Distinctive expression and cellular localisation of zinc homeostasis-related proteins in breast and prostate cancer cells.

BACKGROUND: Zinc transport proteins (ZIP and ZnT), metallothioneins (MT) and protein kinase CK2 are involved in dysregulation of zinc homeostasis in breast and prostate cancer cells. Following up our previous research, we targeted ZIP12, ZnT1, MT2A and CK2 in this study by investigating their expression levels and protein localisation. METHODS: Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunofluorescence confocal microscopy were employed to quantify the expression of ZIP12, ZnT1, MT2A and CK2 subunits in a panel of breast and prostate cell lines without or with extracellular zinc exposure. The cellular localisations of these target proteins were also examined by immunofluorescence confocal microscopy. RESULTS: In response to the extracellular zinc exposure, the gene expression was elevated for SLC39A12 (ZIP12), SLC30A1 (ZnT1) and MT2A (MT2A) in normal prostate epithelial cells (RWPE-1) in contrast to their cancerous counterparts (PC3 and DU145), whilst the gene expression was higher for SLC39A12 (ZIP12) and SLC30A1 (ZnT1) in both normal (MCF10A) and basal breast cancer cells (MDA-MB-231) compared to luminal breast cancer cells (MCF-7). At the protein level, the expression for both ZIP12 and ZnT1 was trending lower in the time course for the breast cancer cells whilst their expression was remained constant in the normal breast epithelial cells. The expression of ZIP12 in prostate cancer cells was higher than the normal prostate cells. The protein expression for CK2 α/αꞌ and CK2ß was markedly higher in prostate cancer cells than the normal prostate cells. Upon extracellular zinc exposure, ZIP12 was, for the first time, conspicuously localised in the plasma membrane of breast cancer cells but not in normal breast epithelial cells and prostate cells. ZnT1 is only localised in the plasma membrane of breast cancer cells. MT2A is distinctively seen close to the plasma membrane in breast cancer cells. CK2 is also for the first time shown to be localised in proximity to the plasma membrane of breast cancer cells. CONCLUSION: The findings, particularly the localisation of ZIP12 and CK2, are novel and significant for our understanding of zinc homeostasis in breast and prostate cancer cells.

Molecular Insights into the Breast and Prostate Cancer Cells in Response to the Change of Extracellular Zinc.

Zinc dyshomeostasis is manifested in breast and prostate cancer cells. This study attempted to uncover the molecular details prodded by the change of extracellular zinc by employing a panel of normal and cancerous breast and prostate cell lines coupled with the top-down proteomics with two-dimensional gel electrophoresis followed by liquid chromatography-tandem mass spectrometry. The protein samples were generated from MCF-7 breast cancer cells, MCF10A normal breast cells, PC3 prostate cancer cells, and RWPE-1 normal prostate cells with or without exogenous zinc exposure in a time course (T0 and T120). By comparing the cancer cells vs respective normal epithelial cells without zinc treatment (T0), differentially expressed proteins (23 upregulated and 18 downregulated in MCF-7 cells; 14 upregulated and 30 downregulated in PC3 cells) were identified, which provides insights into the intrinsic differences of breast and prostate cancer cells. The dynamic protein landscapes in the cancer cells prodded by the extracellular zinc treatment reveal the potential roles of the identified zinc-responsive proteins (e.g., triosephosphate isomerase, S100A13, tumour proteins hD53 and hD54, and tumour suppressor prohibitin) in breast and prostate cancers. This study, for the first time, simultaneously investigated the two kinds of cancer cells related to zinc dyshomeostasis, and the findings shed light on the molecular understanding of the breast and prostate cancer cells in response to extracellular zinc variation.

Encapsulated Rose Bengal Enhances the Photodynamic Treatment of Triple-Negative Breast Cancer Cells.

