Chelators as Antineuroblastomas Agents.

Neuroblastoma represents 8-10 % of all malignant tumors in childhood and is responsible for 15 % of cancer deaths in the pediatric population. Aggressive neuroblastomas are often resistant to chemotherapy. Canonically, neuroblastomas can be classified according to the MYCN (N-myc proto-oncogene protein) gene amplification, a common marker of tumor aggressiveness and poor prognosis. It has been found that certain compounds with chelating properties may show anticancer activity, but there is little evidence for the effect of chelators on neuroblastoma. The effect of new chelators characterized by the same functional group, designated as HLZ (1-hydrazino phthalazine), on proliferation (WST-1 and methylene blue assay), cell cycle (flow cytometry), apoptosis (proliferation assay after use of specific pharmacological inhibitors and western blot analysis) and ROS production (fluorometric assay based on dichlorofluorescein diacetate metabolism) was studied in three neuroblastoma cell lines with different levels of MYCN amplification. The molecules were effective only on MYCN-non-amplified cells in which they arrested the cell cycle in the G0/G1 phase. We investigated the mechanism of action and identified the activation of cell signaling that involves protein kinase C.

Schiff base Cu(II) complexes as inhibitors of proteasome in human cancer cells.

BACKGROUND: It has been demonstrated that proteasome inhibitors might be potential anticancer drugs. The copper complexes can be used as specific proteasome inhibitors in tumor cells able to induce apoptosis by the ubiquitin-proteasome pathway. The goal of our study was to test the cytotoxic and proteasome inhibitory effects of five Schiff base Cu(II) complexes - [Cu2(sal-D,L-glu)2(isoquinoline)2] . 2C2H5OH (1), [Cu(sal-5-met-L-glu)(H2O)].H2O (2), [Cu(ethanol)2(imidazole)4][Cu2(sal-D,L-glu)2(imidazole)2] (3), [Cu(sal-D,L-glu)(2-methylimidazole)] (4) on human lung carcinoma cells A549, cervix carcinoma cells HeLa and glioblastoma cells U-118MG. MATERIAL AND METHODS: For the cytotoxic analysis we used MTT test and for monitoring the proteasome inhibition western blot analysis. RESULTS: We have observed different cytotoxic effects of tested complexes on human cancer cells depending on the ligand present in their structure. Cu(II) complexes 4 and 5 were the most effective against A549 cells; all complexes were cytotoxic against HeLa cells and the complex 4 was the most effective against U-118MG. Moreover, we have detected the inhibition of the proteasome activity in human cancer cells A549 by Cu(II) complexes 1, 2 and 4 at IC50 concentration. CONCLUSION: Results of our study suggest that isoquinoline- and imidazole-based copper complexes could be used as inhibitors of the proteasome system in cancer cells A549 (Tab. 1, Fig. 1, Ref. 26).

Various AKIP1 expression levels affect its subcellular localization but have no effect on NF-kappaB activation.

A-kinase interacting protein 1 (AKIP1) has been shown to interact with a broad range of proteins involved in various cellular processes, including apoptosis, tumorigenesis, and oxidative stress suggesting it might have multiple cellular functions. In this study, we used an epitope-tagged AKIP1 and by combination of immunochemical approaches, microscopic methods and reporter assays we studied its properties. Here, we show that various levels of AKIP1 overexpression in HEK-293 cells affected not only its subcellular localization but also resulted in aggregation. While highly expressed AKIP1 accumulated in electron-dense aggregates both in the nucleus and cytosol, low expression of AKIP1 resulted in its localization within the nucleus as a free, non-aggregated protein. Even though AKIP1 was shown to interact with p65 subunit of NF-kappaB and activate this transcription factor, we did not observe any effect on NF-kappaB activation regardless of various AKIP1 expression level.

Molecular aspects of the interaction between Mason-Pfizer monkey virus matrix protein and artificial phospholipid membrane.

The Mason-Pfizer monkey virus is a type D retrovirus, which assembles its immature particles in the cytoplasm prior to their transport to the host cell membrane. The association with the membrane is mediated by the N-terminally myristoylated matrix protein. To reveal the role of particular residues which are involved in the capsid-membrane interaction, covalent labelling of arginine, lysine and tyrosine residues of the Mason-Pfizer monkey virus matrix protein bound to artificial liposomes containing 95% of phosphatidylcholine and 5% phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2 ) was performed. The experimental results were interpreted by multiscale molecular dynamics simulations. The application of these two complementary approaches helped us to reveal that matrix protein specifically recognizes the PI(4,5)P2 molecule by the residues K20, K25, K27, K74, and Y28, while the residues K92 and K93 stabilizes the matrix protein orientation on the membrane by the interaction with another PI(4,5)P2 molecule. Residues K33, K39, K54, Y66, Y67, and K87 appear to be involved in the matrix protein oligomerization. All arginine residues remained accessible during the interaction with liposomes which indicates that they neither contribute to the interaction with membrane nor are involved in protein oligomerization. Proteins 2016; 84:1717-1727. © 2016 Wiley Periodicals, Inc.

