such as sodium, phosphorus, and iron, while absolute quantification was precluded. We hypothesize that this integrated assay not only advances single-cell metallomics (enabling to link metal accumulation to multi-dimensional characterization of cells/cell populations), but in turn also enhances selectivity in IMC, as in selected cases, labeling strategies can be validated by elemental data. We showcase the power of this integrated single-cell toolbox using an in vivo tumor model in mice and provide mapping of the sodium and iron homeostasis as linked to different cell types and function in mouse organs (such as spleen, kidney, and liver). Phosphorus distribution maps added structural information, paralleled by the DNA intercalator visualizing the cellular nuclei. Overall, iron imaging was the most relevant addition to IMC. In tumor samples, for example, iron-rich regions correlated with high proliferation and/or located blood vessels, which are key for potential drug delivery.

Effects of Salinomycin and Deferiprone on Lead-Induced Changes in the Mouse Brain.

Lead (Pb) is a highly toxic heavy metal that has deleterious effects on the central nervous system. This study aimed to investigate the effects of salinomycin (Sal) and deferiprone (DFP) on brain morphology and on the content of some essential elements in Pb-exposed mice. Adult male Institute of Cancer Research (ICR) mice were exposed to a daily dose of 80 mg/kg body weight ( b.w.) Pb(II) nitrate for 14 days and subsequently treated with Sal (16 mg/kg b.w.) or DFP (19 mg/kg b.w.) for another 14 days. At the end of the experimental protocol, the brains were processed for histological and inductively coupled plasma mass spectrometry (ICP-MS) analyses. Pb exposure resulted in a 50-fold increase in Pb concentration, compared with controls. Magnesium (Mg) and phosphorus (P) were also significantly increased by 22.22% and 17.92%, respectively. The histological analysis of Pb-exposed mice revealed brain pathological changes with features of neuronal necrosis. Brain Pb level remained significantly elevated in Sal- and DFP-administered groups (37-fold and 50-fold, respectively), compared with untreated controls. Treatment with Sal significantly reduced Mg and P concentrations by 22.56% and 18.38%, respectively, compared with the Pb-exposed group. Administration of Sal and DFP ameliorated brain injury in Pb-exposed mice and improved histological features. The results suggest the potential application of Sal and DFP for treatment of Pb-induced neurotoxicity.

Highly Cytotoxic Molybdenocenes with Strong Metabolic Effects Inhibit Tumour Growth in Mice.

A series of six highly lipophilic Cp-substituted molybdenocenes bearing different bioactive chelating ligands was synthesized and characterized by NMR spectroscopy, mass spectrometry and X-ray crystallography. In vitro experiments showed a greatly increased cytotoxic potency when compared to the non-Cp-substituted counterparts. In vivo experiments performed with the dichlorido precursor, (Ph2 C-Cp)2 MoCl2 and the in vitro most active complex, containing the thioflavone ligand, showed an inhibition of tumour growth. Proteomic studies on the same two compounds demonstrated a significant regulation of tubulin-associated and mitochondrial inner membrane proteins for both compounds and a strong metabolic effect of the thioflavone containing complex.

Arene Variation of Highly Cytotoxic Tridentate Naphthoquinone-Based Ruthenium(II) Complexes and In-Depth In Vitro Studies.

The main purpose of this study was to synthesize a new set of naphthoquinone-based ruthenium(II) arene complexes and to develop an understanding of their mode of action. This study systematically reviews the steps of synthesis, aiming to provide a simplified approach using microwave irradiation. The chemical structures and the physicochemical properties of this novel group of compounds were examined by 1H-NMR and 13C-NMR spectroscopy, X-ray diffractometry, HPLC-MS and supporting DFT calculations. Several aspects of the biological activity were investigated in vitro, including short- and long-term cytotoxicity tests, cellular accumulation studies, detection of reactive oxygen species generation, apoptosis induction and NAD(P)H:quinone oxidoreductase 1 (NQO1) activity as well as cell cycle analysis in A549, CH1/PA-1, and SW480 cancer cells. Furthermore, the DNA interaction ability was studied in a cell-free assay. A positive correlation was found between cytotoxicity, lipophilicity and cellular accumulation of the tested complexes, and the results offer some important insights into the effects of the arene. The most obvious finding to emerge from this study is that the usually very chemosensitive CH1/PA-1 teratocarcinoma cells showed resistance to these phthiocol-based organometallics in comparison to the usually less chemosensitive SW480 colon carcinoma cells, which pilot experiments suggest as being related to NQO1 activity.

