Also published as: Sabatella M, Pioli M, Maji M, Juszczak M, Vinoda Rani M, Mariadason, J M, Gallardo M, Böhme M, Kasprzak M, Alfiean M, Zalibera M, Mitrović M, Bartholomä M, Yue M, Ren M, Osmak M, Tian M, Jakubaszek M, Brook M, Gouveia M, Aldrovandi M, Dénes M, Woldeselassie M, Harlos M, Schaier M, Klajner M, Qian M, Schlame M, Albanell-Fernández M, Harkiolaki M, Lucas M, Nomura M, Zeng M, Acharya M, Dickerson M, Hektoen, M, Walczyk M, Muralisankar M, Lekka M, Font-Bardia M, Ionta M, Nieddu M, Nabissi M, Palaniandavar M, Sztiller-Sikorska M, Porchia M, Zheng M, Ranjani M, Aatif A M, Abinaya M, Feelisch M, Devocelle M, Assfalg M, Zienkiewicz-Machnik M, Cecchini M, Subramani M, Lari M, Zoldakova M, Mozzicafreddo M, Milczarek M, Olivar-Villanueva M, Hernaez M, Sá M, Rothemund M, Vilaseca M, Feizi-Dehnayebi M, Ouyang M, Liu M, Bortoluzzi M, Piccioli M, Dürst M, Kostić M, Sedić M, Káplár M, Ahn M, Contel M, Vraneš M, Holtgrewe M, Lv M, Delibašić M, Vaquero M, Dow M, Patra M, Kou M, Lo Bello M, Bian M, Shen M, Dalla Pozza M, Li M, Fogagnolo M, Bosch M, Bazett M, Hadiji M, Muir M, Qin M, Meyer M, Chorilli M, Panigati M, Singh M, Kerou M, Grazul M, Schmid M, Zhang M, Conrad M, Shaloski M, Concepción Gimeno M, Lanznaster M, Međedović M, Xie M, Gao M, Dulović M, Haghdoost M, Miñana M, Hirahara M, Capdevila M, Sanaú M, Létourneau M, Chikuma M, Martínez-Estévez M, Matiková-Mal'arová M, Magrane M, Abovsky M, Rojo de la Vega M, Caraglia M, Sattler M, Folgueira M, Shukla M, Dontenwill M, Camacho-Artacho M, Verma M, Lesser, M, El Sibai M, Sarkar M, Peruzzini M, Chen M, Zou M, Kokoschka M, Martins M, Kandawa-Shultz M, Rusz M, Lange M, Yu M, Markuliak M, Dodds M, Reithofer M, Chesi M, Xiangjun M, Nechay M, Kotlyar M, Helena Garcia M, Guelfi M, Berecka M, Milovanović M, Shee M, Gladkikh M, Savic M, Khater M, Seldin M, Wills M, Myint M, Živanović M, Korb M, Abid M, Zhou M, Feuermann M, Taghizadeh Shool M, Koester M, Bruno, Peter M, Guevara M, Planas M, Block M, Mastore M, Papadakis M, Pellegrino M, Tancredi M, Cocchietto M, Mohanraj M, Zain Aldin M, Arif M, Guerrero M, Aleksandrova M, Borsari M, Huang M, Malček M, Kaplanis M, Zhong M, Lapins M, Park M, Rúbio, Guilherme M D M, Vojtek M, Gazvoda M, Girek M, Piccolo M, Kubanik M, Tharaud M, Shao M, Pan M, Azam M, Nakai M, Pruess M, Siegler M, Piškor M, Jordà-Redondo M, Knopp M, Qasim Warraich M, Bicho M, Mauro M, Ohtsuji M, Piccolella M, Korkmaz M, Yang M, Tang M, Khawar Rauf M, Massi M, Paravatou-Petsotas M, Preiner M, Kaiser M, Karpiel M, Danyel M, Lavaud M, Koronkiewicz M, Sidhoum M, Hanif M, Brabender M, Fernandez M, Peretz M, Matera M, Lin M, Matković M, Łomzik M, Zhao M, Cohen M, Groessl M, Pozzato M, Corsini M, Dobroschke M, Bubrin M, Haukka M, P M, Hammad M, Wagner M, Grätzel M, Raftari M, Lehvaslaiho M, Spehr M, Micksche M, Falasca M, Hannink M, Grigalunas M, Sameni M, Velusamy M, Oleszak M, Carcelli M, Momcilovic M, Kosanić M, Trichet M, Duan M, Trifuoggi M, Gelbcke M, Salamini-Montemurri M, Novak M, de Souza Oliveira M, Xia M, Sun M, He M, Mohanty M, Lutz M, Hejl M, Juhas M, Kim M, Navarro M, Pfeffer M, Więckowska-Szakiel M, Salome M, Krenn M, Melchart M, Yamamoto M, Pongratz M, Jovanović M, Koukouvitaki M, Brown, Lewis M, Fan M, Spink M, Hollenstein M, Chhabra M, Abul Farah M, Zeller M, Ali M, Salmain M, Buczkowska M, Nikhil M, Tourte M, Digman M, Bacac M, Babin M, Paulpandi M, Hu M, Rincón M, Clémancey M, Crestani M, Skreta M, Nieger M, Alagesan M, López Torres M, Scarpi-Luttenauer M, Albrecht M, Murali M, Lesiów M, Dotou M, Pavlović M, Negi M, Dharmasivam M, Richert M, Wühr M, Azmanova