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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

Background

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated.

Methods

We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer.

Results

The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated in common or unique ways.

Conclusions

These two new kinase or "Kin-OMIC" analyses add another dimension of important data about these frequently used breast cancer cell lines. This will assist researchers in selecting the most appropriate cell lines to use for breast cancer studies and provide context for the interpretation of the emerging results. Show less

In this paper a novel ligand debip (2-(4-N,N-diethylbenzenamine)1H-imidazo[4,5-f] [1, 10]phenanthroline) and its Ru(II) polypyridyl complexes [Ru(L)₂(debip)]²⁺, (L = phen (1), bpy (2) and dmb (3)) have been synthesized and characterized by spectroscopic techniques. The DNA binding studies for all these complexes were examined by absorption, emission, quenching studies, viscosity measurements and cyclic voltammetry. The light switching properties of complexes 1-3 have been evaluated. Molecular docking, Density Functional Theory (DFT) and time dependent DFT calculations were performed. The Ru(II) complexes exhibited efficient photocleavage activity against pBR322 DNA upon irradiation and exhibited good antimicrobial activity. Also investigated 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, lactate dehydrogenase (LDH) release assay and reactive oxygen species (ROS) against selected cancer cell lines (HeLa, PC3, Lancap, MCF-7 and MD-MBA 231). Show less

Catalase is well-known as an antioxidant dismutating H2O2 to O2 and H2O. However, catalases evolved when metabolism was largely sulfur-based, long before O2 and reactive oxygen species (ROS) became abundant, suggesting catalase metabolizes reactive sulfide species (RSS). Here we examine catalase metabolism of H2Sn, the sulfur analog of H2O2, hydrogen sulfide (H2S) and other sulfur-bearing molecules using H2S-specific amperometric electrodes and fluorophores to measure polysulfides (H2Sn; SSP4) and ROS (dichlorofluorescein, DCF). Catalase eliminated H2Sn, but did not anaerobically generate H2S, the expected product of dismutation. Instead, catalase concentration- and oxygen-dependently metabolized H2S and in so doing acted as a sulfide oxidase with a P50 of 20mmHg. H2O2 had little effect on catalase-mediated H2S metabolism but in the presence of the catalase inhibitor, sodium azide (Az), H2O2 rapidly and efficiently expedited H2S metabolism in both normoxia and hypoxia suggesting H2O2 is an effective electron acceptor in this reaction. Unexpectedly, catalase concentration-dependently generated H2S from dithiothreitol (DTT) in both normoxia and hypoxia, concomitantly oxidizing H2S in the presence of O2. H2S production from DTT was inhibited by carbon monoxide and augmented by NADPH suggesting that catalase heme-iron is the catalytic site and that NADPH provides reducing equivalents. Catalase also generated H2S from garlic oil, diallyltrisulfide, thioredoxin and sulfur dioxide, but not from sulfite, metabisulfite, carbonyl sulfide, cysteine, cystine, glutathione or oxidized glutathione. Oxidase activity was also present in catalase from Aspergillus niger. These results show that catalase can act as either a sulfide oxidase or sulfur reductase and they suggest that these activities likely played a prominent role in sulfur metabolism during evolution and may continue do so in modern cells as well. This also appears to be the first observation of catalase reductase activity independent of peroxide dismutation. Show less

AbstractWhile NMR and IR spectroscopic signatures and structural characteristics of low‐barrier hydrogen bond (LBHB) formation are well documented in the literature, direct measurement of the LBHB energy is difficult. Here, we show that solid‐state 17O NMR spectroscopy can provide unique information about the energy required to break a LBHB. Our solid‐state 17O NMR data show that the HB enthalpy of the O⋅⋅⋅H⋅⋅⋅N LBHB formed in crystalline nicotinic acid is only 7.7±0.5 kcal mol−1, suggesting that not all LBHBs are particularly strong. Show less

