👤 Cieślak MJ

Ewing sarcoma (EwS) cell line culture largely relies on standard techniques, which do not recapitulate physiological conditions. Here, we report on a feasible and cost-efficient EwS cell culture technique with increased physiological relevance employing an advanced medium composition, reduced fetal calf serum, and spheroidal growth. Improved reflection of the transcriptional activity related to proliferation, hypoxia, and differentiation in EwS patient tumors was detected in EwS cells grown in this refined in vitro condition. Moreover, transcriptional signatures associated with the oncogenic activity of the EwS-specific FET::ETS fusion transcription factors in the refined culture condition were shifted from proliferative toward metabolic gene signatures. The herein-presented EwS cell culture technique with increased physiological relevance provides a broadly applicable approach for enhanced in vitro modeling relevant to advancing EwS research and the validity of experimental results. Show less

The aim of the UniProt Knowledgebase (UniProtKB; https://www.uniprot.org/) is to provide users with a comprehensive, high-quality and freely accessible set of protein sequences annotated with functional information. In this publication, we describe ongoing changes to our production pipeline to limit the sequences available in UniProtKB to high-quality, non-redundant reference proteomes. We continue to manually curate the scientific literature to add the latest functional data and use machine learning techniques. We also encourage community curation to ensure key publications are not missed. We provide an update on the automatic annotation methods used by UniProtKB to predict information for unreviewed entries describing unstudied proteins. Finally, updates to the UniProt website are described, including a new tab linking protein to genomic information. In recognition of its value to the scientific community, the UniProt database has been awarded Global Core Biodata Resource status. Show less

Title: Ruthenium(II) and copper(II) polyamine complexes as promising antitumor agents: synthesis, characterization, and biological evaluation. Abstract: Ruthenium or copper complexes have emerged as some of the most promising alternatives for the treatment of many types of cancer. They have enhanced activity, greater selectivity and reduced side effects compared to their predecessors, cisplatin and its analogues. On the other hand, polyamine metabolism is often deregulated in cancer, leading to increased intracellular concentrations of polyamines that promote cell proliferation, differentiation, and tumorigenesis. In the present work, we report the synthesis and characterization of a family of mono- and binuclear Ru(II) and Cu(II) complexes functionalized with polyamine ligands derived from norspermine. The computer-aided analysis of the electron paramagnetic resonance (EPR) spectra provided magnetic and dynamic parameters, which helped to identify prevalent Cu-N2 coordination in a partially distorted square planar geometry of the Cu(II) complexes and the flexibility of the complexes in solution, slowed down by both the complex size and the hydrophobic interactions between chains. In vitro studies focused on advanced prostate cancer have demonstrated that these new metal complexes present a high level of cytotoxicity against PC3 cells. Furthermore, these metallic compounds exhibit the ability to inhibit cell adhesion and migration while reducing intracellular reactive oxygen species levels, which are key factors of metastasis. Show less

Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Show less

The assumption that there was a "water problem" at the emergence of life-that the Hadean Ocean was simply too wet and salty for life to have emerged in it-is here subjected to geological and experimental reality checks. The "warm little pond" that would take the place of the submarine alkaline vent theory (AVT), as recently extolled in the journal Nature, flies in the face of decades of geological, microbiological and evolutionary research and reasoning. To the present author, the evidence refuting the warm little pond scheme is overwhelming given the facts that (i) the early Earth was a water world, (ii) its all-enveloping ocean was never less than 4 km deep, (iii) there were no figurative "Icelands" or "Hawaiis", nor even an "Ontong Java" then because (iv) the solidifying magma ocean beneath was still too mushy to support such salient loadings on the oceanic crust. In place of the supposed warm little pond, we offer a well-protected mineral mound precipitated at a submarine alkaline vent as life's womb: in place of lipid membranes, we suggest peptides; we replace poisonous cyanide with ammonium and hydrazine; instead of deleterious radiation we have the appropriate life-giving redox and pH disequilibria; and in place of messy chemistry we offer the potential for life's emergence from the simplest of geochemically available molecules and ions focused at a submarine alkaline vent in the Hadean-specifically within the nano-confined flexible and redox active interlayer walls of the mixed-valent double layer oxyhydroxide mineral, fougerite/green rust comprising much of that mound. Show less