M, González-Bártulos M, Angeletti M, Milenković M, Soudani M, Fandzloch M, Cargile M, Sajid Ali M, Tampere M, Casals M, Ganeshpandian M, Pernar M, Hetu M, Machuqueiro M, Lepoivre M, Soula M, Yao M, Iglesias M, Marloye M, Hooshmand M, Tuohan M, M M, Skocic M, Butinar M, Erby M, Riisom M, Monari M, Ibarrola-Villava M, Tanić M, Pizurica M, Chakrabarti M, Koch M, Grujović M, Đorđić Crnogorac M, Scaccaglia M, Wang M, Goldberg M, Audano M, Orts-Arroyo M, Zegke M, Nieminen M, Galanski M, Bette M, Monsalve M, Martínez M, Storch M, Cieslak M, Frik M, El-Sibai M, Ferrer M, Redrado M, Musthafa M, Płotek M, Pérez-Manrique M, Rossi M, Ravi M, Schmidlehner M, Pinto M, Dodson M, Goicuría M, Martínez-Alonso M, Eknæs, M, Řezáčová M, Cuccioloni M
In recent years, Ru(iii) complexes have emerged as a new class of effective anticancer agents against tumors that proved to be resistant to all other chemotherapeutic drugs currently in clinical use. Show more
In recent years, Ru(iii) complexes have emerged as a new class of effective anticancer agents against tumors that proved to be resistant to all other chemotherapeutic drugs currently in clinical use. To extend our previous studies on metal complexes containing sulfur-donor ligands, we report here on the synthesis and characterization, by means of several spectroscopic and analytical techniques, some [Ru(RSDT)(3)] and [Ru(2)(RSDT)(5)]Cl complexes with dithiocarbamato ligands derived from methyl/ethyl/tert-butyl esters of sarcosine. Their electrochemical behaviour was also studied by cyclic voltammetry. All the complexes were tested for their cytotoxicity on a panel of human tumor cell lines showing highly significant antitumor activity. The chemical and biological properties of the newly synthesized complexes, were compared with those of [Ru(DMDT)(3)] and [Ru(2)(DMDT)(5)]Cl species (DMDT = N,N-dimethyldithiocarbamate) whose chemical (not biological) characterization has been already reported in literature. Show less
The complexes mer-[RhCl 3(DMSO-kappa S)(pp)] 1a- 5a may be prepared by reaction of mer,cis-[RhCl 3(DMSO-kappa S) 2(DMSO-kappa O)] with the appropriate polypyridyl ligand (pp = bpy, phen, dpq, dppz, dp Show more
The complexes mer-[RhCl 3(DMSO-kappa S)(pp)] 1a- 5a may be prepared by reaction of mer,cis-[RhCl 3(DMSO-kappa S) 2(DMSO-kappa O)] with the appropriate polypyridyl ligand (pp = bpy, phen, dpq, dppz, dppn) in CH 3OH/H 2O solution at 75 degrees C. The mer isomers of 1a- 5a are stable in chloroform solution but those of 1a and 2a isomerize rapidly to a mixture of fac and mer isomers in DMSO. The complexes are potent in vitro cytotoxic agents and exhibit IC 50 values that are strongly dependent on the size of the polypyridyl ligand. IC 50 values of, respectively, 4.0 (0.5) and 1.9 (0.5), 0.40 (0.06) and 0.19 (0.05), and 0.079 (0.012) and 0.069 (0.021) microM are observed for 1a- 3a against the human cell lines MCF-7 (breast cancer) and HT-29 (colon cancer). Cellular uptake studies showed a rapid and high accumulation of the polypyridyl compounds. Treatment of HT-29 and MCF-7 cells with 3a leads to significant decreases in cellular oxygen consumption and the rate of extracellular acidification. Show less
Ruthenium-arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione-S-transferase (GST) inhibitors with alternate modes of activity through the org Show more