Five heteroleptic tris-diimine ruthenium(II) complexes [RuL(N^N)₂](PF₆)₂ (where L is 3,8-di(benzothiazolylfluorenyl)-1,10-phenanthroline and N^N is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 1,4,8,9-tetraazatriphenylene (tatp) (3), dipyrido[3,2-a:2',3'-c]phenazine (dppz) (4), or benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) (5), respectively) were synthesized. The influence of π-conjugation of the ancillary ligands (N^N) on the photophysical properties of the complexes was investigated by spectroscopic methods and simulated by density functional theory (DFT) and time-dependent DFT. Their ground-state absorption spectra were characterized by intense absorption bands below 350 nm (ligand L localized ¹π,π* transitions) and a featureless band centered at ∼410 nm (intraligand charge transfer (¹ILCT)/¹π,π* transitions with minor contribution from metal-to-ligand charge transfer (¹MLCT) transition). For complexes 4 and 5 with dppz and dppn ligands, respectively, broad but very weak absorption (ε < 800 M^-1 cm^-1) was present from 600 to 850 nm, likely emanating from the spin-forbidden transitions to the triplet excited states. All five complexes showed red-orange phosphorescence at room temperature in CH₂Cl₂ solution with decreased lifetimes and emission quantum yields, as the π-conjugation of the ancillary ligands increased. Transient absorption (TA) profiles were probed in acetonitrile solutions at room temperature for all of the complexes. Except for complex 5 (which showed dppn-localized ³π,π* absorption with a long lifetime of 41.2 μs), complexes 1-4 displayed similar TA spectral features but with much shorter triplet lifetimes (1-2 μs). Reverse saturable absorption (RSA) was demonstrated for the complexes at 532 nm using 4.1 ns laser pulses, and the strength of RSA decreased in the order: 2 ≥ 1 ≈ 5 > 3 > 4. Complex 5 is particularly attractive as a broadband reverse saturable absorber due to its wide optical window (430-850 nm) and long-lived triplet lifetime in addition to its strong RSA at 532 nm. Complexes 1-5 were also probed as photosensitizing agents for in vitro photodynamic therapy (PDT). Most of them showed a PDT effect, and 5 emerged as the most potent complex with red light (EC₅₀ = 10 μM) and was highly photoselective for melanoma cells (selectivity factor, SF = 13). Complexes 1-5 were readily taken up by cells and tracked by their intracellular luminescence before and after a light treatment. Diagnostic intracellular luminescence increased with increased π-conjugation of the ancillary N^N ligands despite diminishing cell-free phosphorescence in that order. All of the complexes penetrated the nucleus and caused DNA condensation in cell-free conditions in a concentration-dependent manner, which was not influenced by the identity of N^N ligands. Although the mechanism for photobiological activity was not established, complexes 1-5 were shown to exhibit potential as theranostic agents. Together the RSA and PDT studies indicate that developing new agents with long intrinsic triplet lifetimes, high yields for triplet formation, and broad ground-state absorption to near-infrared (NIR) in tandem is a viable approach to identifying promising agents for these applications. Show less

Two [Ru(phen)₂ dppz]²⁺ derivatives (phen=1,10-phenantroline, dppz=dipyrido[3,2-a:2',3'-c]phenazine) with different functional groups on the dppz ligand [dppz-7,8-(OMe)₂ (1), dppz-7,8-(OH)₂ (2)] have been synthesized, characterized and investigated as photosensitizers (PSs) for photodynamic therapy (PDT) against cancer. Both complexes showed intense red phosphorescence and promising singlet oxygen (¹ O₂ ) quantum yields of 75 % (1) and 54 % (2) in acetonitrile. Complex 1 (logP_o/w =-0.52, 2.4 nmol Ru per mg protein) was found to be more lipophilic, having also a higher cellular uptake efficiency compared to 2 (logP_o/w =-0.20, 0.9 nmol Ru per mg protein). Complex 1 localized evenly in HeLa cells whereas 2, was mainly visualized in the cell membrane by confocal microscopy. In the dark, complex 1 (IC₅₀ =36.5 μm) was found to be more toxic than complex 2 (IC₅₀ >100 μm) on a HeLa cells monolayer. Importantly, in view of PDT applications, both complexes were found to be non-toxic in the dark towards multicellular HeLa spheroids (IC₅₀ >100 μm). Upon one-photon irradiation (420 nm, 9.27 J cm^-2 ), 1 exhibited higher phototoxicity (IC₅₀ =3.1 μm) than 2 (IC₅₀ =16.7 μm) on HeLa cell monolayers. When two-photon irradiation (800 nm, 9.90 J cm^-2 ) was applied, only 1 (IC₅₀ =9.5 μm) was found to be active toward HeLa spheroids. This study demonstrates that the functional group on the intercalative ligand has a strong influence on the cellular localization and anticancer activity of Ru^II polypyridyl complexes. Show less

Ruthenium(II) arene complexes of 1,4,7-triaza-9-phosphatricyclo[5.3.2.1]tridecane (CAP) were obtained. Cytotoxicity studies against cancer cell lines reveal higher activity than the corresponding PTA analogues and, in comparison to the effects on noncancerous cells, the complexes are endowed with a reasonable degree of cancer cell selectivity. Show less

A fundamental question in cell biology, under investigation for over six decades, is the structural organization of mitochondrial cristae. Long known to harbor electron transport chain proteins, crista membrane integrity is key to establishment of the proton gradient that drives oxidative phosphorylation. Visualization of cristae morphology by electron microscopy/tomography has provided evidence that cristae are tube-like extensions of the mitochondrial inner membrane (IM) that project into the matrix space. Reconciling ultrastructural data with the lipid composition of the IM provides support for a continuously curved cylindrical bilayer capped by a dome-shaped tip. Strain imposed by the degree of curvature is relieved by an asymmetric distribution of phospholipids in monolayer leaflets that comprise cristae membranes. The signature mitochondrial lipid, cardiolipin (~18% of IM phospholipid mass), and phosphatidylethanolamine (34%) segregate to the negatively curved monolayer leaflet facing the crista lumen while the opposing, positively curved, matrix-facing monolayer leaflet contains predominantly phosphatidylcholine. Associated with cristae are numerous proteins that function in distinctive ways to establish and/or maintain their lipid repertoire and structural integrity. By combining unique lipid components with a set of protein modulators, crista membranes adopt and maintain their characteristic morphological and functional properties. Once established, cristae ultrastructure has a direct impact on oxidative phosphorylation, apoptosis, fusion/fission as well as diseases of compromised energy metabolism. Show less