Among breast cancer subtypes, triple-negative breast cancer stands out as the most aggressive, with patients facing a 40% mortality rate within the initial five years. The limited treatment options and unfavourable prognosis for triple-negative patients necessitate the development of novel therapeutic strategies. Photodynamic therapy (PDT) is an alternative treatment that can effectively target triple-negative neoplastic cells such as MDA-MB-231. In this in vitro study, we conducted a comparative analysis of the PDT killing rate of unbound Rose Bengal (RB) in solution versus RB-encapsulated chitosan nanoparticles to determine the most effective approach for inducing cytotoxicity at low laser powers (90 mW, 50 mW, 25 mW and 10 mW) and RB concentrations (50 µg/mL, 25 µg/mL, 10 µg/mL and 5 µg/mL). Intracellular singlet oxygen production and cell uptake were also determined for both treatment modalities. Dark toxicity was also assessed for normal breast cells. Despite the low laser power and concentration of nanoparticles (10 mW and 5 µg/mL), MDA-MB-231 cells experienced a substantial reduction in viability (8 ± 1%) compared to those treated with RB solution (38 ± 10%). RB nanoparticles demonstrated higher singlet oxygen production and greater uptake by cancer cells than RB solutions. Moreover, RB nanoparticles display strong cytocompatibility with normal breast cells (MCF-10A). The low activation threshold may be a crucial advantage for specifically targeting malignant cells in deep tissues.

Photodynamic Treatment of Human Breast and Prostate Cancer Cells Using Rose Bengal-Encapsulated Nanoparticles.

Cancer, a prominent cause of death, presents treatment challenges, including high dosage requirements, drug resistance, poor tumour penetration and systemic toxicity in traditional chemotherapy. Photodynamic therapy, using photosensitizers like rose bengal (RB) with a green laser, shows promise against breast cancer cells in vitro. However, the hydrophilic RB struggles to efficiently penetrate the tumour site due to the unique clinical microenvironment, aggregating around rather than entering cancer cells. In this study, we have synthesized and characterized RB-encapsulated chitosan nanoparticles with a peak particle size of ~200 nm. These nanoparticles are readily internalized by cells and, in combination with a green laser (λ = 532 nm) killed 94-98% of cultured human breast cancer cells (MCF-7) and prostate cancer cells (PC3) at a low dosage (25 µg/mL RB-nanoparticles, fluence ~126 J/cm2, and irradiance ~0.21 W/cm2). Furthermore, these nanoparticles are not toxic to cultured human normal breast cells (MCF10A), which opens an avenue for translational applications.

Genomic and Pathologic Characterization of the First FAdV-C Serotype 4 Isolate from Black-Necked Crane.

Fowl adenoviruses (FAdVs) are distributed worldwide in poultry and incriminated as the etiological agents for several health problems in fowls, and are capable of crossing species barriers between domestic and wild fowls. An FAdV strain was, for the first time, isolated from black-necked crane in this study, and was designated as serotype 4 Fowl aviadenovirus C (abbreviated as BNC2021) according to the phylogenetic analysis of its DNA polymerase and hexon gene. The viral genomic sequence analysis demonstrated that the isolate possessed the ORF deletions that are present in FAdV4 strains circulating in poultry fowls in China and the amino acid mutations associated with viral pathogenicity in the hexon and fiber 2 proteins. A viral challenge experiment with mallard ducks demonstrated systemic viral infection and horizontal transmission. BNC2021 induced the typical clinical signs of hepatitis-hydropericardium syndrome (HHS) with swelling and inflammation in multiple organs and showed significant viral replication in all eight organs tested in the virus-inoculated ducks and their contactees at 6 dpi. The findings highlight the importance of surveillance of FAdVs in wild birds.

Expression profiles of the genes associated with zinc homeostasis in normal and cancerous breast and prostate cells.