Ruthenium-arene complexes conjugated to ethacrynic acid were prepared as part of a strategy to develop novel glutathione-S-transferase (GST) inhibitors with alternate modes of activity through the organometallic fragment, ultimately to provide targeted ruthenium-based anticancer drugs. Enzyme kinetics and electrospray mass spectrometry experiments using GST P1-1 and its cysteine-modified mutant forms revealed that the complexes are effective enzyme inhibitors, but they also rapidly inactivate the enzyme by covalent binding at Cys 47 and, to a lesser extent, Cys 101. They are highly effective against the GST Pi-positive A2780 and A2780cisR ovarian carcinoma cell lines, are among the most effective ruthenium complexes reported so far, and target ubiquitous GST Pi overexpressed in many cancers. Show less
[Ru(tBu2bpy)2(2-appt)](PF6)2 [1.(PF6)2, tBu2bpy = 4,4'-di-tert-butyl-2,2'-bipyridine, 2-appt = 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine] and [Re(CO)3(2-appt)Cl] (2) were prepared and charact Show more
[Ru(tBu2bpy)2(2-appt)](PF6)2 [1.(PF6)2, tBu2bpy = 4,4'-di-tert-butyl-2,2'-bipyridine, 2-appt = 2-amino-4-phenylamino-6-(2-pyridyl)-1,3,5-triazine] and [Re(CO)3(2-appt)Cl] (2) were prepared and characterized by X-ray crystal analysis. The binding of 1.(PF6)2 and 2 to calf thymus DNA (ct DNA) led to increases in the DNA melting temperature (Delta Tm = +12 degrees C), modest hypochromism (29% and 5% of the absorption bands at lambda max = 450 and 376 nm, respectively), and insignificant shifts in the absorption maxima. The binding constants of 1.(PF6)2 and 2 with ct DNA, as determined by absorption titration, are (8.9 +/- 0.5) x 104 and (3.6 +/- 0.1) x 104 dm3 mol-1, respectively. UV-vis absorption titration, DNA melting studies, and competition dialysis using synthetic oligonucleotides [poly(dA-dT)2 and poly(dG-dC)2] revealed that 1.(PF6)2 and 2 exhibit a binding preference for AT sequences. A modeling study on the interaction between 1 or 2 and B-DNA revealed that the minor groove is the most favored binding site and an extensive hydrogen-bonding network is formed. As determined by MTT assays, 1.(PF6)2 and 2 exhibited moderate cytotoxicities toward several human cancer cell lines (KB-3-1, HepG2, and HeLa), as well as a multi-drug-resistant cancer cell line (KB-V-1). According to confocal microscopic and flow cytometric studies, 1.(PF6)2 and 2 induced apoptosis (50-60%) in cancer cells with <5% necrosis detected. Show less
The antitumour activity of the organometallic ruthenium(ii)-arene mixed phosphine complexes, [Ru(eta(6)-p-cymene)Cl(PTA)(PPh(3))]BF(4) and [Ru(eta(6)-C(6)H(5)CH(2)CH(2)OH)Cl(PTA)(PPh(3))]BF(4) (PTA = Show more
The antitumour activity of the organometallic ruthenium(ii)-arene mixed phosphine complexes, [Ru(eta(6)-p-cymene)Cl(PTA)(PPh(3))]BF(4) and [Ru(eta(6)-C(6)H(5)CH(2)CH(2)OH)Cl(PTA)(PPh(3))]BF(4) (PTA = 1,3,5-triaza-7-phosphaadamantane), have been evaluated in vitro and compared to their RAPTA analogues, [Ru(eta(6)-p-cymene)Cl(2)(PTA)] and [Ru(eta(6)-C(6)H(5)CH(2)CH(2)OH)Cl(2)(PTA)] . The results show that the addition of the PPh(3) ligand to increases the cytotoxicity towards the TS/A adenocarcinoma cancer cells, which correlates with increased uptake, but also increases cytotoxicity to non-tumourigenic HBL-100 cells, thus decreasing selectivity. The decrease in selectivity has been correlated to increased DNA interactions relative to proteins, demonstrated by reactivity of the compounds with a 14-mer oligonucleotide and the model proteins ubiquitin and cytochrome-c. Show less
Ru(II) eta6-arene complexes containing p-cymene (p-cym), tetrahydronaphthalene (thn), benzene (bz), or biphenyl (bip), as the arene, phenylazopyridine derivatives (C5H4NN:NC6H5R; R = H (azpy), OH (azp Show more
