👤 Stanković D

[Ru(η(6)-p-cym)Cl{dpa(CH2)4COOEt}][PF6] (cym=cymene; dpa=2,2'-dipyridylamine; complex 2) was prepared and characterized by elemental analysis, IR and multinuclear NMR spectroscopy, as well as ESI-MS and X-ray structural analysis. The structural analog without a side chain [Ru(η(6)-p-cym)Cl(dpa)][PF6] (1) as well as 2 were investigated in vitro against 518A2, SW480, 8505C, A253 and MCF-7 cell lines. Complex 1 is active against all investigated tumor cell lines while the activity of compound 2 is limited only to caspase 3 deficient MCF-7 breast cancer cells, however, both are less active than cisplatin. As CD4(+)Th cells are necessary to trigger all the immune effector mechanisms required to eliminate tumor cells, besides testing the in vitro antitumor activity of 1 and 2, the effect of ruthenium(II) complexes on the cells of the adaptive immune system have also been evaluated. Importantly, complex 1 applied in concentrations which were effective against tumor cells did not affect immune cell viability, nor did exert a general immunosuppressive effect on cytokine production. Thus, beneficial characteristics of 1 might contribute to the overall therapeutic properties of the complex. Show less

Transcription inhibition by platinum anticancer drugs is an important component of their mechanism of action. Phenanthriplatin, a cisplatin derivative containing phenanthridine in place of one of the chloride ligands, forms highly potent monofunctional adducts on DNA having a structure and spectrum of anticancer activity distinct from those of the parent drug. Understanding the functional consequences of DNA damage by phenanthriplatin for the normal functions of RNA polymerase II (Pol II), the major cellular transcription machinery component, is an important step toward elucidating its mechanism of action. In this study, we present the first systematic mechanistic investigation that addresses how a site-specific phenanthriplatin-DNA d(G) monofunctional adduct affects the Pol II elongation and transcriptional fidelity checkpoint steps. Pol II processing of the phenanthriplatin lesion differs significantly from that of the canonical cisplatin-DNA 1,2-d(GpG) intrastrand cross-link. A majority of Pol II elongation complexes stall after successful addition of CTP opposite the phenanthriplatin-dG adduct in an error-free manner, with specificity for CTP incorporation being essentially the same as for undamaged dG on the template. A small portion of Pol II undergoes slow, error-prone bypass of the phenanthriplatin-dG lesion, which resembles DNA polymerases that similarly switch from high-fidelity replicative DNA processing (error-free) to low-fidelity translesion DNA synthesis (error-prone) at DNA damage sites. These results provide the first insights into how the Pol II transcription machinery processes the most abundant DNA lesion of the monofunctional phenanthriplatin anticancer drug candidate and enrich our general understanding of Pol II transcription fidelity maintenance, lesion bypass, and transcription-derived mutagenesis. Because of the current interest in monofunctional, DNA-damaging metallodrugs, these results are of likely relevance to a broad spectrum of next-generation anticancer agents being developed by the medicinal inorganic chemistry community. 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

Three new ruthenium(II) polypyridyl complexes [Ru(phen)2BrIPC](2+) (1), [Ru(bpy)2 BrIPC](2+) (2) and [Ru(dmb)2BrIPC](2+) (3) where, BrIPC = (6-bromo-3-(1H-imidazo[4,5-f] [1,10]-phenanthroline, phen = 1,10-phenanthroline, bpy = 2,2' bipyridine, dmb = 4,4'-dimethyl 2,2' bipyridine, were synthesised and characterised. DNA-binding nature was investigated by spectroscopic titrations and mode of binding was assessed by viscosity measurements. The DNA-binding constants Kb of complexes 1, 2 and 3 were determined to be in the order of 10(5). Experimental results showed that these complexes interact with CT-DNA by intercalative mode. Photocleavage and antimicrobial activities were complex concentration dependent, at high concentration, high activity and vice versa. MTT assay was performed on HeLa cell lines, IC50 values of complexes in the order of 3 > 2 > 1 > cisplatin. From comet assay, cellular uptake studies, we observed that complexes could enter into the cell membrane and accumulate inside the nucleus. Molecular docking studies support the DNA binding affinity with hydrogen bonding and van der Waals attractions between base pairs and phosphate backbone of DNA with metal complexes. Show less