Heme oxygenase is not involved in the anti-proliferative effects of statins on pancreatic cancer cells.

BACKGROUND: Pancreatic cancer is recognized as one of the most fatal tumors due to its aggressiveness and resistance to therapy. Statins were previously shown to inhibit the proliferation of cancer cells via various signaling pathways. In healthy tissues, statins activate the heme oxygenase pathway, nevertheless the role of heme oxygenase in pancreatic cancer is still controversial. The aim of this study was to evaluate, whether anti-proliferative effects of statins in pancreatic cancer cells are mediated via the heme oxygenase pathway. METHODS: In vitro effects of various statins and hemin, a heme oxygenase inducer, on cell proliferation were evaluated in PA-TU-8902, MiaPaCa-2 and BxPC-3 human pancreatic cancer cell lines. The effect of statins on heme oxygenase activity was assessed and heme oxygenase-silenced cells were used for pancreatic cancer cell proliferation studies. Cell death rate and reactive oxygen species production were measured in PA-TU-8902 cells, followed by evaluation of the effect of cerivastatin on GFP-K-Ras trafficking and expression of markers of invasiveness, osteopontin (SPP1) and SOX2. RESULTS: While simvastatin and cerivastatin displayed major anti-proliferative properties in all cell lines tested, pravastatin did not affect the cell growth at all. Strong anti-proliferative effect was observed also for hemin. Co-treatment of cerivastatin and hemin increased anti-proliferative potential of these agents, via increased production of reactive oxygen species and cell death compared to individual treatment. Heme oxygenase silencing did not prevent pancreatic cancer cells from the tumor-suppressive effect of cerivastatin or hemin. Cerivastatin, but not pravastatin, protected Ras protein from trafficking to the cell membrane and significantly reduced expressions of SPP1 (p < 0.05) and SOX2 (p < 0.01). CONCLUSIONS: Anti-proliferative effects of statins and hemin on human pancreatic cancer cell lines do not seem to be related to the heme oxygenase pathway. While hemin triggers reactive oxygen species-induced cell death, cerivastatin targets Ras protein trafficking and affects markers of invasiveness.

Structure, mechanical characteristics and in vitro degradation, cytotoxicity, genotoxicity and mutagenicity of novel biodegradable Zn-Mg alloys.

Zn-(0-1.6)Mg (in wt.%) alloys were prepared by hot extrusion at 300 °C. The structure, mechanical properties and in vitro biocompatibility of the alloys were investigated. The hot-extruded magnesium-based WE43 alloy was used as a control. Mechanical properties were evaluated by hardness, compressive and tensile testing. The cytotoxicity, genotoxicity (comet assay) and mutagenicity (Ames test) of the alloy extracts and ZnCl2 solutions were evaluated with the use of murine fibroblasts L929 and human osteosarcoma cell line U-2 OS. The microstructure of the Zn alloys consisted of recrystallized Zn grains of 12 µm in size and fine Mg2Zn11 particles arranged parallel to the hot extrusion direction. Mechanical tests revealed that the hardness and strength increased with increasing Mg concentration. The Zn-0.8 Mg alloys showed the best combination of tensile mechanical properties (tensile yield strength of 203 MPa, ultimate tensile strength of 301 MPa and elongation of 15%). At higher Mg concentrations the plasticity of Zn-Mg alloys was deteriorated. Cytotoxicity tests with alloy extracts and ZnCl2 solutions proved the maximum safe Zn(2+) concentrations of 120 µM and 80 µM for the U-2 OS and L929 cell lines, respectively. Ames test with extracts of alloys indicated that the extracts were not mutagenic. The comet assay demonstrated that 1-day extracts of alloys were not genotoxic for U-2 OS and L929 cell lines after 1-day incubation.

PTEN sequence analysis in endometrial hyperplasia and endometrial carcinoma in Slovak women.