Ru(II) eta6-arene complexes containing p-cymene (p-cym), tetrahydronaphthalene (thn), benzene (bz), or biphenyl (bip), as the arene, phenylazopyridine derivatives (C5H4NN:NC6H5R; R = H (azpy), OH (azpy-OH), NMe2 (azpy-NMe2)) or a phenylazopyrazole derivative (NHC3H2NN:NC6H5NMe2 (azpyz-NMe2)) as N,N-chelating ligands and chloride as a ligand have been synthesized (1-16). The complexes are all intensely colored due to metal-to-ligand charge-transfer Ru 4d6-pi* and intraligand pi -->pi* transitions (eta = 5000-63 700 M-1 cm-1) occurring in the visible region. In the crystal structures of [(eta6-p-cym)Ru(azpy)Cl]PF6 (1), [(eta6-p-cym)Ru(azpy-NMe2)Cl]PF6 (5), and [(eta6-bip)Ru(azpy)Cl]PF6 (4), the relatively long Ru-N(azo) and Ru-(arene-centroid) distances suggest that phenylazopyridine and arene ligands can act as competitive pi-acceptors toward Ru(II) 4d6 electrons. The pKa* values of the pyridine nitrogens of the ligands are low (azpy 2.47, azpy-OH 3.06 and azpy-NMe2 4.60), suggesting that they are weak sigma-donors. This, together with their pi-acceptor behavior, serves to increase the positive charge on ruthenium, and together with the pi-acidic eta6-arene, partially accounts for the slow decomposition of the complexes via hydrolysis and/or arene loss (t(1/2) = 9-21 h for azopyridine complexes, 310 K). The pKa* of the coordinated water in [(eta6-p-cym)Ru(azpyz-NMe2)OH2]2+ (13A) is 4.60, consistent with the increased acidity of the ruthenium center upon coordination to the azo ligand. None of the azpy complexes were cytotoxic toward A2780 human ovarian or A549 human lung cancer cells, but several of the azpy-NMe2, azpy-OH, and azpyz-NMe2 complexes were active (IC50 values 18-88 microM). Show less
We report structure-activity relationships for organometallic RuII complexes of the type [(eta6-arene)Ru(XY)Cl]Z, where XY is an N,N- (diamine), N,O- (e.g., amino acidate), or O,O- (e.g., beta-diketon Show more
We report structure-activity relationships for organometallic RuII complexes of the type [(eta6-arene)Ru(XY)Cl]Z, where XY is an N,N- (diamine), N,O- (e.g., amino acidate), or O,O- (e.g., beta-diketonate) chelating ligand, the arene ranges from benzene derivatives to fused polycyclic hydrocarbons, and Z is usually PF6. The X-ray structures of 13 complexes are reported. All have the characteristic "piano-stool" geometry. The complexes most active toward A2780 human ovarian cancer cells contained XY=ethylenediamine (en) and extended polycyclic arenes. Complexes with polar substituents on the arene or XY=bipyridyl derivatives exhibited reduced activity. The activity of the O,O-chelated complexes depended strongly on the substituents and on the arene. For arene=p-cymene, XY=amino acidate complexes were inactive. Complexes were not cross-resistant with cisplatin, and cross-resistance to Adriamycin was circumvented by replacing XY=en with 1,2-phenylenediamine. Some complexes were also active against colon, pancreatic, and lung cancer cells. Show less
The antitumor activity of the organometallic ruthenium(II)-arene complexes, RuCl(2)(eta(6)-arene)(PTA), (arene = p-cymene, toluene, benzene, benzo-15-crown-5, 1-ethylbenzene-2,3-dimethylimidazolium te Show more