Zn2+ dyshomeostasis is an intriguing phenomenon in breast and prostate cancers, with breast cancer cells exhibiting higher intracellular Zn2+ level compared to their corresponding normal epithelial cells, in contrast to the low Zn2+ level in prostate cancer cells. In order to gain molecular insights into the zinc homeostasis of breast and prostate cancer cells, this study profiled the expression of 28 genes, including 14 zinc importer genes (SLC39A1-14) that encode Zrt/Irt-like proteins 1-14 to transport Zn2+ into the cytoplasm, 10 zinc exporter genes (SLC30A1-10) which encode Zn2+ transporters 1-10 to transport Zn2+ out of the cytoplasm, and 4 metallothionein genes (MT1B, MT1F, MT1X, MT2A) in breast (MCF10A, MCF-7, MDA-MB-231) and prostate (RWPE-1, PC3, DU145) cell lines in response to extracellular zinc exposures at a mild cytotoxic dosage and a benign dosage. The RNA samples were prepared at 0 min (T0), 30 min (T30), and 120 min (T120) in a time course with or without zinc exposure, which were used for profiling the baseline and dynamic gene expression. The up-regulation of MT genes was observed across the breast and prostate cancer cell lines. The expression landscape of SLC39A and SLC30A was revealed by the quantitative reverse transcription polymerase chain reaction data of this study, which sheds light on the divergence of intracellular Zn2+ levels for breast and prostate cancer cells. Taken together, the findings are valuable in unraveling the molecular intricacy of zinc homeostasis in breast and prostate cancer cells.

Molecular details of aluminium-amyloid ß peptide interaction by nuclear magnetic resonance.

Alzheimer's disease (AD) is a devastating neurodegenerative condition that poses major challenges to human health. Both amyloid ß (Aß) and metal ions such as aluminium are implicated in the development of AD. By the means of NMR, the interactions of Al3+ with Aß1-28 peptide as well as the Aß1-28 analogues were studied, and the key binding sites of Al3+ in Aß determined. NMR data showed Al3+ interacts with Aß1-28 at the NH and αH of numerous residues by exhibiting upfield shifts. Using Aß analogues where His6, His13 and His14 were individually replaced by alanine residue(s), including Aß H6A, Aß H13A, Aß H14A, and Aß H6,13,14A, the results demonstrated that the histidine residues (His6, His13 and His14) and N-terminal Asp1 were involved in the Al3+ coordination. These findings provide, for the first time, the details of the molecular interaction between Al3+ and Aß, which points to the potential role of Al3+ in Aß aggregation, hence in AD development.

Fabrication and characterization of chitosan nanoparticles using the coffee-ring effect for photodynamic therapy.

BACKGROUND AND OBJECTIVES: Biocompatible nanoparticles have been increasingly used in a variety of medical applications, including photodynamic therapy. Although the impact of synthesis parameters and purification methods is reported in previous studies, it is still challenging to produce a reliable protocol for the fabrication, purification, and characterization of nanoparticles in the 200-300 nm range that are highly monodisperse for biomedical applications. STUDY DESIGN/MATERIALS AND METHODS: We investigated the synthesis of chitosan nanoparticles in the 200-300 nm range by evaluating the chitosan to sodium tripolyphosphate (TPP) mass ratio and acetic acid concentration of the chitosan solution. Chitosan nanoparticles were also crosslinked to rose bengal and incubated with human breast cancer cells (MCF-7) to test photodynamic activity using a green laser (λ = 532 nm, power = 90 mW). RESULTS: We established a simple protocol to fabricate and purify biocompatible nanoparticles with the most frequent size occurring between 200 and 300 nm. This was achieved using a chitosan to TPP mass ratio of 5:1 in 1% v/v acetic acid at a pH of 5.5. The protocol involved the formation of nanoparticle coffee rings that showed the particle shape to be spherical in the first approximation. Photodynamic treatment with rose bengal-nanoparticles killed ~98% of cancer cells. CONCLUSION: A simple protocol was established to prepare and purify spherical and biocompatible chitosan nanoparticles with a peak size of ~200 nm. These have remarkable antitumor activity when coupled with photodynamic treatment.