Azocarboxamide (azcH) has been combined for the first time with [Ru-Cym] to generate metal complexes with N,N- and N,O-coordination mode, [(Cym)Ru(azc)Cl] and [(Cym)Ru(azcH)Cl](+) [PF6 ](-). Geometric and electronic structures of the complexes are reported along with their in vitro activities against different tumour cell lines and preliminary results on solution chemistry. Compound [(Cym)Ru(azc)Cl] exhibited remarkable cytotoxic properties. It was cell-type specific and had comparable IC50 values towards both cancer cells and their drug-resistant subline. A tenfold increase in the sensitivity towards [(Cym)Ru(azc)Cl] was noted for the tumour cells with depleted intracellular glutathione (GSH) level, suggesting the essential role of GSH in cell response to this compound. Show less

The reactions of [Ru(NO)Cl5](2-) with glycine (Gly), L-alanine (L-Ala), L-valine (L-Val), L-proline (L-Pro), D-proline (D-Pro), L-serine (L-Ser), L-threonine (L-Thr), and L-tyrosine (L-Tyr) in n-butanol or n-propanol afforded eight new complexes (1-8) of the general formula [RuCl3(AA-H)(NO)](-), where AA = Gly, L-Ala, L-Val, L-Pro, D-Pro, L-Ser, L-Thr, and L-Tyr, respectively. The compounds were characterized by elemental analysis, electrospray ionization mass spectrometry (ESI-MS), (1)H NMR, UV-visible and ATR IR spectroscopy, cyclic voltammetry, and X-ray crystallography. X-ray crystallography studies have revealed that in all cases the same isomer type (from three theoretically possible) was isolated, namely mer(Cl),trans(NO,O)-[RuCl3(AA-H)(NO)], as was also recently reported for osmium analogues with Gly, L-Pro, and D-Pro (see Z. Anorg. Allg. Chem. 2013, 639, 1590-1597). Compounds 1, 4, 5, and 8 were investigated by ESI-MS with regard to their stability in aqueous solution and reactivity toward sodium ascorbate. In addition, cell culture experiments in three human cancer cell lines, namely, A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon carcinoma), were performed, and the results are discussed in conjunction with the lipophilicity of compounds. Show less

Heteroleptic C^N cyclometalated iridium(iii) complexes incorporating a monostyryl/distyryl BODIPY ligand via acetylide bonds of 2,2'-bipyridine (bpy) with both absorption (ca. ε = 8.96 × 10⁴ M^-1 cm^-1, 9.89 × 10⁴ M^-1 cm^-1, and 7.89 × 10⁴ M^-1 cm^-1 at 664 nm, 644 nm, and 729 nm for Ir-2, Ir-3 and Ir-4, respectively) and fluorescence emission bands (ca. 624-794 nm for Ir-1, Ir-2, Ir-3 and Ir-4) in the near infra-red region (NIR) and exceptionally long-lived triplet excited states (τ = 156.5 μs for Ir-2) have been reported. Ir(ppy)₃ (Ir-0; ppy = 2-phenylpyridine) was used as reference, which gives the typical weak absorption in visible range (ε = 1.51 × 10⁴ M^-1 cm^-1 M^-1 cm^-1 at 385 nm). The nanosecond time-resolved transient absorption and DFT calculations proposed that styryl BODIPY-localized long lived ³IL states were populated for Ir-1, Ir-2, Ir-3 and Ir-4 (τ_T = 106.6 μs, 156.5 μs, 92.5 μs and 31.4 μs, respectively) upon photoexcitation. The complexes were used as triplet photosensitizers for singlet oxygen (¹O₂) mediated photooxidation of 1,5-dihydronaphthalene to produce juglone. The ¹O₂ quantum yields (Φ_Δ) of Ir-1 (0.53) and Ir-2 (0.81) are ca. 9-fold of Ir-3 (0.06) and 40-fold of Ir-4 (0.02), respectively. Ir-2 has high molar absorption coefficient at 664 nm, moderate fluorescence in the NIR region, and high singlet oxygen quantum yield (Φ_Δ = 0.81), exhibits predominate photocytotoxicity over dark cytotoxicity in LLC cells (lung cancer cells) upon irradiation, making it potentially suitable for use in in vivo photodynamic therapy (PDT). Our results are useful for preparation of transition metal complexes that show strong absorption of visible light in the NIR region with long-lived triplet excited states and for the application of these complexes in photocatalysis and theranostics such as simultaneous photodynamic therapy (PDT) and luminescent bioimaging. Show less

Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells. Show less

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research. Show less

KP1339 is a promising ruthenium-based anticancer compound in early clinical development. This study aimed to test the effects of KP1339 on the in vitro and in vivo activity of the multi-kinase inhibitor sorafenib, the current standard first-line therapy for advanced hepatoma. Anticancer activity of the parental compounds as compared to the drug combination was tested against a panel of cancer cell lines with a focus on hepatoma. Combination of KP1339 with sorafenib induced in the majority of all cases distinctly synergistic effects, comprising both sorafenib-resistant as well as sorafenib-responsive cell models. Several mechanisms were found to underlie these multifaceted synergistic activities. Firstly, co-exposure induced significantly enhanced accumulation levels of both drugs resulting in enhanced apoptosis induction. Secondly, sorafenib blocked KP1339-mediated activation of P38 signalling representing a protective response against the ruthenium drug. In addition, sorafenib treatment also abrogated KP1339-induced G2/M arrest but resulted in check point-independent DNA-synthesis block and a complete loss of the mitotic cell populations. The activity of the KP1339/sorafenib combination was evaluated in the Hep3B hepatoma xenograft. KP1339 monotherapy led to a 2.4-fold increase in life span and, thus, was superior to sorafenib, which induced a 1.9-fold prolonged survival. The combined therapy further enhanced the mean survival by 3.9-fold. Synergistic activity was also observed in the VM-1 melanoma xenograft harbouring an activating braf mutation. Together, our data indicate that the combination of KP1339 with sorafenib displays promising activity in vitro and in vivo especially against human hepatoma models. Show less

In an endeavor toward the development of metal-based anticancer drugs, we present here the design, synthesis and characterization of three ruthenium(II) functionalized phenanthroline complexes with extended π-conjugation. These complexes have been shown to act as promising CT-DNA intercalators as evidenced by UV-visible, luminescence, emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide and salt dependent studies. All three complexes [Ru(Hdpa)2PPIP](2+) (1), [Ru(Hdpa)2PIP](2+) (2), [Ru(Hdpa)24HEPIP](2+) (3) clearly demonstrated that they can bind to DNA through the intercalation mode. Cell viability experiments indicated that all complexes showed significant dose dependent cytotoxicity in selected cell lines. The apoptosis and cell cycle arrest were also investigated. The complexes were docked into DNA-base-pairs using the 'GOLD' (Genetic Optimization for Ligand Docking), docking program. Show less

An efficient drug delivery strategy is presented for novel anticancer amphiphilic ruthenium anionic complexes, based on the formation of stable nanoparticles with the cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP). This strategy is aimed at ensuring high ruthenium content within the formulation, long half-life in physiological media, and enhanced cell uptake. An in-depth microstructural characterization of the aggregates obtained mixing the ruthenium complex and the phospholipid carrier at 50/50 molar ratio is realized by combining a variety of techniques, including dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR), electron paramagnetic resonance (EPR), and zeta potential measurements. The in vitro bioactivity profile of the Ru-loaded nanoparticles is investigated on human and non-human cancer cell lines, showing IC(50) values in the low μM range against MCF-7 and WiDr cells, that is, proving to be 10-20-fold more active than AziRu, a previously synthesized NAMI-A analog, used for control. Fluorescence microscopy studies demonstrate that the amphiphilic Ru-complex/DOTAP formulations, added with rhodamine-B, are efficiently and rapidly incorporated in human MCF-7 breast adenocarcinoma cells. The intracellular fate of the amphiphilic Ru-complexes was investigated in the same in vitro model by means of an ad hoc designed fluorescently tagged analog, which exhibited a marked tendency to accumulate within or in proximity of the nuclei. Show less

