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1.
Rosana Hudej Damijan Miklavcic Maja Cemazar Vesna Todorovic Gregor Sersa Alberta Bergamo Gianni Sava Anze Martincic Janez Scancar Bernhard K. Keppler Iztok Turel 《The Journal of membrane biology》2014,247(12):1239-1251
To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo. 相似文献
2.
Malgorzata Brindell Dorota Piotrowska Azza A. Shoukry Grażyna Stochel Rudi van Eldik 《Journal of biological inorganic chemistry》2007,12(6):809-818
A systematic study of the reduction of (ImH)[trans-RuCl4(dmso)(Im)] (NAMI-A;
dmso is dimethyl sulfoxide, Im is imidazole), a promising antimetastasing agent
entering phase II clinical trial, by l-ascorbic acid is reported. The rapid reduction of
trans-[RuIIICl4(dmso)(Im)]−
results in formation of trans-[RuIICl4(dmso)(Im)]2−
in acidic medium (pH = 5.0) and is followed by successive dissociation of the
chloride ligands, which cannot be suppressed even in the presence of a large
excess of chloride ions. The reduction of NAMI-A strongly depends on pH and is
accelerated on increasing the pH. Over the small pH range 4.9−5.1, the reaction
is quite pH-independent and the influence of temperature and pressure on the
reaction could be studied. On the basis of the reported activation parameters
and other experimental data, it is suggested that the redox process follows an
outer-sphere electron transfer mechanism. A small contribution from a parallel
reaction ascribed to inner-sphere reduction of aqua derivatives of NAMI-A, was
found to be favored by lower concentrations of the NAMI-A complex and higher
temperature. In the absence of an excess of chloride ions, the reduction
process is catalyzed by the Ru(II) products being formed. The reduction of
NAMI-A is also catalyzed by Cu(II) ions and the apparent catalytic rate
constant was found to be
1.5 × 106 M−2 s−1
at 25 °C.
Electronic supplementary
material The online version of this article
(doi:) contains supplementary material, which is available to
authorized users. 相似文献
3.
The effects of NAMI-A, [H2im][trans-RuCl4(dmso-S)(Him)], a new metal-based agent for treating tumor metastases, have been investigated in vitro on splenocytes, ConA- or LPS-activated T and B lymphoblasts, and thymocytes. Splenocytes and thymocytes exposed for 1 h to 0.01–0.1-mM NAMI-A do not change their mitochondrial functionality, cell cycle distribution, protein synthesis, and CD44 expression in comparison to untreated control samples. Instead, mitochondrial functionality increased 24 h after treatment in a fraction of splenocytes. The same treatment reduced mitochondrial functionality and S phase of the cell cycle in T and B blasts (already after 1 h treatment) and reduced CD44 expression on B blasts, 24 h after treatment. On cocultures of splenocytes and metastatic cells (metGM) (1:1), NAMI-A induces a selective depolarization of mitochondrial membrane potential of metGM cells, while it stimulates splenocytes (mainly lymphocytes), as shown by the increase of the S phase, nitric oxide production, and adhesion onto metastatic cells. This, in turn, reduces the number of metastatic cells and results in the increased ratio between splenocytes and metGM in favor of diploid cells (doubling from one to two). Rosetting of leukocytes onto metastatic cells correlates with induction of CD54 expression on tumor cells after NAMI-A in vivo treatment, which in turn, might contribute to metastasis recognition by cytotoxic lymphocytes. The overall antimetastatic activity displayed by NAMI-A might therefore be the result of complex interactions with tumor cells, on which it displays selective antitumor activity, and with host immune cells through which it promotes activation of host immune defenses involved in tumor suppression. 相似文献
4.