The antitumor activity of the organometallic ruthenium(II)-arene complexes, RuCl(2)(eta(6)-arene)(PTA), (arene = p-cymene, toluene, benzene, benzo-15-crown-5, 1-ethylbenzene-2,3-dimethylimidazolium tetrafluoroborate, ethyl benzoate, hexamethylbenzene; PTA = 1,3,5-triaza-7-phosphaadamantane), abbreviated RAPTA, has been evaluated. In vitro biological experiments demonstrate that these compounds are active toward the TS/A mouse adenocarcinoma cancer cell line whereas cytotoxicity on the HBL-100 human mammary (nontumor) cell line was not observed at concentrations up to 0.3 mM, which indicates selectivity of these ruthenium(II)-arene complexes to cancer cells. Analogues of the RAPTA compounds, in which the PTA ligand is methylated, have also been prepared, and these prove to be cytotoxic toward both cell lines. RAPTA-C and the benzene analogue RAPTA-B were selected for in vivo experiments to evaluate their anticancer and antimetastatic activity. The results show that these complexes can reduce the growth of lung metastases in CBA mice bearing the MCa mammary carcinoma in the absence of a corresponding action at the site of primary tumor growth. Pharmacokinetic studies of RAPTA-C indicate that ruthenium is rapidly lost from the organs and the bloodstream. Show less
Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance Show more
Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium(II) organometallic arene complexes in human ovarian cancer. Br J Cancer 2002;86(10):1652-7]. [(eta6-C6H5C6H5)Ru(en)Cl]+ (as a PF6 salt, where en=ethylenediamine (RM175)) has been evaluated in a 13-cell line panel. Particular sensitivity (approximately 10-fold lower than mean IC50) was noted in breast cancer and non-small cell lung cancer cell lines. In addition, IC50 in the A549 was 2 microM and RM175 (25 mg kg-1, days 1 and 5, i.p.) caused a significant (p=0.004) growth delay in a xenograft model. HC11 [(eta6-tetrahydroanthracene)Ru(en)Cl]PF6 was more potent in the A549 cell line (IC50 0.5 microM). HC11 (25 mg kg-1, days 1, 8 and 15, i.p.) was also active in vivo. Following RM175 25 mg kg-1, days 1 and 5, and 15 mg kg-1, days 1-5, HC11 25 and 40 mg kg-1, day 1, elevated alanine transaminase levels were detected, suggesting hepatotoxicity. No changes were observed in kidney or haematological parameters. In liver sections, multi-focal hepatic necrosis was seen, becoming confluent at high doses of HC11. In vitro studies confirmed that HC11 was more toxic than RM175 to fresh human hepatocytes and equitoxic to mithramycin. Liver toxicity may be related to the arene ligand and modification may reduce the potential for hepatic toxicity, while maintaining the anti-tumour activity seen. Show less
Antineoplastic ruthenium(III) complexes are generally regarded as prodrugs, being activated by reduction. Within a homologous series of ruthenium(III) complexes, cytotoxic potency is therefore expecte Show more
Antineoplastic ruthenium(III) complexes are generally regarded as prodrugs, being activated by reduction. Within a homologous series of ruthenium(III) complexes, cytotoxic potency is therefore expected to increase with increasing ease of reduction. Complexes of the general formula [Ru(III)Cl((6-n))(ind)n](3-n)- (n = 0-4; ind = indazole; counterions = Hind(+) or Cl(-)) and the compound trans-[Ru(II)Cl(2)(ind)(4)] have been prepared and characterized electrochemically. Lever's parametrization method predicts that a higher indazole-to-chloride ratio results in a higher reduction potential, which is confirmed by cyclic voltammetry. In vitro antitumor potencies of these complexes in colon cancer cells (SW480) and ovarian cancer cells (CH1) vary by more than 2 orders of magnitude and increase in the following rank order: [Ru(III)Cl(6)](3-) < [Ru(III)Cl(4)(ind)(2)](-) < [Ru(III)Cl(5)(ind)](2-) << [Ru(III)Cl(3)(ind)(3)] < [Ru(III)Cl(2)(ind)(4)](+) approximately [Ru(II)Cl(2)(ind)(4)]. Thus, the observed differences in potency correlate with reduction potentials largely, though not perfectly, pointing to the influence of additional factors. Differences in the cellular uptake (probably resulting from different lipophilicity) contribute to this correlation but cannot solely account for it. Show less