Clade 2.3.4.4b H5N8 Subtype Avian Influenza Viruses Were Identified from the Common Crane Wintering in Yunnan Province, China.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020-2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020-2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Transcriptomic insights into the zinc homeostasis of MCF-7 breast cancer cells via next-generation RNA sequencing.

A significant gap in the knowledge of zinc homeostasis exists for breast cancer cells. In this study, we investigated the transcriptomic response of the luminal breast cancer cells (MCF-7) to the exposure of extracellular zinc using next-generation RNA sequencing. The dataset was collected for three time points (T0, T30, and T120) in the time course of zinc treatment, which revealed the dramatic increase, up to 869-fold, of the gene expression for metallothioneins (MT1B, MT1F, MT1X, and MT2A) and the zinc exporter ZnT1 (SLC30A1) at T30, continuingly through to T120. The similar dynamic expression pattern was found for the autophagy-related gene (VMP1) and numerous genes for zinc finger proteins (e.g. RNF165, ZNF365, ZBTB2, SNAI1, ZNF442, ZNF547, ZNF563, and ZNF296). These findings point to the all-hands-on-deck strategy adopted by the cancer cells for maintaining zinc homeostasis. The stress responsive genes encoding heat shock proteins (HSPA1A, HSPA1B, HSPA1L, HSPA4L, HSPA6, HSPA8, HSPH1, HSP90AA1, and HSP90AB1) and the MTF-1 biomarker genes (AKR1C2, CLU, ATF3, GDF15, HMOX1, MAP1A, MAFG, SESN2, and UBC) were also differentially up-regulated at T120, suggesting a role of heat shock proteins and the MTF-1 related stress proteins in dealing with zinc exposure. It is for the first time that the gene encoding Polo-like kinase 2 (PLK2) was found to be involved in zinc-related response. The top differentially expressed genes were validated by qRT-PCR and further extended to the basal type breast cancer cells (MDA-MB-231). It was found that the expression level of SLC30A1 in MDA-MB-231 was higher than MCF-7 in response to zinc exposure. Taken together, the findings contribute to our knowledge and understanding of zinc homeostasis in breast cancer cells.

Protein kinase CK2 is involved in zinc homeostasis in breast and prostate cancer cells.

The intracellular zinc profiles of breast and prostate cancer cells are diametrically opposed, with hyper-accumulation of zinc in breast cancer, and low level in prostate cancer. This phenomenon is poorly understood. This study employs two breast and two prostate cancer cell lines to investigate the role of protein kinase CK2 in regulating zinc homeostasis. CK2 was targeted by its specific inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and CX-4945, and by the specific siRNA against each of the three CK2 genes. The effect of zinc exposure after the above CK2 manipulation was observed by MTT [3-(4,5-dimethyliazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] cell viability assay and confocal microscopy for intracellular zinc level. The results demonstrate that CK2 is involved in regulating zinc homeostasis in breast and prostate cancer cells as both TBB and CX-4945 substantially decreased cell viability upon zinc exposure. siRNA-mediated knockdown of the three CK2 subunits (α, α' and ß) revealed their discrete roles in regulating zinc homeostasis in breast and prostate cancer cells. Knockdown of CK2α' decreased the intracellular zinc level of breast cancer cells and in turn increased the cell viability while the opposite findings were obtained for the prostate cancer cells. Knockdown of CK2ß expression substantially increased the zinc level in breast cancer cell lines whilst decreased the zinc level in prostate cancer cells. Taken together, this study shows that CK2 is involved in zinc homeostasis of breast and prostate cancer cells and opens a new avenue for research on these cancers.

The cytotoxicity of some phenanthroline-based antimicrobial copper(II) and ruthenium(II) complexes.

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.

The in vitro renal cell toxicity of some unconventional anticancer phenanthroline-based platinum(II) complexes.