Ravera M Baracco S Cassino C Colangelo D Bagni G Sava G Osella D 《Journal of inorganic biochemistry》2004,98(6):984-990
An electrochemical and biological study of interaction between the prototypical antimetastatic drug imidazolium trans-tetrachlorodimethylsulfoxideimidazoleruthenate (III) complex, [ImH][RuCl(4)(DMSO)(Im)] (DMSO = dimethylsulfoxide, Im = imidazole), nicknamed NAMI-A, and several biomolecules, namely DNA, bovine (BSA) and human (HSA) serum albumin, is reported. Electrochemistry offers great advantages over the existing devices based on optical techniques, since it provides rapid, simple, and low-cost information whether the interaction occurs or not. Moreover, we describe some biochemical assays to test the interaction of NAMI-A with ribonucleoprotein telomerase and protein Taq polymerase. All the data confirm the preferential interaction of NAMI-A with proteins with respect to nucleotides, especially when compared with the behaviour of the well-known alkylating drug cisplatin in the presence of the same targets. 相似文献
5.
Trávníček Z Matiková-Mal'arová M Novotná R Vančo J Stěpánková K Suchý P 《Journal of inorganic biochemistry》2011,105(7):937-948
A series of novel octahedral ruthenium(III) complexes involving 6-benzylaminopurine (L) derivatives as N-donor ligands has been prepared by the reaction of [(DMSO)2H][trans-RuCl4(DMSO)2] with the corresponding L derivative. The complexes 1-12 have the general compositions trans-[RuCl4(DMSO)(n-Cl-LH)] ⋅ xSol (1-3), trans-[RuCl4(DMSO)(n-Br-LH)] · xSol (4-6), trans-[RuCl4(DMSO)(n-OMe-LH)] · xSol (7-9) and trans-[RuCl4(DMSO)(n-OH-LH)] · xSol (10-12); n = 2, 3, and 4, x = 0-1.5; and Sol = H2O, DMSO, EtOH and/or (Me)2CO. The complexes have been thoroughly characterized by elemental analysis, UV-visible, FTIR, Raman, and EPR spectroscopy, ES + (positive ionization electrospray) mass spectrometry, thermal analysis, cyclic voltammetry, magnetic and conductivity measurements. The X-ray molecular structure of trans-[RuCl4(DMSO)(3-Br-LH)] ⋅ (Me)2CO (5) revealed the distorted octahedral coordination in the vicinity of the central atom, and also confirmed that the 3-Br-L ligand is present as the N3-protonated N7-H tautomer and is coordinated to Ru(III) through the N9 atom of the purine moiety. The tested complexes have been found to be in vitro non-cytotoxic against K562, G361, HOS and MCF7 human cancer cell lines with IC50 > 100 μM in contrast to the moderate results regarding the antiradical activity with IC50 ≈ 10− 3 M. On the contrary, in vivo antitumor activity screening showed that the prepared Ru(III) complexes possess higher pro-apoptotic activity than NAMI-A. The reduction of Ru(III) to Ru(II) and Ru(II)-species formation in tumor tissues was confirmed by means of a simple method of detection and visualization of intracellular Ru(II) by fluorescence microscopy. The originality of this method is based on the preparation of a Ru(II)-bipyridine complex in situ. 相似文献
6.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,759(1):81-89
Ruthenium(III) complexes of the general formula HL[RuCl4L2], with two trans-standing heterocyclic ligands L bound to ruthenium via nitrogen, show remarkable activity in different tumor models. To obtain a deeper insight into the mode of action of this class of anticancer compounds, we investigated the interaction of HIm trans-[RuCl4(im)2] (im, imidazole) and HInd trans-[RuCl4(ind)2] (ind, indazole) with all four nucleoside monophosphates in buffered solution by means of capillary electrophoresis. A preference for GMP- and AMP-coordination was found. A decrease of the pH resulted in a significantly increased amount of bound nucleotide. This feature seems to be interesting with regard to the lower pH values in solid tumors. 相似文献
7.
