Preclinical Evaluation of Zn(II) Self-Assemblies with Selective Cytotoxic Activity Against Cancer Cells In Vitro and In Ovo.

Dipodal pyridylthiazole amine ligands L1 and L2 both form different metallo-supramolecular self-assemblies with Zn2+ and Cu2+ and these are shown to be toxic and selective towards cancer cell lines in vitro. Furthermore, potency and selectivity are highly dependent upon the metal ions, ligand system and bound anion, with significant changes in chemosensitivity and selectivity dependent upon which species are employed. Importantly, significant anti-tumor activity was observed in ovo at doses that are non-toxic.

The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets.

Bladder cancer is the 10th most common cancer worldwide. For muscle-invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is generally poor. For non-muscle-invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial-mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH-A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH-B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH-A is expressed in normal urothelial cells, LDH-A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected-identifying LDH-A as a cancer-selective therapeutic target for bladder cancers. LDH-A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.

Bis(bipyridine)ruthenium(II) Ferrocenyl ß-Diketonate Complexes: Exhibiting Nanomolar Potency against Human Cancer Cell Lines.

The synthesis and characterization of new bis(bipyridine)ruthenium(II) ferrocenyl ß-diketonate complexes, [(bpy)2 Ru(Fc-acac)][PF6 ] (bpy=2,2'-bipyridine; Fc-acac=functionalized ferrocenyl ß-diketonate ligand) are reported. Alongside clinical platinum drugs, these bimetallic ruthenium-iron complexes have been screened for their cytotoxicity against MIA PaCa-2 (human pancreatic carcinoma), HCT116 p53+/+ (human colon carcinoma, p53-wild type) and ARPE-19 (human retinal pigment epithelial) cell lines. With the exception of one complex, the library exhibit nanomolar potency against cancerous cell lines, and their relative potencies are up to 40x, 400x and 72x more cytotoxic than cisplatin, carboplatin and oxaliplatin, respectively. Under hypoxic conditions, the complexes remain cytotoxic (sub-micromolar range), highlighting their potential in targeting hypoxic tumor regions. The Comet assay was used to determine their ability to damage DNA, and results show dose dependent damage which correlates well with the cytotoxicity results. Their potential to treat bacterial and fungal strains has been determined, and highlight complexes have selective growth inhibition of up to 87-100 % against Staphylococcus aureus and Candida albicans.

An Efficient Method for the Isolation of Toxins from Pteridium aquilinum and Evaluation of Ptaquiloside Against Cancer and Non-cancer Cells.

The common fern, bracken (Pteridium aquilinum), is well known for its toxic effects on livestock due principally to the carcinogenic constituent ptaquiloside ( 1: ), although other toxins are present including the cyanogenic glycoside, prunasin ( 2: ). Here, we report an improved and relatively "green" process for the isolation of 1: and 2: from fresh bracken fronds and the evaluation of 1: for cytotoxicity against several cancer cell lines. The results indicate that 1: displays selective toxicity against cancer cells relative to noncancer retinal epithelial cells, and the improved method for the isolation of 1: is expected to facilitate further exploration of its pharmacological properties.

Glycoconjugated Metallohelices have Improved Nuclear Delivery and Suppress Tumour Growth In Vivo.

Monosaccharides are added to the hydrophilic face of a self-assembled asymmetric FeII metallohelix, using CuAAC chemistry. The sixteen resulting architectures are water-stable and optically pure, and exhibit improved antiproliferative selectivity against colon cancer cells (HCT116 p53+/+ ) with respect to the non-cancerous ARPE-19 cell line. While the most selective compound is a glucose-appended enantiomer, its cellular entry is not mainly glucose transporter-mediated. Glucose conjugation nevertheless increases nuclear delivery ca 2.5-fold, and a non-destructive interaction with DNA is indicated. Addition of the glucose units affects the binding orientation of the metallohelix to naked DNA, but does not substantially alter the overall affinity. In a mouse model, the glucose conjugated compound was far better tolerated, and tumour growth delays for the parent compound (2.6 d) were improved to 4.3 d; performance as good as cisplatin but with the advantage of no weight loss in the subjects.

Ruthenium-Containing Linear Helicates and Mesocates with Tuneable p53-Selective Cytotoxicity in Colorectal Cancer Cells.