Phosphatase and tensin homolog (PTEN) is a protein that acts as a tumor suppressor by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate. Loss of PTEN function has been implicated in the pathogenesis of a number of different tumors, particularly endometrial carcinoma (ECa). ECa is the most common neoplasia of the female genital tract. Our study evaluates an association between the morphological appearance of endometrial hyperplasia and endometrial carcinoma and the degree of PTEN alterations. A total of 45 endometrial biopsies from Slovak women were included in present study. Formalin-fixed and paraffin-embedded tissue samples with simple hyperplasia (3), complex hyperplasia (5), atypical complex hyperplasia (7), endometrioid carcinomas G1 (20) and G3 (5), and serous carcinoma (5) were evaluated for the presence of mutations in coding regions of PTEN gene, the most frequently mutated tumor suppressor gene in endometrial carcinoma. 75% of the detected mutations were clustered in exons 5 and 8. Out of the 39 mutations detected in 24 cases, 20 were frameshifts and 19 were nonsense, missense, or silent mutations. Some specimens harboured more than one mutation. The results of current study on Slovak women were compared to a previous study performed on Polish population. The two sets of results were similar.

Striking antitumor activity of a methinium system with incorporated quinoxaline unit obtained by spontaneous cyclization.

A novel pentamethinium salt was synthesized with an unforeseen expanded conjugated quinoxaline unit directly incorporated into a pentamethinium chain. The compound exhibited high fluorescence intensity, selective mitochondrial localization, high cytotoxicity, and selectivity toward malignant cell lines, and resulted in remarkable in vivo suppression of tumor growth in mice.

Structure, mechanical properties, corrosion behavior and cytotoxicity of biodegradable Mg-X (X=Sn, Ga, In) alloys.

As-cast Mg-Sn, Mg-Ga and Mg-In alloys containing 1-7 wt.% of alloying elements were studied in this work. Structural and chemical analysis of the alloys was performed by using light and scanning electron microscopy, energy dispersive spectrometry, x-ray diffraction, x-ray photoelectron spectroscopy and glow discharge spectrometry. Mechanical properties were determined by Vickers hardness measurements and tensile testing. Corrosion behavior in a simulated physiological solution (9 g/l NaCl) was studied by immersion tests and potentiodynamic measurements. The cytotoxicity effect of the alloys on human osteosarcoma cells (U-2 OS) was determined by an indirect contact assay. Structural investigation revealed the dendritic morphology of the as-cast alloys with the presence of secondary eutectic phases in the Mg-Sn and Mg-Ga alloys. All the alloying elements showed hardening and strengthening effects on magnesium. This effect was the most pronounced in the case of Ga. All the alloying elements at low concentrations of approximately 1 wt.% were also shown to positively affect the corrosion resistance of Mg. But at higher concentrations of Ga and Sn the corrosion resistance worsened due to galvanic effects of secondary phases. Cytotoxicity tests indicated that Ga had the lowest toxicity, followed by Sn. The most severe toxicity was observed in the case of In.

Atomic force microscopy investigation of Mason-Pfizer monkey virus and human immunodeficiency virus type 1 reassembled particles.

Particles of DeltaProCANC, a fusion of capsid (CA) and nucleocapsid (NC) protein of Mason-Pfizer monkey virus (M-PMV), which lacks the amino terminal proline, were reassembled in vitro and visualized by atomic force microscopy (AFM). The particles, of 83-84 nm diameter, exhibited ordered domains based on trigonal arrays of prominent rings with center to center distances of 8.7 nm. Imperfect closure of the lattice on the spherical surface was affected by formation of discontinuities. The lattice is consistent only with plane group p3 where one molecule is shared between contiguous rings. There are no pentameric clusters nor evidence that the particles are icosahedral. Tubular structures were also reassembled, in vitro, from two HIV fusion proteins, DeltaProCANC and CANC. The tubes were uniform in diameter, 40 nm, but varied in length to a maximum of 600 nm. They exhibited left handed helical symmetry based on a p6 hexagonal net. The organization of HIV fusion proteins in the tubes is significantly different than for the protein units in the particles of M-PMV DeltaProCANC.

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.

Analysis of Mason-Pfizer monkey virus Gag domains required for capsid assembly in bacteria: role of the N-terminal proline residue of CA in directing particle shape.

Mason-Pfizer monkey virus (M-PMV) preassembles immature capsids in the cytoplasm prior to transporting them to the plasma membrane. Expression of the M-PMV Gag precursor in bacteria results in the assembly of capsids indistinguishable from those assembled in mammalian cells. We have used this system to investigate the structural requirements for the assembly of Gag precursors into procapsids. A series of C- and N-terminal deletion mutants progressively lacking each of the mature Gag domains (matrix protein [MA]-pp24/16-p12-capsid protein [CA]-nucleocapsid protein [NC]-p4) were constructed and expressed in bacteria. The results demonstrate that both the CA and the NC domains are necessary for the assembly of macromolecular arrays (sheets) but that amino acid residues at the N terminus of CA define the assembly of spherical capsids. The role of these N-terminal domains is not based on a specific amino acid sequence, since both MA-CA-NC and p12-CA-NC polyproteins efficiently assemble into capsids. Residues N terminal of CA appear to prevent a conformational change in which the N-terminal proline plays a key role, since the expression of a CA-NC protein lacking this proline results in the assembly of spherical capsids in place of the sheets assembled by the CA-NC protein.