Stimulation of plasma membrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR), locally increases the abundance of reactive oxygen species (ROS). These ROS then oxidize cysteine residues in proteins to potentiate downstream signaling. Spatial confinement of ROS is an important regulatory mechanism of redox signaling that enables the stimulation of different RTKs to oxidize distinct sets of downstream proteins. To uncover additional mechanisms that specify cysteines that are redox regulated by EGF stimulation, we performed time-resolved quantification of the EGF-dependent oxidation of 4200 cysteine sites in A431 cells. Fifty-one percent of cysteines were statistically significantly oxidized by EGF stimulation. Furthermore, EGF induced three distinct spatiotemporal patterns of cysteine oxidation in functionally organized protein networks, consistent with the spatial confinement model. Unexpectedly, protein crystal structure analysis and molecular dynamics simulations indicated widespread redox regulation of cryptic cysteine residues that are solvent exposed only upon changes in protein conformation. Phosphorylation and increased flux of nucleotide substrates served as two distinct modes by which EGF specified the cryptic cysteine residues that became solvent exposed and redox regulated. Because proteins that are structurally regulated by different RTKs or cellular perturbations are largely unique, these findings suggest that solvent exposure and redox regulation of cryptic cysteine residues contextually delineate redox signaling networks. Show less

Title: Photoactivated Osmium Arene Anticancer Complexes. Abstract: Half-sandwich Os-arene complexes exhibit promising anticancer activity, but their photochemistry has hardly been explored. To exploit the photocytotoxicity and photochemistry of Os-arenes, O,O-chelated complexes [Os(η6-p-cymene)(Curc)Cl] (OsCUR-1, Curc = curcumin) and [Os(η6-biphenyl)(Curc)Cl] (OsCUR-2), and N,N-chelated complexes [Os(η6-biphenyl)(dpq)I]PF6 (OsDPQ-2, dpq = pyrazino[2,3-f][1,10]phenanthroline) and [Os(η6-biphenyl)(bpy)I]PF6 (OsBPY-2, bpy = 2,2'-bipyridine), have been investigated. The Os-arene curcumin complexes showed remarkable photocytotoxicity toward a range of cancer cell lines (blue light IC50: 2.6-5.8 μM, photocytotoxicity index PI = 23-34), especially toward cisplatin-resistant cancer cells, but were nontoxic to normal cells. They localized mainly in mitochondria in the dark but translocated to the nucleus upon photoirradiation, generating DNA and mitochondrial damage, which might contribute toward overcoming cisplatin resistance. Mitochondrial damage, apoptosis, ROS generation, DNA damage, angiogenesis inhibition, and colony formation were observed when A549 lung cancer cells were treated with OsCUR-2. The photochemistry of these Os-arene complexes was investigated by a combination of NMR, HPLC-MS, high energy resolution fluorescence detected (HERFD), X-ray adsorption near edge structure (XANES) spectroscopy, total fluorescence yield (TFY) XANES spectra, and theoretical computation. Selective photodissociation of the arene ligand and oxidation of Os(II) to Os(III) occurred under blue light or UVA excitation. This new approach to the design of novel Os-arene complexes as phototherapeutic agents suggests that the novel curcumin complex OsCUR-2, in particular, is a potential candidate for further development as a photosensitizer for anticancer photoactivated chemotherapy (PACT). Show less

Life cannot emerge on a planet or moon without the appropriate electrochemical disequilibria and the minerals that mediate energy-dissipative processes. Here, it is argued that four minerals, olivine ([Mg>Fe]2SiO4), bridgmanite ([Mg,Fe]SiO3), serpentine ([Mg,Fe,]2-3Si2O5[OH)]4), and pyrrhotite (Fe(1-x)S), are an essential requirement in planetary bodies to produce such disequilibria and, thereby, life. Yet only two minerals, fougerite ([Fe2+6xFe3+6(x-1)O12H2(7-3x)]2+·[(CO2-)·3H2O]2-) and mackinawite (Fe[Ni]S), are vital-comprising precipitate membranes-as initial "free energy" conductors and converters of such disequilibria, i.e., as the initiators of a CO2-reducing metabolism. The fact that wet and rocky bodies in the solar system much smaller than Earth or Venus do not reach the internal pressure (≥23 GPa) requirements in their mantles sufficient for producing bridgmanite and, therefore, are too reduced to stabilize and emit CO2-the staple of life-may explain the apparent absence or negligible concentrations of that gas on these bodies, and thereby serves as a constraint in the search for extraterrestrial life. The astrobiological challenge then is to search for worlds that (i) are large enough to generate internal pressures such as to produce bridgmanite or (ii) boast electron acceptors, including imported CO2, from extraterrestrial sources in their hydrospheres. Show less