The ligands L1 and L2 both form separable dinuclear double-stranded helicate and mesocate complexes with RuII . In contrast to clinically approved platinates, the helicate isomer of [Ru2 (L1 )2 ]4+ was preferentially cytotoxic to isogenic cells (HCT116 p53-/- ), which lack the critical tumour suppressor gene. The mesocate isomer shows the reverse selectivity, with the achiral isomer being preferentially cytotoxic towards HCT116 p53+/+ . Other structurally similar RuII -containing dinuclear complexes showed very little cytotoxic activity. This study demonstrates that alterations in ligand or isomer can have profound effects on cytotoxicity towards cancer cells of different p53 status and suggests that selectivity can be "tuned" to either genotype. In the search for compounds that can target difficult-to-treat tumours that lack the p53 tumour suppressor gene, [Ru2 (L1 )2 ]4+ is a promising compound for further development.

Bis-picolinamide Ruthenium(III) Dihalide Complexes: Dichloride-to-Diiodide Exchange Generates Single trans Isomers with High Potency and Cancer Cell Selectivity.

A library of new bis-picolinamide ruthenium(III) dihalide complexes of the type [RuX2 L2 ] (X=Cl or I, L=picolinamide) have been synthesised and characterised. The complexes exhibit different picolinamide ligand binding modes, whereby one ligand is bound (N,N) and the other bound (N,O). Structural studies revealed a mixture of cis and trans isomers for the [RuCl2 L2 ] complexes but upon a halide exchange reaction to yield [RuI2 L2 ], only single trans isomers were detected. High cytotoxic activity against human cancer cell lines was observed, with the potencies of some complexes similar to or better than cisplatin. The conversion to [RuI2 L2 ] substantially increased the activity towards cancer cell lines by more than twelvefold. The [RuI2 L2 ] complexes displayed potent activity against the A2780cis (cisplatin-resistant human ovarian cancer) cell line, with a more than fourfold higher potency than cisplatin. Equitoxic activity was observed against normoxic and hypoxic cancer cells, which indicates the potential to eradicate both the hypoxic and aerobic fractions of solid tumours with similar efficiency. The activity of selected complexes against non-cancer ARPE-19 cells was also tested. The [RuI2 L2 ] complexes were found to be more potent than the [RuCl2 L2 ] analogues and also more selective towards cancer cells with a selectivity factor in excess of sevenfold.

Rhodium, iridium, and ruthenium half-sandwich picolinamide complexes as anticancer agents.

Novel rhodium, iridium, and ruthenium half-sandwich complexes containing (N,N)-bound picolinamide ligands have been prepared for use as anticancer agents. The complexes show promising cytotoxicities, with the presence, position, and number of halides having a significant effect on the corresponding IC50 values. One ruthenium complex was found to be more cytotoxic than cisplatin on HT-29 and MCF-7 cells after 5 days and 1 h, respectively, and it remains active with MCF-7 cells even under hypoxic conditions, making it a promising candidate for in vivo studies.

Carbophilic 3-component cascades: access to complex bioactive cyclopropyl diindolylmethanes.

Naturally occurring indole-3-carbinol and 3,3-diindolylmethane show bioactivity in a number of disparate disease areas, including cancer, prompting substantial synthetic analogue activity. We describe a new approach to highly functionalised derivatives that starts from allene gas and proceeds via the combination of a three-component Pd(0)-catalysed cascade with a one-pot, three-component carbophilic Pt(II) cascade linked to a stereoselective acid-catalysed Mannich-Michael reaction that generates complex cyclopropyl diindolylmethanes which show selective activity against prostate cancer cell lines.

Cytotoxicity of Ruthenium(II) Arene Complexes Containing Functionalized Ferrocenyl ß-Diketonate Ligands.

The synthesis and characterization of 24 ruthenium(II) arene complexes of the type [(p-cym)RuCl(Fc-acac)] (where p-cym = p-cymene and Fc-acac = functionalized ferrocenyl ß-diketonate ligands) are reported, including single-crystal X-ray diffraction for 21 new complexes. Chemosensitivity studies have been conducted against human pancreatic carcinoma (MIA PaCa-2), human colorectal adenocarcinoma p53-wildtype (HCT116 p53+/+) and normal human retinal epithelial cell lines (APRE-19). The most active complex, which contains a 2-furan-substituted ligand (4), is 5x more cytotoxic than the analogs 3-furan complex (5) against MIA PaCa-2. Several complexes were screened under hypoxic conditions and at shorter-time incubations, and their ability to damage DNA was determined by the comet assay. Compounds were also screened for their potential to inhibit the growth of both bacterial and fungal strains.