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 ≫ 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides. Show less

In the present study, the interaction between GC-rich sequence of bcl-2 gene P1 promoter (Pu39) and two ruthenium (II) polypyridyl complexes, [Ru(bpy)₂(tip)]²⁺ (1) and [Ru(phen)₂(tip)]²⁺ (2), was investigated by UV-Visible, fluorescence spectroscopy, circular dichroism, fluorescence resonance energy transfer melting assay and polymerase chain reaction stop assay. Those experimental results indicated that the two complexes can effectively stabilize the G-quadruplex of Pu39. It was found that the complex 2 exhibited greater cytotoxic activity than 1 against human Hela cells and can enter into Hela cells in a short period of time to effectively induce apoptosis of cells. Further experiments found that complexes 1 and 2 had as potent inhibitory effects on ECV-304 cell migration as suramin. Those noteworthy results provide new insights into the development of anticancer agents for targeting G-quadruplex DNA. Show less

Cisplatin forms intrastrand cross-links on DNA and is a widely used chemotherapy agent. Among human translesion DNA polymerases, Pol-η can bypass cisplatin adducts. The crystal structure of human Pol-η in complex with a DNA template with a cisplatin lesion is now presented. In addition to the larger active site, the structure reveals specific interactions with the adduct by residues that are not conserved in other translesion polymerases. Show less

The in vitro antiproliferative activity of the title compound on five tumor cell lines shows preference for the colon-rectal tumor HCT116, IC(50) = 13.98 μM, followed by breast MCF7 (19.58 μM) and ovarian A2780 (23.38 μM) cell lines; human glioblastoma U-87 and lung carcinoma A549 are less sensitive. A commercial curcumin reagent, also containing demethoxy and bis-demethoxy curcumin, was used to synthesize the title compound, and so (p-cymene)Ru(demethoxy-curcuminato)chloro was also isolated and chemically characterized. The crystal structure of the title compound shows (1) the chlorine atom linking two neighboring complexes through H-bonds with two O(hydroxyl), forming an infinite two-step network; (2) significant twist in the curcuminato, 20° between the planes of the two phenyl rings. This was also seen in the docking of the Ru-complex onto a rich guanine B-DNA decamer, where a Ru-N7(guanine) interaction is detected. This Ru-N7(guanine) interaction is also seen with ESI-MS on a Ru-complex-guanosine derivative. Show less

RuII(arene) complexes have been shown to be promising anticancer agents, capable of overcoming major drawbacks of currently used chemotherapeutics. We have synthesized RuII(η6-arene) compounds carrying bioactive flavonol ligands with the aim to obtain multitargeted anticancer agents. To validate this concept, studies on the mode of action of the complexes were conducted which indicated that they form covalent bonds to DNA, have only minor impact on the cell cycle, but inhibit CDK2 and topoisomerase IIα in vitro. The cytotoxic activity was determined in human cancer cell lines, resulting in very low IC50 values as compared to other RuII(arene) complexes and showing a structure-activity relationship dependent on the substitution pattern of the flavonol ligand. Furthermore, the inhibition of cell growth correlates well with the topoisomerase inhibitory activity. Compared to the flavonol ligands, the RuII(η6-p-cymene) complexes are more potent antiproliferative agents, which can be explained by potential multitargeted properties. Show less