The development of both chemotherapeutic drug resistance as well as adverse side effects suggest that the current chemotherapeutic drugs remain ineffective in treating the various types of cancers. The development of new metallodrugs presenting anti-cancer activity is therefore needed. Ruthenium complexes have gained a great deal of interest due to their promising anti-tumour properties and reduced toxicity in vivo. This study highlighted the effective induction of cell death in a malignant melanoma cell by two novel bis-amino-phosphine ruthenium(II) complexes referred to as GA105 and GA113. The IC₅₀ concentrations were determined for both the complexes, the ligand and cisplatin, for comparison. Both complexes GA105 and GA113 displayed a high anti-cancer selectivity profile as they exhibited low IC₅₀ values of 6.72 µM and 8.76 µM respectively, with low toxicity towards a non-malignant human cell line. The IC₅₀ values obtained for both complexes were lower than that of cisplatin. The new complexes were more effective compared to the free ligand, GA103 (IC₅₀ = >20 µM). Morphological studies on treated cells induced apoptotic features, which with further studies could indicate an intrinsic cell death pathway. Additionally, flow cytometric analysis revealed that the mode of cell death of complex GA113 was apoptosis. The outcomes herein give further insight into the potential use of selected Ru(II) complexes as alternative chemotherapeutic drugs in the future. Show less

Functionalized carbon nanotubes are interesting, promising and unique delivery systems for anticancer drugs, which are now in the spotlight of nanomedicine. Connecting nanotubes with anticancer drugs or new compounds with anticancer properties aims at improving their stability, efficiency and reduces the toxic side effects of cancer treatment. In our research, we are interested in connecting functionalized MWCNTs-NH2 with [InH][trans-RuCl4(In)2], (KP1019) which is one of the most promising anticancer ruthenium(iii) drug candidates, known mainly as a cytotoxic agent for the treatment of platinum-resistant colorectal cancers. As a result of the amidation of MWCNTs (1), MWCNTs-NH2 (2) were obtained. Then, they were modified with [InH][RuCl4(In)2] (4) and the nanosystem [MWCNT-NH3+][RuCl4(In)2-] (3) was obtained. The characterization of the resulting products was performed using IR, Raman spectroscopy, thermal gravimetric, XRD, STEM-EDX, ESI-MS, ICP-MS, and XPS analyses. The cytotoxic activity has been tested on human lung carcinoma (A549), chronic myelogenous leukemia (K562) and human cervix carcinoma (HeLa) cells which showed the higher toxicity of the nanosystem than the ruthenium complex. Show less

Current anticancer drug discovery efforts focus on the identification of first-in-class compounds with a mode-of-action distinct from conventional DNA-targeting agents for chemotherapy. An emerging trend is the identification of endoplasmic reticulum (ER) targeting compounds that induce ER stress in cancer cells, leading to cell death. However, a limited pool of such compounds has been identified to date, and there are limited studies done on such compounds to allow for the rational design of ER stress-inducing agents. In our present study, we present a series of highly cytotoxic, ER stress-inducing Ru(II)-arene Schiff-Base (RAS) complexes, bearing iminoquinoline chelate ligands. We demonstrate that by structural modification to the iminoquinoline ligand, we could tune its π-acidity and influence reactive oxygen species (ROS) induction, switching between a ROS-mediated ER stress pathway activation and one that is not mediated by ROS induction. Our current study adds to the available ER stress inducers and shows how structural tuning could be used as a means to modulate the mode-of-action of such compounds. Show less