Infrared and Raman Diagnostic Modeling of Phosphate Adsorption on Ceria Nanoparticles.

Cerium dioxide (CeO2; ceria) nanoparticles (CeNPs) are promising nanozymes that show a variety of biological activity. Effective nanozymes need to retain their activity in the face of surface speciation in biological environments, and characterizing surface speciation is therefore critical to understanding and controlling the therapeutic capabilities of CeNPs. In particular, adsorbed phosphates can impact the enzymatic activity exploited to convert phosphate prodrugs into therapeutics in vivo and also define the early stages of the phosphate-scavenging processes that lead to the transformation of active CeO2 into inactive CePO4. In this work, we utilize ab initio lattice-dynamics calculations to study the interaction of phosphates with the three major surfaces of ceria and to predict the infrared (IR) and Raman spectral signatures of adsorbed phosphate species. We find that phosphates adsorb strongly to CeO2 surfaces in a range of stable binding configurations, of which 5-fold coordinated P species in a trigonal bipyramidal coordination may represent a stable intermediate in the early stages of phosphate scavenging. We find that the phosphate species show characteristic spectral fingerprints between 500 and 1500 cm-1, whereas the bare CeO2 surfaces show no active modes above 600 cm-1, and the 5-fold coordinated P species in particular show potential diagnostic P-O stretching modes between 650 and 700 cm-1 in both IR and Raman spectra. This comprehensive exploration of different binding modes for phosphates on CeO2 and the set of reference spectra provides an important step toward the experimental characterization of phosphate speciation and, ultimately, control of its impact on the performance of ceria nanozymes.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Investigation of the cytotoxicity induced by didocosahexaenoin, an omega 3 derivative, in human prostate carcinoma cell lines.

The aim of the present study was to investigate the cytotoxicity induced by an omega-3 derivative, didocosahexaenoin (Dido) on human prostate carcinoma cells and to compare the cytotoxicity to that of docosahexaenoic acid (DHA). Different carcinoma- and non-carcinoma cells were exposed to various concentrations of omega-3 compounds at varying exposure times and the cytotoxicity was measured by MTT assay. The mechanism of Dido-induced apoptosis was investigated in prostate carcinoma cells. Dido induced stronger cytotoxicity than DHA in human prostate carcinoma cells in a dose- and time-dependent manner. Dido was also more selective and potent in inducing cytotoxicity in prostate carcinoma cells than other carcinoma cell lines tested. Pre-treatment with Dido increased the level of reactive oxygen species (ROS) in prostate carcinoma cells. Pre-treatment with various antioxidants reduced the cytotoxicity induced by Dido. Pre-treatment with Dido ≥30 â€‹µM also induced apoptosis which was suggested to involve an externalisation of phosphatidyl serine, a significant increase in the mitochondrial membrane potential (p â€‹< â€‹0.01) and the level of activated caspase 3/7 (p â€‹< â€‹0.05) in prostate carcinoma cells. This study is the first to show that Dido induced cytotoxicity with high selectivity and higher potency than DHA in human prostate carcinoma cells. The mechanism of action is likely to involve an increase in the level of ROS, loss in the mitochondrial membrane potential as well as externalisation of phosphatidyl serine and increase in the caspase 3/7 activity. Dido may have potential to be used for the adjuvant therapy or combination therapy with conventional chemotherapeutic drugs.

Self-assembly of an anion receptor with metal-dependent kinase inhibition and potent in vitro anti-cancer properties.

One topical area of supramolecular chemistry is the binding of anionic species but despite the importance of anions in diverse cellular processes and for cancer development, anion receptors or 'binders' have received little attention as potential anti-cancer therapeutics. Here we report self-assembling trimetallic cryptands (e.g. [L2(Metal)3]6+ where Metal = Cu2+, Zn2+ or Mn2+) which can encapsulate a range of anions and which show metal-dependent differences in chemical and biological reactivities. In cell studies, both [L2Cu3]6+ and [L2Zn3]6+ complexes are highly toxic to a range of human cancer cell lines and they show significant metal-dependent selective activity towards cancer cells compared to healthy, non-cancerous cells (by up to 2000-fold). The addition of different anions to the complexes (e.g. PO43-, SO42- or PhOPO32-) further alters activity and selectivity allowing the activity to be modulated via a self-assembly process. The activity is attributed to the ability to either bind or hydrolyse phosphate esters and mechanistic studies show differential and selective inhibition of multiple kinases by both [L2Cu3]6+ and [L2Zn3]6+ complexes but via different mechanisms.