Effects of subacute cadmium exposure and subsequent deferiprone treatment on cadmium accumulation and on the homeostasis of essential elements in the mouse brain.

INTRODUCTION: Cadmium (Cd) is а hazardous multi-organ toxin. In this study, we provide the first results about the effect of oral administration of deferiprone (DFP) on Cd accumulation and on the homeostasis of essential elements in the brain of Cd-exposed mice. METHODS: Adult Institute of Cancer Research (ICR) male mice were randomized into four experimental groups: untreated controls - administered distilled water for 28 days; Cd-exposed group - exposed to 18 mg/kg body weight (b.w.) Cd(II) acetate for 14 days followed by the administration of distilled water for two weeks; Cd + DFP (low dose) - Cd-intoxicated mice subsequently treated with 19 mg/kg b.w. DFP for two weeks; and Cd + DFP (high dose) - Cd-exposed mice administered high-dose DFP (135 mg/kg b.w.) for 14 days. Brains were subjected to inductively coupled plasma-mass spectrometry (ICP-MS) and histological analysis. RESULTS: The results revealed that exposure of mice to Cd for 14 days significantly increased Cd concentration and significantly decreased magnesium (Mg), phosphorus (P), and zinc (Zn) contents in the brain compared to untreated controls. This effect was accompanied by necrotic-degenerative changes in both the cerebrum and cerebellum. Oral administration of low-dose DFP to Cd-exposed mice decreased the concentration of the toxic metal in the brain by 16.37% and restored the concentration of the essential elements to normal control values. Histological analysis revealed substantially improved cerebral and cerebellar histoarchitectures. In contrast, oral administration of high-dose DFP increased Cd content and significantly decreased selenium (Se) concentration in the brain. Necrotic neurons and Purkinje cells were still observed in the cerebral and cerebellar cortices. CONCLUSION: The results demonstrated that oral administration of DFP at low doses has a better therapeutic potential for the treatment of Cd-induced brain damage compared to high doses.

Comparative Effects of Deferiprone and Salinomycin on Lead-Induced Disturbance in the Homeostasis of Intrarenal Essential Elements in Mice.

Lead (Pb) exposure induces severe nephrotoxic effects in humans and animals. Herein, we compare the effects of two chelating agents, salinomycin and deferiprone, on Pb-induced renal alterations in mice and in the homeostasis of essential elements. Adult male mice (Institute of Cancer Research (ICR)) were randomized into four groups: control (Ctrl)-untreated mice administered distilled water for 28 days; Pb-exposed group (Pb)-mice administered orally an average daily dose of 80 mg/kg body weight (BW) lead (II) nitrate (Pb(NO3)2) during the first two weeks of the experimental protocol followed by the administration of distilled water for another two weeks; salinomycin-treated (Pb + Sal) group-Pb-exposed mice, administered an average daily dose of 16 mg/kg BW salinomycin for two weeks; deferiprone-treated (Pb + Def) group-Pb-exposed mice, administered an average daily dose of 20 mg/kg BW deferiprone for 14 days. The exposure of mice to Pb induced significant accumulation of the toxic metal in the kidneys and elicited inflammation with leukocyte infiltrations near the glomerulus. Biochemical analysis of the sera revealed that Pb significantly altered the renal function markers. Pb-induced renal toxicity was accompanied by a significant decrease in the endogenous renal concentrations of phosphorous (P), calcium (Ca), copper (Cu) and selenium (Se). In contrast to deferiprone, salinomycin significantly improved renal morphology in Pb-treated mice and decreased the Pb content by 13.62% compared to the Pb-exposed group. There was also a mild decrease in the renal endogenous concentration of magnesium (Mg) and elevation of the renal concentration of iron (Fe) in the salinomycin-treated group compared to controls. Overall, the results demonstrated that salinomycin is a more effective chelating agent for the treatment of Pb-induced alterations in renal morphology compared to deferiprone.