Four mononuclear [(L-L)₂ Ru(tatpp)]²⁺ and two dinuclear [(L-L)₂ Ru(tatpp)Ru(L-L)₂ ]⁴⁺ ruthenium(II) polypyridyl complexes (RPCs) containing the 9,11,20,22-tetraazatetrapyrido[3,2-a:2',3'-c:3'',2''-l:2''',3'''-n]pentacene (tatpp) ligand were synthesized, in which L-L is a chelating diamine ligand such as 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me₄ phen) or 4,7-diphenyl-1,10-phenanthroline (Ph₂ phen). These Ru-tatpp analogues all undergo reduction reactions with modest reducing agents, such as glutathione (GSH), at pH 7. These, plus several structurally related but non-redox-active RPCs, were screened for DNA cleavage activity, cytotoxicity, acetylcholinesterase (AChE) inhibition, and acute mouse toxicity, and their activities were examined with respect to redox activity and lipophilicity. All of the redox-active RPCs show single-strand DNA cleavage in the presence of GSH, whereas none of the non-redox-active RPCs do. Low-micromolar cytotoxicity (IC₅₀ ) against malignant H358, CCL228, and MCF7 cultured cell lines was mainly restricted to the redox-active RPCs; however, they were substantially less toxic toward nonmalignant MCF10 cells. The IC₅₀ values for AChE inhibition in cell-free assays and the acute toxicity of RPCs in mice revealed that whereas most RPCs show potent inhibitory action against AChE (IC₅₀ values <15 μm), Ru-tatpp complexes as a class are surprisingly well tolerated in animals relative to other RPCs. Show less

The in vitro cytotoxic properties of antimicrobial copper(II) complexes with 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-dipyridyl-1,10-phenanthroline (DIP) ligands and ruthenium(II) complexes coordinated with TMP or 2,9-dimethyl-1,10-phenanthroline ligands were investigated. Both copper(II) complexes were found to have similar inhibitory concentrations (IC₅₀~2-2.5μM). Their cytotoxicity was found to be necrotic, associated with cytoplasmic vacuolisation, rounding, detachment and lack of apoptosis-associated DNA fragmentation, in comparison to the apoptotic effects of cisplatin which demonstrate adherent cell enlargement or detachment, membrane blebbing and condensation. Antimicrobial ruthenium(II) complexes demonstrated a lower renal cytotoxicity than copper(II) complexes or cisplatin (IC₅₀>60μM). [Cu(DIP)(dach)](ClO₄)₂ and [Cu(TMP)(dach)](ClO₄)₂ (where dach=1,2-diaminocyclohexane) induced dihydroethidium-sensitive ROS and the cytotoxicity of both TMP and DIP coordinated copper(II) complexes was mitigated by catalase, highlighting a role of H₂O₂ generation in their mode of action. The cytotoxicity of either copper(II) complex was not affected by coincubation with organic cation transporter (OCT) inhibitors cimetidine or disopyramide, in contrast to cisplatin, suggesting a non-OCT dependent mode of uptake for the copper(II) complexes in human cells. Coincubation with copper sulfate reduced the cytotoxicity of [Cu(TMP)(dach)](ClO₄)₂ (3-6×). The TMP complex induced a greater degree of G2/M accumulation and micronuclei generation than the DIP complex, possibly attributable to its greater DNA binding affinity. These results highlight the potentially low genotoxicity of copper(II) complexes coordinated with TMP or DIP and polypyridyl ruthenium(II) complexes as potential antimicrobial agents. Show less

Multidrug resistance is a major impediment to chemotherapy and limits the efficacies of conventional anticancer drugs. A strategy to bypass multidrug resistance is to develop new drug candidates capable of inducing apoptosis-independent programmed cell death. However, cellular pathways implicated in alternative programmed cell death are not well-elucidated and multifactorial, making a target-based discovery approach a challenge. Here, we show that a coordination-directed three-component assembly and phenotypic screening strategy could be employed as a viable alternative for the identification of apoptosis-independent organoruthenium anticancer agents. Through an on-plate synthesis and screening of 195 organoruthenium complexes against apoptosis-sensitive and -resistant cancers, we identified two apoptosis-independent hits. Subsequent validation of the two hits showed a lack of induction of apoptotic biomarkers, a caspase-independent activity and an equal efficacy in both apoptosis-sensitive and -resistant colorectal cancers. This validated their apoptosis-independent modes-of-action, paving the way as potential candidates for the treatment of highly-refractory cancer phenotypes. Show less

