Synthesis, Chemical Characterization, and Biological Evaluation of Hydrophilic Gold(I) and Silver(I) N-Heterocyclic Carbenes as Potential Anticancer Agents.

A series of new gold(I) and silver(I) N-heterocyclic carbenes bearing a 1-thio-ß-d-glucose tetraacetate moiety was synthesized and chemically characterized. The compounds' stability and solubility in physiological conditions were investigated employing a multitechnique approach. Interaction studies with biologically relevant proteins, such as superoxide dismutase (SOD) and human serum albumin (HSA), were conducted via UV-vis absorption spectroscopy and high-resolution ESI mass spectrometry. The biological activity of the compounds was evaluated in the A2780 and A2780R (cisplatin-resistant) ovarian cancer cell lines and the HSkMC (human skeletal muscle) healthy cell line. Inhibition studies of the selenoenzyme thioredoxin reductase (TrxR) were also carried out. The results highlighted that the gold complexes are more stable in aqueous environment and capable of interaction with SOD and HSA. Moreover, these carbenes strongly inhibited the TrxR activity. In contrast, the silver ones underwent structural alterations in the aqueous medium and showed greater antiproliferative activity.

Unlocking the Power of Human Ferritin: Enhanced Drug Delivery of Aurothiomalate in A2780 Ovarian Cancer Cells.

Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provided a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.

Cytotoxic auranofin analogues bearing phosphine, arsine and stibine ligands: A study on the possible role of the ligand on the biological activity.

Three gold(I) linear compounds, sharing the general formula [AuI(LPh3)], have been synthesized and characterized. The nature of the ligand has been modified by moving down among some of the elements of group 15, i.e. phosphorus, arsenic and antimony. The structures of derived compounds have been solved through XRD and the reactivity behaviour towards selected biomolecules has been investigated through a multi-technique approach involving NMR, high-resolution mass spectrometry and IR. Moreover, the biological activity of the investigated compounds has been comparatively analyzed through classical methodologies and the disclosed differences are discussed in detail.

STAT3 Signalling Drives LDH Up-Regulation and Adiponectin Down-Regulation in Cachectic Adipocytes.

Cachexia is a devastating pathology that worsens the quality of life and antineoplastic treatment outcomes of oncologic patients. Herein, we report that the secretome from murine colon carcinoma CT26 induces cachectic features in both murine and human adipocytes that are associated with metabolic alterations such as enhanced lactate production and decreased oxygen consumption. The use of oxamate, which inhibits lactate dehydrogenase activity, hinders the effects induced by CT26 secretome. Interestingly, the CT26 secretome elicits an increased level of lactate dehydrogenase and decreased expression of adiponectin. These modifications are driven by the STAT3 signalling cascade since the inhibition of STAT3 with WP1066 impedes the formation of the cachectic condition and the alteration of lactate dehydrogenase and adiponectin levels. Collectively, these findings show that STAT3 is responsible for the altered lactate dehydrogenase and adiponectin levels that, in turn, could participate in the worsening of this pathology and highlight a step forward in the comprehension of the mechanisms underlying the onset of the cachectic condition in adipocytes.

The effects of two cytotoxic gold(i) carbene compounds on the metabolism of A2780 ovarian cancer cells: mechanistic inferences through NMR analysis.

NMR metabolomics is a powerful tool to characterise the changes in cancer cell metabolism elicited by anticancer drugs. Here, the large metabolic alterations produced by two cytotoxic gold carbene compounds in A2780 ovarian cancer cells are described and discussed in comparison to auranofin, in the frame of the available mechanistic knowledge.

A complex bearing TSPO PIGA ligand coordinated to the [Au(PEt3)]+ pharmacophore is highly cytotoxic against ovarian cancer cells.

Auranofin ([1-(thio-κS)-ß-D-glucopyranose-2,3,4,6-tetraacetato](triethylphosphine)-gold) is a leading gold-based drug clinically used to treat arthritis. In the last years, it entered various drug reprofiling programs, and it has been found promising against various forms of tumor, including ovarian cancer. Evidence showed as its antiproliferative profile mainly depends on the inhibition of thioredoxin reductase (TrxR), being this mitochondrial system its main target. In this context, we report here the synthesis and biological evaluation of a novel complex designed as auranofin analogue obtained through the conjugation of a phenylindolylglyoxylamide ligand (which belongs to the so-called PIGA TSPO ligand family) with the auranofin-derived cationic fragment [Au(PEt3)]+. This complex is characterized by two parts. The phenylindolylglyoxylamide moiety, owing to its high affinity for TSPO (in the low nM range) should drive the compound to target mitochondria, whereas the [Au(PEt3)]+ cation is the actual anticancer-active molecular fragment. Overall, we wanted to offer the proof-of-concept that by coupling PIGA ligands to anticancer gold active moieties, it is possible to preserve and even improve anticancer effects, opening the avenue to a reliable approach for targeted therapy.

Chemical Modification of Auranofin Yields a New Family of Anticancer Drug Candidates: The Gold(I) Phosphite Analogues.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Pyruvate prevents the onset of the cachectic features and metabolic alterations in myotubes downregulating STAT3 signaling.