The Anticancer Ruthenium Compound BOLD-100 Targets Glycolysis and Generates a Metabolic Vulnerability towards Glucose Deprivation.

Cellular energy metabolism is reprogrammed in cancer to fuel proliferation. In oncological therapy, treatment resistance remains an obstacle and is frequently linked to metabolic perturbations. Identifying metabolic changes as vulnerabilities opens up novel approaches for the prevention or targeting of acquired therapy resistance. Insights into metabolic alterations underlying ruthenium-based chemotherapy resistance remain widely elusive. In this study, colon cancer HCT116 and pancreatic cancer Capan-1 cells were selected for resistance against the clinically evaluated ruthenium complex sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (BOLD-100). Gene expression profiling identified transcriptional deregulation of carbohydrate metabolism as a response to BOLD-100 and in resistance against the drug. Mechanistically, acquired BOLD-100 resistance is linked to elevated glucose uptake and an increased lysosomal compartment, based on a defect in downstream autophagy execution. Congruently, metabolomics suggested stronger glycolytic activity, in agreement with the distinct hypersensitivity of BOLD-100-resistant cells to 2-deoxy-d-glucose (2-DG). In resistant cells, 2-DG induced stronger metabolic perturbations associated with ER stress induction and cytoplasmic lysosome deregulation. The combination with 2-DG enhanced BOLD-100 activity against HCT116 and Capan-1 cells and reverted acquired BOLD-100 resistance by synergistic cell death induction and autophagy disturbance. This newly identified enhanced glycolytic activity as a metabolic vulnerability in BOLD-100 resistance suggests the targeting of glycolysis as a promising strategy to support BOLD-100 anticancer activity.

Micro-droplet-based calibration for quantitative elemental bioimaging by LA-ICPMS.

In this work, a novel standardization strategy for quantitative elemental bioimaging is evaluated. More specifically, multi-element quantification by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) is performed by multi-point calibration using gelatin-based micro-droplet standards and validated using in-house produced reference materials. Fully automated deposition of micro-droplets by micro-spotting ensured precise standard volumes of 400 ± 5 pL resulting in droplet sizes of around 200 µm in diameter. The small dimensions of the micro-droplet standards and the use of a low-dispersion laser ablation setup reduced the analysis time required for calibration by LA-ICPMS significantly. Therefore, as a key advance, high-throughput analysis (pixel acquisition rates of more than 200 Hz) enabled to establish imaging measurement sequences with quality control- and standardization samples comparable to solution-based quantification exercises by ICP-MS. Analytical figures of merit such as limit of detection, precision, and accuracy of the calibration approach were assessed for platinum and for elements with biological key functions from the lower mass range (phosphorus, copper, and zinc). As a proof-of-concept application, the tool-set was employed to investigate the accumulation of metal-based anticancer drugs in multicellular tumor spheroid models at clinically relevant concentrations. Graphical abstract.

Accurate characterization of ß-amyloid (Aß40, Aß42) standards using species-specific isotope dilution by means of HPLC-ICP-MS/MS.