The cytotoxicity of platinum(II) complexes coordinated to a chiral diamine, 1S,2S-diaminocyclohexane or 1R,2R-diaminocyclohexane and 1,10-phenanthroline or 3,4,7,8-tetramethyl-1,10-phenanthroline has been investigated in the renal proximal tubule HK-2 cell line. All platinum(II) complexes exhibited lower cytotoxicity in HK-2 cells (IC50 1.7-25µM) than in A2780 ovarian cancer cells or cisplatin-resistant A2780cisR cells (IC50 0.2-2.1µM) (at 48h). PHENSS ([Pt(1,10-phenanthroline)(1S,2S-dach)]2+) induced apoptosis and necrosis in ovarian cancer cells at concentrations that are relatively cytostatic to renal cells. Cisplatin was similarly or more cytotoxic to renal cells than ovarian cancer cells. Similar trends were reflected with shorter term exposure (1.5h). PHENSS demonstrated a comparatively cytostatic mode of action in renal cell cultures than cisplatin, as demonstrated by lower toxicity at higher concentrations (90µM). PHENSS induced an elongated renal cell morphology, cytoskeletal stress fibre thickening, and increased ß-galactosidase activity, but no detectable change in reactive oxygen species generation or cell cycle distribution. In contrast, cisplatin treatment was associated with increased oxidative stress, cellular enlargement, G2/M arrest and apoptosis. The cytotoxicity of all platinum(II) complexes in both renal and ovarian cell lines were reduced in the presence of organic cation transporter (OCT) inhibitors cimetidine, disopyramide and amantadine. PHENSS and analogues demonstrated low level genotoxicity in an in vitro micronuclei assay compared to cisplatin or etoposide. These findings highlight PHENSS and other phen-based unconventional platinum(II) complexes as promising anticancer agents with alternative modes of action that induce lower kidney cell toxicity and genotoxicity, while demonstrating greater cisplatin-resistant ovarian cancer cell toxicity.

Proteomic response to linoleic acid hydroperoxide in Saccharomyces cerevisiae.

Yeast AP-1 transcription factor (Yap1p) and the enigmatic oxidoreductases Oye2p and Oye3p are involved in counteracting lipid oxidants and their unsaturated breakdown products. In order to uncover the response to linoleic acid hydroperoxide (LoaOOH) and the roles of Oye2p, Oye3p and Yap1p, we carried out proteomic analysis of the homozygous deletion mutants oye3Δ, oye2Δ and yap1Δ alongside the diploid parent strain BY4743. The findings demonstrate that deletion of YAP1 narrowed the response to LoaOOH, as the number of proteins differentially expressed in yap1Δ was 70% of that observed in BY4743. The role of Yap1p in regulating the major yeast peroxiredoxin Tsa1p was demonstrated by the decreased expression of Tsa1p in yap1Δ. The levels of Ahp1p and Hsp31p, previously shown to be regulated by Yap1p, were increased in LoaOOH-treated yap1Δ, indicating their expression is also regulated by another transcription factor(s). Relative to BY4743, protein expression differed in oye3Δ and oye2Δ under LoaOOH, underscored by superoxide dismutase (Sod1p), multiple heat shock proteins (Hsp60p, Ssa1p, and Sse1p), the flavodoxin-like protein Pst2p and the actin stabiliser tropomyosin (Tpm1p). Proteins associated with glycolysis were increased in all strains following treatment with LoaOOH. Together, the dataset reveals, for the first time, the yeast proteomic response to LoaOOH, highlighting the significance of carbohydrate metabolism, as well as distinction between the roles of Oye3p, Oye2p and Yap1p.

Unravelling the role of protein kinase CK2 in metal toxicity using gene deletion mutants.