Some of the largest improvements in clinical outcomes for patients with solid cancers observed over the past 3 decades have been from concurrent treatment with chemotherapy and radiotherapy (RT). The lethal effects of RT on cancer cells arise primarily from damage to DNA. Ruthenium (Ru) is a transition metal of the platinum group, with potentially less toxicity than platinum drugs. We postulated that ruthenium-arene complexes are radiosensitisers when used in combination with RT. We screened 14 ruthenium-arene complexes and identified AH54 and AH63 as supra-additive radiosensitisers by clonogenic survival assays and isobologram analyses. Both complexes displayed facial chirality. At clinically relevant doses of RT, radiosensitisation of cancer cells by AH54 and AH63 was p53-dependent. Radiation enhancement ratios for 5-10 micromolar drug concentrations ranged from 1.19 to 1.82. In p53-wildtype cells, both drugs induced significant G2 cell cycle arrest and apoptosis. Colorectal cancer cells deficient in DNA damage repair proteins, EME1 and MUS81, were significantly more sensitive to both agents. Both drugs were active in cancer cell lines displaying acquired resistance to oxaliplatin or cisplatin. Our findings broaden the potential scope for these drugs for use in cancer therapy, including combination with radiotherapy to treat colorectal cancer. Show less

p53 is a key tumor suppressor gene involved in key cellular processes and implicated in cancer therapy. However, it is inactivated in more than 50% of all cancers due to mutation or overexpression of its negative regulators. This leads to drug resistance and poor chemotherapeutic outcome as most clinical drugs act via a p53-dependent mechanism of action. An attractive strategy to circumvent this resistance would be to identify new anticancer drugs that act via p53-independent mode of action. In the present study, we identified 9 Ru (II)-Arene Schiff-base (RAS) complexes able to induce p53-independent cytotoxicity and discuss structural features that are required for their p53-independent activity. Increasing hydrophobicity led to an increase in cellular accumulation in cells with a corresponding increase in efficacy. We further showed that all nine complexes demonstrated p53-independent activity. This was despite significant differences in their physicochemical properties, suggesting that the iminoquinoline ligand, a common structural feature for all the complexes, is required for the p53-independent activity. Show less

Multidrug resistance (MDR) is a major impediment to the success of chemotherapy in many cancer types. One particular MDR mechanism is the inherent or acquired adaptation of the cellular survival pathways that render malignant cells resistant to apoptotic cell death. Since most drugs act through apoptosis, compounds capable of inducing alternative forms of programmed cell death (PCD) can potentially be harnessed to bypass MDR. We investigated two organoruthenium complexes, RAS-1H and RAS-1T, and demonstrated that although they both induced non-apoptotic PCD through ER stress pathways, their modes-of-action were drastically different despite modest structural variations. RAS-1T acted through ROS-mediated ER stress while RAS-1H was ROS-independent. We further showed that they were more efficacious against apoptosis-resistant cells compared to clinical drugs including oxaliplatin. This work provides the basis for underpinning ER stress modulation using metal complexes to bypass apoptosis resistance. Show less

The organometallic "half-sandwich" compound [Os(η(6)-p-cymene)(4-(2-pyridylazo)-N,N-dimethylaniline)I]PF6 is 49× more potent than the clinical drug cisplatin in the 809 cancer cell lines that we screened and is a candidate drug for cancer therapy. We investigate the mechanism of action of compound 1 in A2780 epithelial ovarian cancer cells. Whole-transcriptome sequencing identified three missense mutations in the mitochondrial genome of this cell line, coding for ND5, a subunit of complex I (NADH dehydrogenase) in the electron transport chain. ND5 is a proton pump, helping to maintain the coupling gradient in mitochondria. The identified mutations correspond to known protein variants (p.I257V, p.N447S, and p.L517P), not reported previously in epithelial ovarian cancer. Time-series RNA sequencing suggested that osmium-exposed A2780 cells undergo a metabolic shunt from glycolysis to oxidative phosphorylation, where defective machinery, associated with mutations in complex I, could enhance activity. Downstream events, measured by time-series reverse-phase protein microarrays, high-content imaging, and flow cytometry, showed a dramatic increase in mitochondrially produced reactive oxygen species (ROS) and subsequent DNA damage with up-regulation of ATM, p53, and p21 proteins. In contrast to platinum drugs, exposure to this organo-osmium compound does not cause significant apoptosis within a 72-h period, highlighting a different mechanism of action. Superoxide production in ovarian, lung, colon, breast, and prostate cancer cells exposed to three other structurally related organo-Os(II) compounds correlated with their antiproliferative activity. DNA damage caused indirectly, through selective ROS generation, may provide a more targeted approach to cancer therapy and a concept for next-generation metal-based anticancer drugs that combat platinum resistance. Show less