Revisiting Bromohexitols as a Novel Class of Microenvironment-Activated Prodrugs for Cancer Therapy.

Bromohexitols represent a potent class of DNA-alkylating carbohydrate chemotherapeutics that has been largely ignored over the last decades due to safety concerns. The limited structure-activity relationship data available reveals significant changes in cytotoxicity with even subtle changes in stereochemistry. However, no attempts have been made to improve the therapeutic window by rational drug design or by using a prodrug approach to exploit differences between tumour physiology and healthy tissue, such as acidic extracellular pH and hypoxia. Herein, we report the photochemical synthesis of highly substituted endoperoxides as key precursors for dibromohexitol derivatives and investigate their use as microenvironment-activated prodrugs for targeting cancer cells. One endoperoxide was identified to have a marked increased activity under hypoxic and low pH conditions, indicating that endoperoxides may serve as microenvironment-activated prodrugs.

Triazole-based, optically-pure metallosupramolecules; highly potent and selective anticancer compounds.

Functionalised triazole aldehydes are used in the highly selective self-assembly of water-compatible, optically pure, low symmetry Fe(ii)- and Zn(ii)-based metallohelices. Sub-micromolar antiproliferative activity is observed against various cancerous cell lines, accompanied by excellent selectivity versus non-cancerous cells and potential for synergistic combinatorial therapy with cisplatin.

Hypoxia-selective targeting by the bioreductive prodrug AQ4N in patients with solid tumors: results of a phase I study.

PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.

Inactivation of apaziquone by haematuria: implications for the design of phase III clinical trials against non-muscle invasive bladder cancer.

PURPOSE: Despite positive responses in phase II clinical trials, the bioreductive prodrug apaziquone failed to achieve statistically significant activity in non-muscle invasive bladder cancer in phase III trials. Apaziquone was administered shortly after transurethral resection and here we test the hypothesis that haematuria inactivates apaziquone. METHODS: HPLC analysis was used to determine the ability of human whole blood to metabolise apaziquone ex vivo. An in vitro model of haematuria was developed and the response of RT112 and EJ138 cells following a 1-h exposure to apaziquone was determined in the presence of urine plus or minus whole blood or lysed whole blood. RESULTS: HPLC analysis demonstrated that apaziquone is metabolised by human whole blood with a half-life of 78.6 ± 23.0 min. As a model for haematuria, incubation of cells in media containing up to 75% buffered (pH 7.4) urine and 25% whole blood was not toxic to cells for a 1-h exposure period. Whole blood (5% v/v) significantly (p < 0.01) reduced the potency of apaziquone in this experimental model. Lysed whole blood also significantly (p < 0.05) reduced cell growth, although higher concentrations were required to achieve an effect (15% v/v). CONCLUSIONS: The results of this study demonstrate that haematuria can reduce the potency of apaziquone in this experimental model. These findings impact upon the design of further phase III clinical trials and strongly suggest that apaziquone should not be administered immediately after transurethral resection of non-muscle invasive bladder cancer when haematuria is common.

Discovery of selective, antimetastatic and anti-cancer stem cell metallohelices via post-assembly modification.

Helicates and related metallofoldamers, synthesised by dynamic self-assembly, represent an area of chemical space inaccessible by traditional organic synthesis, and yet with potential for discovery of new classes of drug. Here we report that water-soluble, optically pure Fe(ii)- and even Zn(ii)-based triplex metallohelices are an excellent platform for post-assembly click reactions. By these means, the in vitro anticancer activity and most importantly the selectivity of a triplex metallohelix Fe(ii) system are dramatically improved. For one compound, a remarkable array of mechanistic and pharmacological behaviours is discovered: inhibition of Na+/K+ ATPase with potency comparable to the drug ouabain, antimetastatic properties (including inhibition of cell migration, re-adhesion and invasion), cancer stem cell targeting, and finally colonosphere inhibition competitive with the drug salinomycin.

Chemical synthesis and biological evaluation of a NAD(P)H:quinone oxidoreductase-1 targeted tripartite quinone drug delivery system.

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.