Metal ions, biologically essential or toxic, are present in the surrounding environment of living organisms. Understanding their uptake, homeostasis or detoxification is critical in cell biology and human health. In this study, we investigated the role of protein kinase CK2 in metal toxicity using gene deletion strains of Saccharomyces cerevisiae against a panel of six metal ions. The deletion of CKA2, the yeast orthologue of mammalian CK2α', leads to a pronounced resistant phenotype against Zn2+ and Al3+, whilst the deletion of CKB1 or CKB2 results in tolerance to Cr6+ and As3+. The individual deletion mutants of CK2 subunits (CKA1, CKA2, CKB1 and CKB2) did not have any benefit against Co2+ and Cd2+. The metal ion content in the treated cells was then measured by inductively coupled plasma mass spectrometry. Two contrasting findings were obtained for the CKA2 deletion mutant (cka2Δ) against Al3+ or Zn2+. Upon exposure to Al3+, cka2Δ had markedly lower Al3+ content than the wild type and other CK2 mutants, congruous to the resistant phenotype of cka2Δ against Al3+, indicating that CKA2 is responsible for Al3+ uptake. Upon zinc exposure the same mutant showed similar Zn2+ content to the wild type and cka1Δ. Strikingly, the selective inhibitor of CK2 TBB (4,5,6,7-tetrabromo-1H-benzotriazole) abolished the resistant phenotype of cka2Δ against Zn2+. Hence, the CK2 subunit CKA1 plays a key role in Zn2+ sequestration of the cell. Given that both zinc and CK2 are implicated in cancer development, the findings herein are of significance to cancer research and anticancer drug development.

The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport.

The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2's regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport.

The antimicrobial efficacy and DNA binding activity of the copper(II) complexes of 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline and 1,2-diaminocyclohexane.

Four copper(II) complexes of the general structure [Cu(L1)(L2)]2+, where L1 is (1S,2S)-diaminocyclohexane or (1R,2R)-diaminocyclohexane and L2 is 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-diphenyl-1,10-phenanthroline (DIP), have been investigated in this study for their antimicrobial activity, short-term antimicrobial efficacy, and in vitro DNA-binding affinity. Against an expanded panel of bacterial and fungal strains in 12 species, minimal inhibitory concentrations (MIC) for these metallocomplexes were determined. The data confirmed our previous finding that they are effective against Gram-positive bacteria (MIC 5.6-13.1µM), with DIP coordinated complexes more so than TMP counterparts. Additionally, novel and significant findings were obtained here for these copper(II) complexes. While the four metallocomplexes exhibited high anti-Candida yeast activity (MIC 13.1-26.1µM), they demonstrated stronger anti-fungal activity against the drug-resistant Candida krusei (MIC 13.1µM and 22.6µM for TMP and DIP complexes, respectively) than the anti-fungal agent, 5-fluorocytosine. Fluorescence cell viability assays revealed that these complexes exert faster antibacterial effect than ampicillin as their inhibition against Staphylococcus aureus and Enterococcus faecalis were significantly evident within 0.5h of exposure compared to ampicillin. Similarly, these complexes but not ampicillin demonstrated bactericidal activity in non-proliferating conditions. All complexes exhibited DNA binding affinities similar to that of the known DNA intercalator, ethidium bromide (Ka ~105M-1) in linear dichroism binding studies and fluorescent dye displacement assays. Taken together, these findings imply that the four copper(II) complexes have different modes of action to the established antibiotics such as ampicillin and 5-fluorocytosine, and provide further insight into development of effective antimicrobial metallocomplexes.

Revelation of molecular basis for chromium toxicity by phenotypes of Saccharomyces cerevisiae gene deletion mutants.

Chromium toxicity is increasingly relevant to living organisms such as humans, due to the environmental contamination of chromium and the application of stainless steel-based medical devices like hip prostheses. Despite the investigations in past years, the molecular details for chromium toxicity remain to be delineated. In this study, we seek to gain insights into the molecular aspects of chromium toxicity by screening a genome-wide deletion set of individual genes in Saccharomyces cerevisiae against hexavalent chromium [Cr(vi)] using chromium trioxide. From the primary data collected in this study, two lists of deletion mutants in response to Cr(vi) exposure were obtained, one for the sensitive phenotype and the other for the resistant phenotype. The functional analysis of the genes corresponding to the sensitive mutants reveals the key features of Cr(vi) toxicity, which include genotoxicity, protein damage, disruption of energy and sulfur metabolisms. DNA repair, ubiquitination-mediated protein degradation, iron homeostasis and growth attenuation are the intrinsic facets of the cell's detoxification mechanisms. Protein kinase CK2 is, for the first time, found to be involved in regulating chromium toxicity by reducing the uptake of Cr(vi). Taken together, the findings provide meaningful details into the basic understanding of chromium toxicity in terms of its uptake, modes of action, cellular detoxification and molecular regulatory mechanisms.