Reactive oxygen species (ROS) are well known to be involved in oncogene-mediated cellular transformation. However, the regulatory mechanisms underlying ROS generation in oncogene-transformed cells are unclear. In the present study, we found that oncogenic K-Ras induces ROS generation through activation of NADPH oxidase 1 (NOX1), which is a critical regulator for the K-Ras-induced cellular transformation. NOX1 was activated by K-Ras-dependent translocation of p47(phox), a subunit of NOX1 to plasma membrane. Of note, PKCδ, when it was activated by PDPK1, directly bound to the SH3-N domain of p47(phox) and catalyzed the phosphorylation on Ser348 and Ser473 residues of p47(phox) C-terminal in a K-Ras-dependent manner, finally leading to its membrane translocation. Notably, oncogenic K-Ras activated all MAPKs (JNK, ERK and p38); however, only p38 was involved in p47(phox)-NOX1-dependent ROS generation and consequent transformation. Importantly, K-Ras-induced activation of p38 led to an activation of PDPK1, which then signals through PKCδ, p47(phox) and NOX1. In agreement with the mechanism, inhibition of p38, PDPK1, PKCδ, p47(phox) or NOX1 effectively blocked K-Ras-induced ROS generation, anchorage-independent colony formation and tumor formation. Taken together, our findings demonstrated that oncogenic K-Ras activates the signaling cascade p38/PDPK1/PKCδ/p47(phox)/NOX1 for ROS generation and consequent malignant cellular transformation. Show less

The structural diversity of metal scaffolds makes them a viable alternative to traditional organic scaffolds for drug design. Combinatorial chemistry and multicomponent reactions, coupled with high-throughput screening, are useful techniques in drug discovery, but they are rarely used in metal-based drug design. We report the optimization and validation of a new combinatorial, metal-based, three-component assembly reaction for the synthesis of a library of 442 Ru-arene Schiff-base (RAS) complexes. These RAS complexes were synthesized in a one-pot, on-a-plate format using commercially available starting materials under aqueous conditions. The library was screened for their anticancer activity, and several cytotoxic lead compounds were identified. In particular, [(η6-1,3,5-triisopropylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)aniline)]Cl (4) displayed low micromolar IC50 values in ovarian cancers (A2780, A2780cisR), breast cancer (MCF7), and colorectal cancer (HCT116, SW480). The absence of p53 activation or changes in IC50 value between p53+/+ and p53-/- cells suggests that 4 and possibly the other lead compounds may act independently of the p53 tumor suppressor gene frequently mutated in cancer. Show less

The effect of the PPh3 group in the antitumor activity of some new organometallic ruthenium(II) complexes has been investigated. Several complexes of the type [Ru((II))(Cl)(PPh3)(Lig-N)], [Ru((II))(Cl)2(Lig-N)] (where Lig-N=pyridine derivate) and [Ru((II))(Cl)(PPh3)2], have been synthesized and characterized. A noticeable increment of the antitumor activity and cytotoxicity of the complexes due to the presence of PPh3 moiety has also been demonstrated, affording IC50 values of 5.2 μM in HL-60 tumor cell lines. Atomic force microscopy, circular dichroism and electrophoresis experiments have proved that these complexes can bind DNA resulting in a distortion of both secondary and tertiary structures. Ethidium bromide displacement fluorescence spectroscopy studies and viscosity measurements support that the presence of PPh3 group induces intercalation interactions with DNA. Indeed, crystallographic analysis, suggest that intra-molecular π-π interactions could be involved in the intercalation within DNA base pairs. Furthermore, high performance liquid chromatography mass spectrometry (HPLC-MS) studies have confirmed a strong interaction between ruthenium complexes and proteins (ubiquitin and potato carboxypeptidase inhibitor - PCI) including slower kinetics due to the presence of PPh3 moiety, which could have an important role in detoxification mechanism and others. Finally, ion mobility mass spectrometry (IMMS) experiments have proved that there is no significant change in the gas phase structural conformation of the proteins owing to their bonding to ruthenium complexes. Show less