Mitochondria extrude protons across their inner membrane to generate the mitochondrial membrane potential (ΔΨ(m)) and pH gradient (ΔpH(m)) that both power ATP synthesis. Mitochondrial uptake and efflux of many ions and metabolites are driven exclusively by ΔpH(m), whose in situ regulation is poorly characterized. Here, we report the first dynamic measurements of ΔpH(m) in living cells, using a mitochondrially targeted, pH-sensitive YFP (SypHer) combined with a cytosolic pH indicator (5-(and 6)-carboxy-SNARF-1). The resting matrix pH (∼7.6) and ΔpH(m) (∼0.45) of HeLa cells at 37 °C were lower than previously reported. Unexpectedly, mitochondrial pH and ΔpH(m) decreased during cytosolic Ca(2+) elevations. The drop in matrix pH was due to cytosolic acid generated by plasma membrane Ca(2+)-ATPases and transmitted to mitochondria by P(i)/H(+) symport and K(+)/H(+) exchange, whereas the decrease in ΔpH(m) reflected the low H(+)-buffering power of mitochondria (∼5 mm, pH 7.8) compared with the cytosol (∼20 mm, pH 7.4). Upon agonist washout and restoration of cytosolic Ca(2+) and pH, mitochondria alkalinized and ΔpH(m) increased. In permeabilized cells, a decrease in bath pH from 7.4 to 7.2 rapidly decreased mitochondrial pH, whereas the addition of 10 μm Ca(2+) caused a delayed and smaller alkalinization. These findings indicate that the mitochondrial matrix pH and ΔpH(m) are regulated by opposing Ca(2+)-dependent processes of stimulated mitochondrial respiration and cytosolic acidification. Show less

The synthesis and characterization of a number of organometallic ruthenium(II) complexes containing a series of bidentate thiosemicarbazone ligands derived from piperonal is reported. The structure of compounds have been confirmed by spectroscopic analysis (IR and NMR) as well as X-ray crystallographic analysis of [(η⁶-p-cymene)Ru(pPhTSC)Cl]Cl (4) (pPhTSC is piperonal-N(4)-phenylthiosemicarbazone). The interaction of the complexes ([(η⁶-p-cymene)Ru(pEtTSC)Cl]Cl) (3) (pEtTSC is piperonal-N(4)-ethylthiosemicarbazone) and 4 with calf thymus DNA, human serum albumin (HSA) and pBR322 plasmid DNA were studied by spectroscopic, gel electrophoresis and hydrodynamic methods. The apparent binding constant for the interaction with DNA was determined to be 3.97×10³ M⁻¹ and 4.07×10³ M⁻¹ at 293 K for 3 and 4 respectively. The complexes bind strongly to HSA with binding constants of 2.94×10⁴ M⁻¹ and 12.2×10⁴ M⁻¹ at 296 K for 3 and 4 respectively. The in vitro anticancer activity of 3 and 4 has been evaluated against two human colon cancer cell line (HCT-116 and Caco-2) with IC50 values in the range of 26–150 μM. Both 3 and 4 show good activity as a catalytic inhibitor of human topoisomerase II at concentrations as low as 20 μM. The proficiency of 3 and 4 to act as antibacterial agents was also evaluated against six pathogenic bacterial strains with the best activity seen against Gram-positive strains. Show less

In this study, two isomeric ruthenium(II) complexes [Ru(bpy)(2)(p-mopip)](2+) (1) and [Ru(bpy)(2)(o-mopip)](2+) (2) (bpy = 2, 2-bipyridine; L: p-mopip = 2-(4-methoxylphenyl) imidazo [4,5-f][1,10]phenanthroline, o-mopip = 2-(2-methoxylphenyl) imidazo[4,5-f][1,10] phenan-throline) contained -OCH(3) at different positions on the phenyl ring and their enantiomers Λ-1, -2 and Δ-1, -2 displayed different properties. The cell viability of these ruthenium(II) complexes was evaluated by MTT, and complex Λ-1 has shown significant higher anticancer potency than Δ-1 against all the cell lines screened. Fluorescence microscopy and flow cytometric analyses demonstrated that complex Λ-1 was able to induce apoptosis. The interactions of complexes Λ-1, 1, and Δ-1 with bovine serum albumin (BSA) were investigated by fluorescence and circular dichroism (CD) measurements. The fluorescence quenching mechanism of BSA by complexes Λ-1, 1, and Δ-1 was determined to be a static process, and the apparent binding constant K(a) values is as follows: Λ-1 >1 > Δ-1. The number of binding sites n for all these complexes was 1. The result of CD showed that the secondary structure of BSA molecules was changed in the presence of the ruthenium(II) complex. Show less