Protein kinase CK2 regulates metal toxicity in neuronal cells.

Protein kinase CK2 is a pleiotropic tetrameric enzyme, regulating numerous biological processes from cell proliferation to stress response. This study demonstrates for the first time that CK2 is involved in the regulation of metal uptake and toxicity in neuronal cells. After the determination of inhibitory concentrations (IC50) for a range of metal salts (ZnSO4, Al(mal)3, CoCl2, CrO3, NaAsO2 and CaCl2) in Neuro-2a mouse neuroblastoma cells, the effect of CK2 on metal toxicity was investigated by three lines of experiments using CK2 inhibitors, metal ion specific fluorophores and siRNA-mediated knockdown of CK2 expression. The results showed that both CK2 inhibitors, 4,5,6,7-tetrabromobenzotriazole (TBB) and quinalizarin, markedly reduced the toxicity of Zn(ii), Al(iii), Co(ii), Cr(vi) and As(iii). Confocal microscopy imaging revealed that Zn(ii) uptake was accompanied by the increase of intracellular Ca(ii) in Neuro-2a cells treated with IC50 of ZnSO4 (240 µM), and such concurrent elevation of intracellular Zn(ii) and Ca(ii) was blocked by TBB and quinalizarin. The role of CK2 in metal uptake was further characterised using specific siRNA against each of the three subunits (CK2α, α' and ß) and the data demonstrate that CK2α' is the prominent subunit regulating the metal toxicity. Finally, the role of CK2 in metal toxicity was found to be conserved in the distant species-Saccharomyces cerevisiae by employing the complete deletion mutants of CK2 (cka1Δ, cka2Δ, ckb1Δ and ckb2Δ). Taken together, these findings shed light on a new facet of CK2 functionality and provide a basis for further research on the regulation of Zn(ii) and Ca(ii) homeostasis by CK2.

Molecular insight into arsenic toxicity via the genome-wide deletion mutant screening of Saccharomyces cerevisiae.

Arsenic is omnipresent in soil, air, food and water. Chronic exposure to arsenic is a serious problem to human health. In-depth understanding of this metalloid's toxicity is a fundamental step towards development of arsenic-free foods and measures for bioremediation. By screening the complete set of gene deletion mutants (4873) of Saccharomyces cerevisiae, this study uncovered 75 sensitive and 39 resistant mutants against arsenite [As(III)]. Functional analysis of the corresponding genes revealed the molecular details for its uptake, toxicity and detoxification. On the basis of the hypersensitivity of yap3Δ, the transcription factor, Yap3p, is for the first time linked to the cell's detoxification against As(III). Apart from confirming the previously described role of the mitogen-activated protein kinase (MAPK) Hog1 pathway in combating arsenic toxicity, the results show that the regulatory subunits (Ckb1p and Ckb2p) of protein kinase CK2 are also involved in the process, suggesting possible crosstalk between the two key protein kinases. The sensitivity to As(III) conferred by deletion of the genes involved in protein degradation and chromatin remodelling demonstrates protein damage is the key mode of toxicity for the metalloid. Furthermore, the resistant phenotype of fps1Δ, snf3Δ and pho81Δ against As(III) links arsenic uptake with the corresponding plasma membrane-bound transporters-aquaglyceroporin (Fps1p), hexose (Snf3p) and phosphate transporters. The molecular details obtained in this screen for As(III) uptake, detoxification and toxicity provide the basis for future investigations into arsenic-related problems in the environment, agriculture and human health.