The amyloid ß peptide, as one of the main components in senile plaque, represents a defining pathological feature for Alzheimer's disease, and is therefore commonly used as a biomarker for this disease in clinical analysis. However, the selection of suitable standards is limited here, since only a few are commercially available, and these suffer from varying purity. Hence, the accurate characterization of these standards is of great importance. In this study, we developed a method for the traceable quantification of the peptide content using species-specific isotope dilution and ICP-MS/MS detection. It is based on the separation of the sulfur-containing amino acids methionine and cysteine after oxidation and hydrolysis of the peptide. Using a strong anion exchange column, both amino acids could be separated from each other, as well as from their oxidized forms and sulfate. The sulfur content was determined via ICP-MS/MS using oxygen as reaction gas. Species-specific isotope dilution was enabled by using a 34S-labeled yeast hydrolysate, containing methionine sulfone and cysteic acid with different isotopic composition. The peptide contents of Aß standards (Aß40,42), as well as myoglobin and lysozyme with different degrees of purity, were determined. For validation purposes, the standard reference material NIST 2389a, which contains the amino acids in a similar concentration, was subjected to the developed sample preparation and analysis method. In addition to accounting for errors during sample preparation, high levels of accuracy and precision could be obtained using this method, making it fit-for-purpose for the characterization of peptide standards.

Cisplatin Uptake in Macrophage Subtypes at the Single-Cell Level by LA-ICP-TOFMS Imaging.

A high-throughput laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.

Ameliorative effects of deferiprone and tetraethylammonium salt of salinomycinic acid on lead-induced toxicity in mouse testes.

In this study, we compare the effects of deferiprone (Def) and tetraethylammonium salt of salinomycinic acid (Sal) on lead (Pb)-induced toxicity in testes of Pb-exposed mice. Mature male ICR mice were allocated into four groups as follows: untreated control mice (ctrl)-received distilled water for 4 weeks; Pb-exposed mice (Pb)-subjected to 14-day Pb (II) nitrate administration at dose 80 mg/kg body weight (b.w.); Pb + Def group-Pb-exposed mice, treated with 20 mg/kg b.w. Def for 2 weeks; and Pb + Sal group-Pb-intoxicated mice, treated with 16 mg/kg b.w. Sal for 14 days. The results demonstrated that Pb exposure significantly increased blood and testicular Pb concentrations, decreased testicular calcium (Ca) content, significantly elevated testicular levels of magnesium (Mg), zinc (Zn), and selenium (Se) but did not significantly affect the endogenous contents of phosphorous (P) and iron (Fe) compared with untreated controls. Pb intoxication induced disorganization of the seminiferous epithelium. Def or Sal administration reduced blood Pb and testicular Pb concentrations in Pb-exposed mice compared with the Pb-intoxicated group. Mg, Zn, and Se concentrations in testes of Pb-exposed mice, treated with Def or Sal, remained higher compared with the untreated controls. Sal significantly increased testicular P concentration compared with untreated controls and significantly elevated the testicular Ca and Fe concentrations compared with the toxic control group. Both chelating agents improved testicular morphology to a great extent. The results demonstrate the potential of both compounds as antidotes for treatment of Pb-induced impairment of male reproductive function.

An Organometallic Gold(I) Bis-N-Heterocyclic Carbene Complex with Multimodal Activity in Ovarian Cancer Cells.

The organometallic AuI bis-N-heterocyclic carbene complex [Au(9-methylcaffeine-8-ylidene)2 ]+ (AuTMX2 ) was previously shown to selectively and potently stabilise telomeric DNA G-quadruplex (G4) structures. This study sheds light on the molecular reactivity and mode of action of AuTMX2 in the cellular context using mass spectrometry-based methods, including shotgun proteomics in A2780 ovarian cancer cells. In contrast to other metal-based anticancer agents, this organogold compound is less prone to form coordinative bonds with biological nucleophiles and is expected to exert its drug effects mainly by non-covalent interactions. Global protein expression changes of treated cancer cells revealed a multimodal mode of action of AuTMX2 by alterations in the nucleolus, telomeres, actin stress-fibres and stress-responses, which were further supported by pharmacological assays, fluorescence microscopy and cellular accumulation experiments. Proteomic data are available via ProteomeXchange with identifier PXD020560.