Cleavage of vimentin by different retroviral proteases.

Proteases (PRs) of retroviruses cleave viral polyproteins into their mature structural proteins and replication enzymes. Besides this essential role in the replication cycle of retroviruses, PRs also cleave a variety of host cell proteins. We have analyzed the in vitro cleavage of mouse vimentin by proteases of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), bovine leukemia virus (BLV), Mason-Pfizer monkey virus (M-PMV), myeloblastosis-associated virus (MAV), and two active-site mutants of MAV PR. Retroviral proteases display significant differences in specificity requirements. Here, we show a comparison of substrate specificities of several retroviral proteases on vimentin as a substrate. Vimentin was cleaved by all the proteases at different sites and with different rates. The results show that the physiologically important cellular protein vimentin can be degraded by different retroviral proteases.

Enhanced bioaccumulation of heavy metal ions by bacterial cells due to surface display of short metal binding peptides.

Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane of E. coli. Isolated cell envelopes of E. coli bearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+ accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells.

Testing of the effects of xenobiotics in cultured cells often requires the use of organic solvents to effect suspension of the test agents in cell culture media. However, the toxic effects of the solvents themselves may introduce artifacts, which obscure interpretation of the experimental results. In this article, the toxicity of different solvents commonly used for solvation of a variety of xenobiotic agents was studied. We show that ethanol, acetone, isooctane, methanol, and hexane were considerably less toxic than the more commonly used solvent, DMSO, when ATP content and growth rates of HeLa cells exposed to these solvents was measured.

Three active forms of aspartic proteinase from Mason-Pfizer monkey virus.

Mason-Pfizer monkey virus (M-PMV) proteinase, released by the autocatalytic cleavage of Gag-Pro and Gag-Pro-Pol polypeptide precursors, catalyzes the processing of viral precursors to yield the structural proteins and enzymes of the virion. In retroviruses, usually only one proteolytically active form of proteinase exists. Here, we describe an unusual feature of M-PMV, the existence of three active forms of a retroviral proteinase with molecular masses of 17, 13, and 12 kDa as determined by mass spectroscopy. These forms arise in vitro by self-processing of a 26-kDa proteinase precursor. We have developed a process for isolation of each truncated product and demonstrate that all three forms display proteolytic activity. Amino acid analyses, as well as the determination of N- and C-terminal sequences, revealed that the N-termini of all three forms are identical, confirming that in vitro autoprocessing of the 17-kDa form occurs at the C-terminus to yield the truncated forms. The 17-kDa form and the newly described 13-kDa form of proteinase were identified in virions collected from the rhesus monkey CMMT cell line chronically infected with M-PMV, confirming that multiple forms exist in vivo.

The three-dimensional solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer monkey virus, and implications for the morphology of retroviral assembly.

The Mason-Pfizer monkey virus (M-PMV) is the prototype of the type D retroviruses. In type B and D retroviruses, the Gag protein pre-assembles before association with the membrane, whereas in type C retroviruses (lentiviruses, BLV/HTLV group) Gag is targeted efficiently to the plasma membrane, where the particle formation occurs. The N-terminal domain of Gag, the matrix protein (MA), plays a critical role in determining this morphogenic difference. We have determined the three-dimensional solution structure of the M-PMV MA by heteronuclear nuclear magnetic resonance. The protein contains four alpha-helices that are structurally similar to the known type C MA structures. This similarity implies possible common assembly units and membrane-binding mechanisms for type C and B/D retroviruses. In addition to this, the interpretation of mutagenesis data has enabled us to identify, for the first time, the structural basis of a putative intracellular targeting motif.

Characterization of a 1.8-kb DNA segment located upstream from the human Ha-ras protooncogene and possibly regulating its function.

We have cloned a 1.8-kb segment of DNA just adjacent to the 5' end of the 6.4-kb BamHI fragment which contains the Ha-ras oncogene (ras1; GenBank notation HUMRASH). Electrophoretic mobility shift assays (EMSA) indicate the presence of multiple nuclear protein-binding sites within the 1.8-kb segment. At least three putative regulatory protein-binding sites have been identified in a 206-bp subfragment by DNase I footprint analysis. Within the subfragment containing the DNase-protected regions, there is also a segment containing a number of consensus sequences for DNA-binding proteins and two sub-sequences that exhibit strong sequence homology to at least two previously characterized enhancers. These data suggest that a novel set of regulatory elements may lie as far as 2 kb upstream from the normal Ha-ras transcription start points.

Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria.

The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.