We report on the exploitation of a new tetrazole-substituted 1,10-phenanthroline and a 2,2′-bipyridine (bpy) ancillary ligand modified with an electron-donating group in cationic ruthenium complexes. This complex, placed in between two electrodes without any polymer, demonstrates high efficiency near-infrared (NIR) electroluminescence (EL). The comparison between bpy and its methyl-substituted ancillary ligand shows that the cationic Ru tetrazolate complex containing methyl groups exhibits a red shift in the EL wavelength from 620 to 800 nm compared to [Ru(bpy)3]2+ and an almost twofold reduction in the turn-on voltage, i.e., from 5 to 3 V, with respect to 5-tetrazole-1,10-phenanthroline. An external quantum efficiency of 0.95% for the dimethyl derivative is demonstrated, which is a remarkable result for non-doped NIR light electrochemical cells based on ruthenium polypyridyl. Show less

Herein we present a series of DCA-Ir(iii) co-drug complexes that preferentially accumulate in mitochondria and selectively cause cancer cell metabolic alterations and were found to act in synergy by sensitizing cancer cells for PDT to achieve cancer-specific enhanced two-photon PDT in the hypoxic environment of multicellular tumor spheroids. Show less

AbstractThe nucleotide excision repair system removes a wide variety ofDNAlesions from the human genome, including photoproducts induced by ultraviolet (UV) wavelengths of sunlight. A defining feature of nucleotide excision repair is its dual incision mechanism, in which two nucleolytic incision events on the damaged strand ofDNAat sites bracketing the lesion generate a damage‐containingDNAoligonucleotide and a single‐strandedDNAgap approximately 30 nucleotides in length. Although the early events of nucleotide excision repair, which include lesion recognition and the dual incisions, have been explored in detail and are reasonably well understood, the fate of the single‐strandedDNAgaps and excised oligonucleotide products of repair have not been as extensively examined. In this review, recent findings that address these less‐explored aspects of nucleotide excision repair are discussed and support the concept that postincision gap and excised oligonucleotide processing are critical steps in the cellular response toDNAdamage induced byUVlight and other environmental carcinogens. Defects in these latter stages of repair lead to cell death and otherDNAdamage signaling responses and may therefore contribute to a number of human disease states associated with exposure toUVwavelengths of sunlight, including skin cancer, aging and autoimmunity. Show less

Hypoxia inducible factor 1 alpha (HIF-1) orchestrates cellular adaptation to low oxygen and nutrient deprived environment and drives progression to malignancy in human solid cancers. Its canonical regulation involves prolyl hydroxylases (PHDs), which in normoxia induce degradation, whereas in hypoxia allow stabilization of HIF-1. However, in certain circumstances HIF-1 regulation goes beyond the actual external oxygen levels and involves PHD-independent mechanisms. Here we gather and discuss the evidence on the non-canonical HIF-1 regulation, focusing in particular on the consequences of mitochondrial respiratory complexes damage on stabilization of this pleiotropic transcription factor. Show less

Two luminescent iridium(iii) complexes, 1 and 2, were synthesized and evaluated for their ability to probe COX-2 in human cancer cells. This is the first application of iridium(iii) complexes as imaging agents for COX-2. We demonstrate that complex 1 differentiates cancer cells from normal cells with high stability and low cytotoxicity. Show less

Necroptosis is a form of regulated cell death that critically depends on receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) and generally manifests with morphological features of necrosis. The molecular mechanisms that underlie distinct instances of necroptosis have just begun to emerge. Nonetheless, it has already been shown that necroptosis contributes to cellular demise in various pathophysiological conditions, including viral infection, acute kidney injury, and cardiac ischemia/reperfusion. Moreover, human tumors appear to obtain an advantage from the downregulation of key components of the molecular machinery for necroptosis. Although such an advantage may stem from an increased resistance to adverse microenvironmental conditions, accumulating evidence indicates that necroptosis-deficient cancer cells are poorly immunogenic and hence escape natural and therapy-elicited immunosurveillance. Here, we discuss the molecular mechanisms and relevance to disease of necroptosis. Show less

The redox properties of both metals and ligands in transition metal complexes offer unusual routes for new mechanisms of anticancer therapy. Metal complexes can introduce artificial reductive and oxidative stress into cancer cells, including behavior as photoactivatable agents and catalysts. Relatively inert metal complexes (“prodrugs”) can be activated by redox processes within cancer cells. Examples of pharmaceuticals activated by bioreduction include three PtIV and two RuIII compounds that have already entered clinical trials. More recently, novel CoIII, FeIII, PtIV, Ru(III/II), OsII, and IrIII complexes have been reported to exhibit redox‐mediated anticancer activity. Redox activation strategies can introduce new methods to increase cancer cell selectivity and combat drug resistance. Using combination therapy together with redox modulators to increase potency is also possible. This essay focuses on metal complexes that are activated in the reducing environment of cancer cells. Show less