Various interaction modes between a group of six ruthenium polypyridyl complexes and DNA have been studied using a number of spectroscopic techniques. Five mononuclear species were selected with formula [Ru(tpy)L(1)L(2)]((2-n)+), and one closely related dinuclear cation of formula [{Ru(apy)(tpy)}(2){mu-H(2)N(CH(2))(6)NH(2)}](4+). The ligand tpy is 2,2':6',2''-terpyridine and the ligand L(1) is a bidentate ligand, namely, apy (2,2'-azobispyridine), 2-phenylazopyridine, or 2-phenylpyridinylmethylene amine. The ligand L(2) is a labile monodentate ligand, being Cl(-), H(2)O, or CH(3)CN. All six species containing a labile L(2) were found to be able to coordinate to the DNA model base 9-ethylguanine by (1)H NMR and mass spectrometry. The dinuclear cationic species, which has no positions available for coordination to a DNA base, was studied for comparison purposes. The interactions between a selection of four representative complexes and calf-thymus DNA were studied by circular and linear dichroism. To explore a possible relation between DNA-binding ability and toxicity, all compounds were screened for anticancer activity in a variety of cancer cell lines, showing in some cases an activity which is comparable to that of cisplatin. Comparison of the details of the compound structures, their DNA binding, and their toxicity allows the exploration of structure-activity relationships that might be used to guide optimization of the activity of agents of this class of compounds. Show less

The diaminocyclohexane platinum (Pt(DACH)) derivatives ormaplatin and oxaliplatin have caused severe and dose-limiting peripheral sensory neurotoxicity in a clinical trial. We hypothesized that this toxicity could vary in relation to the biotransformation and stereochemistry of these Pt(DACH) derivatives. We prepared pure R,R and S,S enantiomers of ormaplatin (Pt(DACH)Cl4), oxaliplatin (Pt(DACH)oxalato) and their metabolites (Pt(DACH)Cl2 and Pt(DACH)methionine) and assessed their peripheral sensory neurotoxicity and tissue distribution in the rat and in vitro anti-tumour activity in human ovarian carcinoma cell lines. The R,R enantiomers of Pt(DACH)Cl4, Pt(DACH)oxalato and Pt(DACH)Cl2, induced peripheral sensory neurotoxicity at significantly lower cumulative doses (18 +/- 5.7 vs 32 +/- 2.3 micromol kg(-1); P < 0.01) and at earlier times (4 +/- 1 vs 6.7 +/- 0.6 weeks; P = 0.016) during repeat-dose treatment than the S,S enantiomers. Pt(DACH)methionine enantiomers showed no biological activity. There was no difference between Pt(DACH) enantiomers in the platinum concentration in sciatic nerve, dorsal root ganglia, spinal cord, brain or blood at the end of each experiment. Three human ovarian carcinoma cell lines (41 M, 41 McisR and SKOV-3) showed no (or inconsistent) chiral discrimination in their sensitivity to Pt(DACH) enantiomers, whereas two cell lines (CH-1 and CH-1cisR) showed modest enantiomeric selectivity favouring the R,R isomer (more active). In conclusion, Pt(DACH) derivatives exhibit enantiomeric-selective peripheral sensory neurotoxicity during repeated dosing in rats favouring S,S isomers (less neurotoxic). They exhibited less chiral discrimination in their accumulation within peripheral nerves and in vitro anti-tumour activity. Show less

The use of molecular biological methodologies has provided a greater understanding of the cytotoxic effects of cisplatin and the underlying mechanisms of tumour cell resistance. Resistance to cisplatin is often multifocal with plasma membrane, cytosolic and nuclear components. Cisplatin-DNA adducts appear to be recognised by specific damage recognition proteins. Proteins associated with the transport of platinum through plasma membranes and genes associated with cisplatin resistance appear to be close to being elucidated. Current Phase I and Phase II clinical trials with platinum-containing complexes largely focus on the 1,2 diaminocyclohexane (DACH) carrier ligand, the dicarboxylatocyclobutane leaving group and complexes which circumvent cisplatin resistance in murine leukaemia models. At present, the trials are at too early a stage to allow comment on their clinical utility and, consequently, the relevance of the murine leukaemia-based preclinical observations. On the horizon, orally active platinum (IV) ammine/amine dicarboxylate dichloride coordination complexes with preclinical toxicological profiles similar to carboplatin should enter clinical trial in the next year. Show less