Mauro Ravera Elisabetta Gabano Sara Baracco Manuele Sardi Domenico Osella 《Inorganica chimica acta》2008,361(9-10):2879-2886
A study of the electrochemical behavior of a series of antimetastatic mono- and di-ruthenium complexes, namely [Na][trans-RuIIICl4(DMSO)(L)] and [Na]2[{trans-RuIIICl4(DMSO)}2(μ-L)], L = pyrazine (pyz), pyrimidine (pym), 4,4′-bipyridine (bipy), and 1,2-bis-(4-pyridyl)ethylene (etbipy), is reported. The results obtained show that in all dimeric Ru(III) complexes linked by heterocyclic non-chelating N-donor bridges, the two redox centers behave independently (with no remarkable electrochemical interaction), thus conferring no advantage in the likely hypothesis they act as pro-drugs (activation by reduction). Moreover, electrochemical evaluation of interaction between albumin and the title complexes confirms that this protein can act as the vehicle for drugs of this type in blood. 相似文献
8.
Darren Griffith Sara Cecco Ennio Zangrando Alberta Bergamo Gianni Sava Celine J. Marmion 《Journal of biological inorganic chemistry》2008,13(4):511-520
Reaction of 3-pyridinehydroxamic acid and 4-pyridinehydroxamic acid (3-pyha and 4-pyha) with either [NBu4][RuCl4(dmso-S)2] or [(dmso)2H][RuCl4(dmso-S)2] (dmso is dimethyl sulfoxide) in acetone afforded three new ruthenium(III) dimethyl sulfoxide pyridinehydroxamic acid complexes:
[NBu4][trans-RuCl4(dmso-S)(4-pyha)]·CH3COCH3 (1), [3-pyhaH][trans-RuCl4(dmso-S)(3-pyha)] (2) and [4-pyhaH][trans-RuCl4(dmso-S)(4-pyha)] (3). The solid-state structure of [NBu4][trans-RuCl4(dmso-S)(4-pyha)]·CH3COCH3 (1) was determined by X-ray crystallography. 2 and 3 were pharmacologically evaluated for their in vitro cytotoxicity, their ability to inhibit cell invasion and their gelatinase
activity. 2 and 3 were devoid of cytotoxicity against the cell lines tested. 2 inhibited invasion of the highly invasive MDA-MB-231 cells to a much greater extent than 3. Contrary to expectations, neither 2 nor 3 had any inhibitory effect on matrix metalloproteinase (MMP) production and/or activity and in fact 3 was found to enhance the production and/or activity of both MMP-2 and MMP-9. 相似文献
9.
10.
Gianfranco Pintus Bruna Tadolini Anna Maria Posadino Bastiano Sanna Marcella Debidda Federico Bennardini Gianni Sava Carlo Ventura 《European journal of biochemistry》2002,269(23):5861-5870
Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation. 相似文献
11.
A novel effect of the inhibition of the decomposition of amino acids to carbonates on addition of imidazole (HIm) to a reacting system containing equimolar amounts of copper and zinc metal powders, an amino acid [glycine (Hgly), aspartic acid (H2Asp) or glycylglycine (H2gg)] (1:1:2) and excess hydrogen peroxide (H2O2) resulting in formation of a mixed metal mixed ligand peroxo complex compound was observed, because in the absence of imidazole the corresponding reaction system yields only a mixed metal peroxo carbonate. For the resulting complex compounds, the homogeneity, i.e. [Cu(Zn)(O2
2–)(Gly)2(HIm)(H2O)], [Cu(Zn)(O2
2–)(Asp)(HIm)(H2O)2] or [Cu(Zn)2(O2
2–)2(gg)(HIm)(H2O)4], molecular formula, presence of peroxo group and coordination environment were established by combined physicochemical evidence from elemental and thermogravimetric analysis in air and argon atmospheres, electron spin resonance and electronic and IR spectral data. It is noteworthy to mention that the corresponding carboxylic acids of the above-mentioned amino acids, i.e. acetic and succinic acids, either do not decompose to carbonates in the absence of imidazole or form novel homogeneous peroxo mixed metal mixed ligand complex compounds as described above in the presence of imidazole. This suggests an important and significant mutual influence (in vitro) of biologically active chromophores like peroxo ions, imidazole and amino groups in the above-mentioned chemical reactions containing bioactive metals such as copper and zinc. 相似文献
12.