Cachexia is a systemic disease associated with several pathologies, including cancer, that leads to excessive weight loss due to enhanced protein degradation. Previously, we showed that cachectic features in myotubes are provoked by a metabolic shift toward lactic fermentation. Our previous results led us to hyphotesise that increasing pyruvate concentration could impede the metabolic modifications responsible for induction of cachexia in myotubes. Here, we demonstrated that the addition of sodium pyruvate in conditioned media from CT26 colon cancer cells (CM CT26) prevents the onset of either phenotypic and metabolic cachectic features. Myotubes treated with CM CT26 containing sodium pyruvate show a phenotype similar to the healthy counterpart and display lactate production, oxygen consumption, and pyruvate dehydrogenase activity as control myotubes. The use of the Mitochondrial Pyruvate Carrier inhibitor UK5099, highlights the importance of mitochondrial pyruvate amount in the prevention of cachexia. Indeed, UK5099-treated myotubes show cachectic features as those observed in myotubes treated with CM CT26. Finally, we found that sodium pyruvate is able to decrease STAT3 phosphorylation level, a signaling pathway involved in the induction of cachexia in myotubes. Collectively, our results show that cachexia in myotubes could be prevented by the utilization of sodium pyruvate which impedes the metabolic modifications responsible for the acquisition of the cachectic features.

A Metabolic Change towards Fermentation Drives Cancer Cachexia in Myotubes.

Cachexia is a disorder associated with several pathologies, including cancer. In this paper, we describe how cachexia is induced in myotubes by a metabolic shift towards fermentation, and the block of this metabolic modification prevents the onset of the cachectic phenotype. Cachectic myotubes, obtained by the treatment with conditioned medium from murine colon carcinoma cells CT26, show increased glucose uptake, decreased oxygen consumption, altered mitochondria, and increased lactate production. Interestingly, the block of glycolysis by 2-deoxy-glucose or lactate dehydrogenase inhibition by oxamate prevents the induction of cachexia, thus suggesting that this metabolic change is greatly involved in cachexia activation. The treatment with 2-deoxy-glucose or oxamate induces positive effects also in mitochondria, where mitochondrial membrane potential and pyruvate dehydrogenase activity became similar to control myotubes. Moreover, in myotubes treated with interleukin-6, cachectic phenotype is associated with a fermentative metabolism, and the inhibition of lactate dehydrogenase by oxamate prevents cachectic features. The same results have been achieved by treating myotubes with conditioned media from human colon HCT116 and human pancreatic MIAPaCa-2 cancer cell lines, thus showing that what has been observed with murine-conditioned media is a wide phenomenon. These findings demonstrate that cachexia induction in myotubes is linked with a metabolic shift towards fermentation, and inhibition of lactate formation impedes cachexia and highlights lactate dehydrogenase as a possible new tool for counteracting the onset of this pathology.

The Adipokines in Cancer Cachexia.

Cachexia is a devastating pathology induced by several kinds of diseases, including cancer. The hallmark of cancer cachexia is an extended weight loss mainly due to skeletal muscle wasting and fat storage depletion from adipose tissue. The latter exerts key functions for the health of the whole organism, also through the secretion of several adipokines. These hormones induce a plethora of effects in target tissues, ranging from metabolic to differentiating ones. Conversely, the decrease of the circulating level of several adipokines positively correlates with insulin resistance, metabolic syndrome, diabetes, and cardiovascular disease. A lot of findings suggest that cancer cachexia is associated with changed secretion of adipokines by adipose tissue. In agreement, cachectic patients show often altered circulating levels of adipokines. This review reported the findings of adipokines (leptin, adiponectin, resistin, apelin, and visfatin) in cancer cachexia, highlighting that to study in-depth the involvement of these hormones in this pathology could lead to the development of new therapeutic strategies.

Glucose Metabolic Reprogramming of ER Breast Cancer in Acquired Resistance to the CDK4/6 Inhibitor Palbociclib.

The majority of breast cancers express the estrogen receptor (ER) and are dependent on estrogen for their growth and survival. Endocrine therapy (ET) is the standard of care for these tumors. However, a superior outcome is achieved in a subset of ER positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) metastatic breast cancer patients when ET is administrated in combination with a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor, such as palbociclib. Moreover, CDK4/6 inhibitors are currently being tested in ER+/HER2+ breast cancer and reported encouraging results. Despite the clinical advances of a combinatorial therapy using ET plus CDK4/6 inhibitors, potential limitations (i.e., resistance) could emerge and the metabolic adaptations underlying such resistance warrant further elucidation. Here we investigate the glucose-dependent catabolism in a series of isogenic ER+ breast cancer cell lines sensitive to palbociclib and in their derivatives with acquired resistance to the drug. Importantly, ER+/HER2- and ER+/HER2+ cell lines show a different degree of glucose dependency. While ER+/HER2- breast cancer cells are characterized by enhanced aerobic glycolysis at the time of palbociclib sensitivity, ER+/HER2+ cells enhance their glycolytic catabolism at resistance. This metabolic phenotype was shown to have prognostic value and was targeted with multiple approaches offering a series of potential scenarios that could be of clinical relevance.