We report the synthesis and characterisation of 32 half sandwich phenylazopyridine Os(II) arene complexes [Os(η(6)-arene)(phenylazopyridine)X](+) in which X is chloride or iodide, the arene is p-cymene or biphenyl and the pyridine and phenyl rings contain a variety of substituents (F, Cl, Br, I, CF(3), OH or NO(2)). Ten X-ray crystal structures have been determined. Cytotoxicity towards A2780 human ovarian cancer cells ranges from high potency at nanomolar concentrations to inactivity. In general the introduction of an electron-withdrawing group (e.g. F, Cl, Br or I) at specific positions on the pyridine ring significantly increases cytotoxic activity and aqueous solubility. Changing the arene from p-cymene to biphenyl and the monodentate ligand X from chloride to iodide also increases the activity significantly. Activation by hydrolysis and DNA binding appears not to be the major mechanism of action since both the highly active complex [Os(η(6)-bip)(2-F-azpy)I]PF(6) (9) and the moderately active complex [Os(η(6)-bip)(3-Cl-azpy)I]PF(6) (23) are very stable and inert towards aquation. Studies of octanol-water partition coefficients (log P) and subcellular distributions of osmium in A2780 human ovarian cancer cells suggested that cell uptake and targeting to cellular organelles play important roles in determining activity. Although complex 9 induced the production of reactive oxygen species (ROS) in A2780 cells, the ROS level did not appear to play a role in the mechanism of anticancer activity. This class of organometallic osmium complexes has new and unusual features worthy of further exploration for the design of novel anticancer drugs. Show less

Cationic ruthenium(II) pentamethylcyclopentadienyl benzenesulfonamide sandwich complexes have been synthesized and screened for enzymatic inhibition of the physiologically dominant carbonic anhydrase (CA) isozymes: human CA I and II, mitochondrial isozymes VA and VB, and the cancer-associated isozyme IX. The complexes demonstrated weaker binding to CAs compared with typical aromatic sulfonamides, inhibiting the enzyme at high nanomolar concentrations. An in vitro cytotoxic evaluation of the complexes was also undertaken against a range of tumorigenic cell lines and a healthy human cell line. Complexes inhibited the growth of cancerous cells at low micromolar concentrations while expressing lower levels of toxicity towards the normal human cell line. Factors influencing the synthesis, cytotoxicity, and enzyme affinity for this series of organometallic complexes are discussed. Show less

Reaction of 3-pyridinehydroxamic acid and 4-pyridinehydroxamic acid (3-pyha and 4-pyha) with either [NBu4][RuCl4(dmso-S)2] or [(dmso)2H][RuCl4(dmso-S)2] (dmso is dimethyl sulfoxide) in acetone afforded three new ruthenium(III) dimethyl sulfoxide pyridinehydroxamic acid complexes: [NBu4][trans-RuCl4(dmso-S)(4-pyha)] x CH3CO CH3 (1), [3-pyhaH][trans-RuCl4(dmso-S)(3-pyha)] (2) and [4-pyhaH][trans-RuCl4(dmso-S)(4-pyha)] (3). The solid-state structure of [NBu4][trans-RuCl4(dmso-S)(4-pyha)] x CH3COCH3 (1) was determined by X-ray crystallography. 2 and 3 were pharmacologically evaluated for their in vitro cytotoxicity, their ability to inhibit cell invasion and their gelatinase activity. 2 and 3 were devoid of cytotoxicity against the cell lines tested. 2 inhibited invasion of the highly invasive MDA-MB-231 cells to a much greater extent than 3. Contrary to expectations, neither 2 nor 3 had any inhibitory effect on matrix metalloproteinase (MMP) production and/or activity and in fact 3 was found to enhance the production and/or activity of both MMP-2 and MMP-9. Show less

The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin. Show less

Intracellular pH (pHi) has an important role in the maintenance of normal cell function, and hence this parameter has to be tightly controlled within a narrow range, largely through the activity of transporters located at the plasma membrane. These transporters can be modulated by endogenous or exogenous molecules as well as, in some pathological situations, leading to pHi changes that have been implicated in both cell proliferation and cell death. Whereas intracellular alkalinization seems to be a common feature of proliferative processes, the precise role of pHi in apoptosis is still unclear. The present review gathers the most recent advances along with previous data on both the origin and the role of pHi alterations in apoptosis and highlights the major concerns that merit further research in the future. Special attention is given to the possible role played by pHi-regulating transporters. Show less

Purpose

The cytotoxicity, intracellular accumulation and DNA adduct formation of the ruthenium complex imidazolium trans-imidazoledimethylsulfoxide tetrachlororuthenate (ImH[ trans-RuCl(4)(DMSO)Im], Nami-A) were compared in vitro with those of cisplatin in four human tumor cell lines: Igrov-1, 2008, MCF-7, and T47D.