Kuo-Hui Su Kuan-I Lee Song-Kun Shyue Hsiang-Ying Chen Jeng Wei Tzong-Shyuan Lee 《International journal of biological sciences》2014,10(9):990-996
14,15-epoxyeicosatrienoic acid (14,15-EET) is implicated in regulating physiological functions of endothelial cells (ECs), yet the potential molecular mechanisms underlying the beneficial effects in ECs are not fully understood. In this study, we investigated whether transient receptor potential vanilloid receptor type 1 (TRPV1) is involved in 14,15-EET-mediated Ca2+ influx, nitric oxide (NO) production and angiogenesis. In human microvascular endothelial cells (HMECs), 14,15-EET time-dependently increased the intracellular level of Ca2+. Removal of extracellular Ca2+, pharmacological inhibition or genetic disruption of TRPV1 abrogated 14,15-EET-mediated increase of intracellular Ca2+ level in HMECs or TRPV1-transfected HEK293 cells. Furthermore, removal of extracellular Ca2+ or pharmacological inhibition of TRPV1 decreased 14,15-EET-induced NO production. 14,15-EET-mediated tube formation was abolished by TRPV1 pharmacological inhibition. In an animal experiment, 14,15-EET-induced angiogenesis was diminished by inhibition of TRPV1 and in TRPV1-deficient mice. TRPV1 may play a crucial role in 14,15-EET-induced Ca2+ influx, NO production and angiogenesis. 相似文献
13.
F. Frank Isik Nicole S. Gibran Young-Chul Jang Linda Sandell Stephen M. Schwartz 《Journal of cellular physiology》1998,175(2):149-155
Angiogenesis after tissue injury occurs in a matrix environment consisting of fibrin, fibronectin, and vitronectin as the major extracellular matrix (ECM) constituents. ECM-integrin interactions is critical for angiogenesis and failure to bind a ligand to certain integrin receptors (αvβ3 or αvβ5) inhibits angiogenesis. The ligand that binds to αvβ3 or αvβ5 integrin receptors during microvascular angiogenesis has not been identified. Our hypothesis is that provisional matrix molecules provide the environmental context cues to microvascular endothelial cells and promote angiogenesis by decreased programmed cell death. Using cultured human microvascular endothelial cells, we show that vitronectin, in comparison to growth on alternative provisional matrix molecules (fibronectin, fibrinogen plus thrombin), collagen I, and basement membrane molecules (collagen IV), significantly reduces microvascular endothelial cell death in vitro. This reduction was observed using morphologic criteria, TdT-mediated dUTP nick end labeling (TUNEL) assay, histone release into the cytoplasm, and thymidine release into the supernatant. Though our data confirm that vitronectin may bind to more than one integrin receptor to reduce MEC apoptosis, binding to the αv component appears to be the critical integrin subcomponent for reducing apoptosis. J. Cell. Physiol. 175:149–155, 1998. © 1998 Wiley-Liss, Inc. 相似文献
14.
Alethia A. Hostetter Michelle L. Miranda Victoria J. DeRose Karen L. McFarlane Holman 《Journal of biological inorganic chemistry》2011,16(8):1177-1185
[ImH][trans-RuIIICl4(DMSO)(Im)] (where DMSO is dimethyl sulfoxide and Im is imidazole) (NAMI-A) is an antimetastatic prodrug currently in phase
II clinical trials. The mechanisms of action of this and related Ru-based anticancer agents are not well understood, but several
cellular targets have been suggested. Although Ru has been observed to bind to DNA following in vitro NAMI-A exposure, little
is known about Ru–DNA interactions in vivo and even less is known about how this or related metallodrugs might influence cellular
RNA. In this study, Ru accumulation in cellular RNA was measured following treatment of Saccharomyces cerevisiae with NAMI-A. Drug-dependent growth and cell viability indicate relatively high tolerance, with approximately 40% cell death
occurring at 6 h for 450 μM NAMI-A. Significant dose-dependent accumulation of Ru in cellular RNA was observed by inductively coupled plasma mass spectrometry
measurements on RNA extracted from yeast treated with NAMI-A. In vitro, binding of Ru species to drug-treated model DNA and
RNA oligonucleotides at pH 6.0 and 7.4 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry in the presence and absence of the reductant ascorbate. The extent of Ru–nucleotide interactions increases slightly
with lower pH and significantly in the presence of ascorbate, with differences in observed species distribution. Taken together,
these studies demonstrate the accumulation of aquated and reduced derivatives of NAMI-A on RNA in vitro and in cellulo, and
enhanced binding with nucleic acid targets in a tumorlike acidic, reducing environment. To our knowledge, this is also the
first study to characterize NAMI-A treatment of S. cerevisiae, a genetically tractable model organism. 相似文献
15.
