Effect of tamoxifen on the enzymatic activity of human cytochrome CYP2B6

Tamoxifen is primarily used in the treatment of breast cancer. It has been approved as a chemopreventive agent for individuals at high risk for this disease. Tamoxifen is metabolized to a number of different products by cytochrome P450 enzymes. The effect of tamoxifen on the enzymatic activity of bacterially expressed human cytochrome CYP2B6 in a reconstituted system has been investigated. The 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity of purified CYP2B6 was inactivated by tamoxifen in a time- and concentration-dependent manner. Enzymatic activity was lost only in samples that were incubated with both tamoxifen and NADPH. The inactivation was characterized by a K-l of 0.9 muM, a k(inact) of 0.02 min(-1), and a t(1/2) of 34 min. The loss in the 7-ethoxy-4-(trifluoromethyl) coumarin O-deethylation activity did not result in a similar percentage loss in the reduced carbon monoxide spectrum, suggesting that the heme moiety was not the major site of modification. The activity of CYP2B6 was not recovered after removal of free tamoxifen using spin column gel filtration. The loss in activity seemed to be due to a modification of the CYP2B6 and not reductase because adding fresh reductase back to the inactivated samples did not restore enzymatic activity. A reconstituted system containing purified CYP2B6, NADPH-reductase, and NADPH-generating system was found to catalyze tamoxifen metabolism to 4-OH-tamoxifen, 4'-OH-tamoxifen, and N-desmethyl-tamoxifen as analyzed by high-performance liquid chromatography analysis. Preliminary studies showed that tamoxifen had no effect on the activities of CYP1B1 and CYP3A4, whereas CYP2D6 and CYP2C9 exhibited a 25% loss in enzymatic activity.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Studies of the Antiproliferative Activity of Ruthenium (II) Cyclopentadienyl-Derived Complexes with Nitrogen Coordinated Ligands

Four cationic ruthenium(II) complexes with the formula [Ru(eta(5)-C5H5)(PPh3)(2)](+), with L = 5-phenyl-1H-tetrazole (TzH) 1, imidazole (ImH) 2, benzo[1,2-b; 4,3-b'] dithio-phen-2-carbonitrile (Bzt) 3, and [5-(2-thiophen-2-yl)-vinyl]-thiophene-2-carbonitrile] (Tvt) 4 were prepared and characterized in view to evaluate their potentialities as antitumor agents. Studies by Circular Dichroism indicated changes in the secondary structure of ct-DNA. Changes in the tertiary structure of pBR322 plasmid DNA were also observed in gel electrophoresis experiment and the images obtained by atomic force microscopy (AFM) suggest strong interaction with pBR322 plasmid DNA; the observed decreasing of the viscosity with time indicates that the complexes do not intercalate between DNA base pairs. Compounds 1, 2, and 3 showed much higher cytotoxicity than the cisplatin against human leukaemia cancer cells (HL-60 cells).

Synthesis, Antimicrobial and Antiproliferative Activity of Novel Silver(I) Tris(pyrazolyl)methanesulfonateand 1,3,5-Triaza-7-phosphadamantane Complexes

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesized and fully characterized by elemental analyses, H-1, C-13, and P-31 NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N-3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA1S9), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA EB system on addition of Ag complexes shows that the fluorescence quenching of DNA EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3 >> 4.

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.

Electrocatalytic evaluation of DNA damage by superoxide radical for antioxidant capacity assessment

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

Insights into the mechanims underlyng the antiproliferative potential of a Co(II) coordination compound bearing 1,10-Phenanthroline-5,6-Dione: DNA and protein interaction studies

The very high antiproliferative activity of [Co(Cl)(H2O)(phendione)(2)][BF4] (phendione is 1,10-phenanthroline-5,6-dione) against three human tumor cell lines (half-maximal inhibitory concentration below 1 mu M) and its slight selectivity for the colorectal tumor cell line compared with healthy human fibroblasts led us to explore the mechanisms of action underlying this promising antitumor potential. As previously shown by our group, this complex induces cell cycle arrest in S phase and subsequent cell death by apoptosis and it also reduces the expression of proteins typically upregulated in tumors. In the present work, we demonstrate that [Co(Cl)(phendione)(2)(H2O)][BF4] (1) does not reduce the viability of nontumorigenic breast epithelial cells by more than 85 % at 1 mu M, (2) promotes the upregulation of proapoptotic Bax and cell-cycle-related p21, and (3) induces release of lactate dehydrogenase, which is partially reversed by ursodeoxycholic acid. DNA interaction studies were performed to uncover the genotoxicity of the complex and demonstrate that even though it displays K (b) (+/- A standard error of the mean) of (3.48 +/- A 0.03) x 10(5) M-1 and is able to produce double-strand breaks in a concentration-dependent manner, it does not exert any clastogenic effect ex vivo, ruling out DNA as a major cellular target for the complex. Steady-state and time-resolved fluorescence spectroscopy studies are indicative of a strong and specific interaction of the complex with human serum albumin, involving one binding site, at a distance of approximately 1.5 nm for the Trp214 indole side chain with log K (b) similar to 4.7, thus suggesting that this complex can be efficiently transported by albumin in the blood plasma.

New iron(II) cyclopentadienyl derivative complexes: synthesis and antitumor activity against human leucemia cancer cells

A new family of "Fe-II(eta(5)-C5H5)" half sandwich compounds bearing a N-heteroaromatic ligand coordinated to the iron center by a nitrile functional group has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for complexes 1 and 3, which crystallized in the monoclinic P2(1)/c and monoclinic P2(1)/n space groups, respectively. Studies of interaction of these five new complexes with plasmid pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC50 values revealed excellent antiproliferative activity compared to cisplatin. (C) 2014 Elsevier B.V. All rights reserved.

Effects of UV radiation exposure on DNA and DNA repair enzymes

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Regulation of PLK4 levels and activity to ensure centriole number control

Dissertation presented to obtain a Ph.D degree in Cellular Biology

The role of electron transfer in DNA building blocks: evaluation of strand breaks and their implications

Dissertação para obtenção do Grau de Doutor em Engenharia Física

Development of elastin-like recombinamer films with antimicrobial activity

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Methylation at the CpG island shore region upregulates Nr3c1 promoter activity after early-life stress

Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore.

A glycine-arginine domain in control of the human MRE11 DNA repair protein.

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.