Methods

Cytotoxicity was assessed in vitro using a growth inhibition assay. Accumulation was determined by flameless atomic absorption spectroscopy (AAS). GG and AG intrastrand adducts were measured using the (32)P-postlabeling assay.

Results

Nami-A was on average 1053 times less cytotoxic than cisplatin. The cytotoxicity of cisplatin was linearly related to both intracellular platinum accumulation and DNA binding, while the cytotoxicity of Nami-A was significantly related only to DNA binding and not to intracellular ruthenium accumulation. The levels of accumulation of Nami-A measured as ruthenium and of cisplatin measured as platinum were correlated linearly with the incubation concentration over a concentration range of 0 to 600 micro M of both drugs. Ruthenium intracellular accumulation and DNA binding were on average 4.8 and 42 times less, respectively, than those of cisplatin. In addition, the numbers of GG and AG intrastrand adducts induced by Nami-A were 418 and 51 times fewer, respectively. Nami-A and cisplatin had the same binding capacity to calf thymus DNA. Nami-A was 25-40% less bound to cellular proteins than cisplatin.

Conclusions

There was no saturation of the uptake and DNA binding capacity of either Nami-A or cisplatin. Furthermore, the low binding of Nami-A to cellular DNA cannot simply be explained by a lower capacity to bind to DNA, because the absolute level of binding in vitro to calf thymus DNA was the same for Nami-A and cisplatin. Finally, the lower cytotoxicity of Nami-A on a molar basis than that of cisplatin can at least partly be explained by its reduced reactivity to DNA in intact cells. Show less

Previous work has shown platinum drugs to differ in their effects on the peripheral nervous system. To test whether their differential toxicity was due to differences in their partitioning into the peripheral nervous system, we correlated the hydrophobicity, reactivity, tissue accumulation and neurotoxicity of a series of eight platinum analogues. Neurotoxicity was detected by measuring sensory nerve conduction velocity (SNCV) in Wistar rats treated twice per week at the maximum tolerated dose. Tissue platinum concentrations were measured by inductively coupled plasma mass spectrometry. Hydrophobicity (log P) was measured using an octanol-aqueous shake-flask method. The half-life of platinum drug binding to plasma proteins in vitro was determined. The cumulative dose causing altered SNCV ranged from 15 to > 2050 μmol kg–1. Ranking of the compounds by their neurotoxic potency in rats (oxaliplatin >R,R -(DACH)PtC4> ormaplatin >S,S -(DACH)PtCl4>S,S -(DACH)Pt oxalato > cisplatin > carboplatin > JM216) correlated with the frequency of neurotoxicity in patients (r> 0.99;P< 0.05). Ranking the compounds by their peripheral nerve accumulation was cisplatin > carboplatin > oxaliplatin >R,R -(DACH)PtCl4≈S,S -(DACH)PtCl4and did not correlate with neurotoxicity. Log P ranged from – 2.53 to –0.16 but did not correlate with neurotoxicity. Log P correlated inversely with platinum accumulation in dorsal root ganglia (r2= 0.99;P = 0.04), sural nerve (r2= 0.85;P = 0.025), sciatic nerve (r2= 0.98;P = 0.0012), spinal cord (r2= 0.97, P = 0.018) and brain (r2= 0.98, P = 0.001). Reactivity correlated with neurotoxicity potency in rats (r2= 0.89, P = 0.0005) and with the frequency of neurotoxicity in patients (r2= 0.99, P = 0.0002). The hydrophilicity of platinum drugs correlates with platinum sequestration in the peripheral nervous system but not with neurotoxicity. Differences in the reactivity of platinum complexes accounts for some of the variation in their neurotoxicity. © 2000 Cancer Research Campaign Show less

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization. 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