Hydrogen sulfide (H2S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs‐926) with NaHS (a H2S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2S had little effect on eNOS protein expression in ECs. L‐cysteine, a precursor of H2S, stimulated NO production whereas blockage of the activity of H2S‐generating enzyme, cystathionine gamma‐lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3‐K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS‐specific siRNA or nitro‐L‐arginine methyl ester (L‐NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2S on ECs. Our results suggest that H2S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt‐dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2S‐induced angiogenesis. 相似文献
16.
Alessio E Iengo E Zorzet S Bergamo A Coluccia M Boccarelli A Sava G 《Journal of inorganic biochemistry》2000,79(1-4):173-177
A novel class of dianionic Ru(III) dimers of formula Na2[[trans-RuCl4(Me2SO)]2(mu-L)], with L = pyrazine (pyz, 1), pyrimidine (pym, 2), 4,4'-bipyridine (bipy, 3), and 1,2-bis(4-pyridine) ethane (etbipy, 4), was developed by us with the specific aim of assessing their antitumor properties. The dimers are in fact structurally related to the antimetastatic mononuclear compound (ImH) [trans-RuCl4(Me2SO)(Im)] (NAMI-A, Im = imidazole). Preliminary results concerning the antineoplastic activity of 1-4 against the murine MCa carcinoma model, a tumor which spontaneously metastasizes in the lungs, are reported. Similarly to what is normally observed with NAMI-A, the treatment with the dimeric complexes was scarcely effective against the growth of the primary tumor. However, dimers 1, 2, and 4 reduced very effectively the number and, in particular, the weight of lung metastases (to about 5% with respect to controls); in particular, Na2[[trans-RuCl4(Me2SO)]2(mu-etbipy)] (4) was as effective as NAMI-A in reducing the spontaneous metastases at a dosage which, in terms of moles of ruthenium, is about 3.5 times lower compared to that normally used for NAMI-A. Furthermore, in vitro tests showed that dimers 1-4 are capable of forming interstrand cross-links with linearized plasmidic DNA in a time-dependent manner. All the dimeric species are more active in inducing cross-links compared to NAMI-A, and the dimer bridged by the etbipy ligand (4) is the most effective among those tested. 相似文献
17.
R. I. Viji V. B. Sameer Kumar M. S. Kiran P. R. Sudhakaran 《Molecular and cellular biochemistry》2009,323(1-2):91-100
Nitric oxide (NO) produced by the action of endothelial nitric oxide synthase (eNOS) plays an important role in the regulation of vascular tone, cell survival, and angiogenesis. Interaction of endothelial cells (ECs) with a fibronectin (FN) rich matrix is important in the regulation of EC function and survival during angiogenesis. The present study was carried out to examine if FN can regulate eNOS and thereby NO levels in ECs. The activity and the levels of mRNA and protein of eNOS were significantly low in HUVECs maintained in culture on FN. Inhibition of p38 MAPK and blocking the interaction of FN with α5β1 integrin using antibody caused the reversal of the FN effect. Immunoblot analysis of Ser/Thr phosphorylation of purified eNOS suggested that FN downregulates post-translational phosphorylation of eNOS at Ser residues. These results suggest that FN negatively modulates eNOS in an α5β1 integrin-p38 MAPK-dependent pathway. 相似文献
18.
Maria S. Novak Gabriel E. Büchel Bernhard K. Keppler Michael A. Jakupec 《Journal of biological inorganic chemistry》2016,21(3):347-356
Since the discovery that nitric oxide (NO) is a physiologically relevant molecule, there has been great interest in the use of metal nitrosyl compounds as antitumor pharmaceuticals. Particularly interesting are those complexes which can deliver NO to biological targets. Ruthenium- and osmium-based compounds offer lower toxicity compared to other metals and show different mechanisms of action as well as different spectra of activity compared to platinum-based drugs. Novel ruthenium- and osmium-nitrosyl complexes with azole heterocycles were studied to elucidate their cytotoxicity and possible interactions with DNA. Apoptosis induction, changes of mitochondrial transmembrane potential and possible formation of reactive oxygen species were investigated as indicators of NO-mediated damage by flow cytometry. Results suggest that ruthenium- and osmium-nitrosyl complexes with the general formula (indazolium)[cis/trans-MCl4(NO)(1H-indazole)] have pronounced cytotoxic potency in cancer cell lines. Especially the more potent ruthenium complexes strongly induce apoptosis associated with depolarization of mitochondrial membranes, and elevated reactive oxygen species levels. Furthermore, a slight yet not unequivocal trend to accumulation of intracellular cyclic guanosine monophosphate attributable to NO-mediated effects was observed. 相似文献
19.
Clyde A. Smith Andrew J. Sutherland-Smith Felix Kratz E. N. Baker B. H. Keppler 《Journal of biological inorganic chemistry》1996,1(5):424-431
The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm2Cl4], HInd[RuInd2Cl4] and (HInd)2 [RuIndCl5], where Im?=?imidazole and Ind?=?indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6?Å, 2.2?Å and 2.4?Å respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3?Å data) and 0.192 (for 8.0 to 2.4?Å data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins. 相似文献
20.
Cristina Bardita Dan Predescu Matthew J. Justice Irina Petrache Sanda Predescu 《Apoptosis : an international journal on programmed cell death》2013,18(1):57-76
Intersectin-1s (ITSN-1s) is a general endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, via MEK/Erk1/2MAPK signaling. To investigate the in vivo effects of ITSN-1s deficiency and the resulting ECs apoptosis on pulmonary vasculature and lung homeostasis, we used an ITSN-1s knocked-down (KDITSN) mouse generated by repeated delivery of a specific siRNA targeting ITSN-1 gene (siRNAITSN). Biochemical and histological analyses as well as electron microscopy (EM) revealed that acute KDITSN [3-days (3d) post-siRNAITSN treatment] inhibited Erk1/2MAPK pro-survival signaling, causing significant ECs apoptosis and lung injury; at 10d of KDITSN, caspase-3 activation was at peak, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive ECs showed 3.4-fold increase, the mean linear intercept (MLI) showed 48 % augment and pulmonary microvessel density as revealed by aquaporin-1 staining (AQP-1) decreased by 30 %, all compared to controls; pulmonary function was altered. Concomitantly, expression of several growth factors known to activate Erk1/2MAPK and suppress Bad pro-apoptotic activity increased. KDITSN altered Smads activity, downstream of the transforming growth factor beta-receptor-1 (TβR1), as shown by subcellular fractionation and immunoblot analyses. Moreover, 24d post-siRNAITSN, surviving ECs became hyper-proliferative and apoptotic-resistant against ITSN-1s deficiency, as demonstrated by EM imaging, 5-bromo-deoxyuridine (BrdU) incorporation and Bad-Ser112/155 phosphorylation, respectively, leading to increased microvessel density and repair of the injured lungs, as well as matrix deposition. In sum, ECs endocytic dysfunction and apoptotic death caused by KDITSN contribute to the initial lung injury and microvascular loss, followed by endothelial phenotypic changes and microvascular remodeling in the remaining murine pulmonary microvascular bed. 相似文献