The metallic compound cisplatin has been used for many years to treat various human cancers. Here, we describe the cytostatic and cytotoxic properties of a new class of organometallic compounds that c Show more
The metallic compound cisplatin has been used for many years to treat various human cancers. Here, we describe the cytostatic and cytotoxic properties of a new class of organometallic compounds that contain a ruthenium (II) atom covalently linked to carbon and nitrogen atoms. We found that several ruthenium-derived compounds (RDCs) led to G1 arrest and induced apoptosis in tumor cell lines derived from glioblastomas, neuroblastomas, and lymphoid tumors at least as efficiently as cisplatin. We further analyzed the signaling pathways underlying these effects, and we showed that both RDCs and cisplatin induced p53 and p73 protein levels but with different intensities and kinetics. This accumulation of p53 and p73 proteins correlated with an increase in p21 and Bax expression, two p53 target genes linked to cell growth arrest and apoptosis. However, in contrast to cisplatin-induced apoptosis, overexpression of DeltaNp73, a p53 and p73 dominant-negative isoform, only partly reduced RDC-induced apoptosis, suggesting p53-dependent and p53-independent modes of action. This observation was further confirmed by the ability of RDC to induce apoptosis in p53-/- cells. Altogether, this study highlights key cellular and molecular features of RDCs and suggests that further development of this new class of compounds may contribute to improve future chemotherapeutic protocols. Show less
Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of Show more
Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system. Show less
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regu Show more
The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress. Show less
The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of o Show more
The platinum compound oxaliplatin has been shown to be an effective chemotherapeutic agent for the treatment of colorectal cancer. In this study, we investigate the molecular mechanisms of action of oxaliplatin to identify means of predicting response to this agent. Exposure of colon cancer cells to oxaliplatin resulted in G2/M arrest and apoptosis. Immunofluorescent staining demonstrated that the apoptotic cascade initiated by oxaliplatin is characterised by translocation of Bax to the mitochondria and cytochrome c release into the cytosol. Oxaliplatin treatment resulted in caspase 3 activation and oxaliplatin-induced apoptosis was abrogated by inhibition of caspase activity with z-VAD-fmk, but was independent of Fas/FasL association. Targeted inactivation of Bax or p53 in HCT116 cells resulted in significantly increased resistance to oxaliplatin. However, the mutational status of p53 was unable to predict response to oxaliplatin in a panel of 30 different colorectal cancer cell lines. In contrast, the expression profile of these 30 cell lines, assessed using a 9216-sequence cDNA microarray, successfully predicted the apoptotic response to oxaliplatin. A leave-one-out cross-validation approach was used to demonstrate a significant correlation between experimentally observed and expression profile predicted apoptosis in response to clinically achievable doses of oxaliplatin (R=0.53; P=0.002). In addition, these microarray experiments identified several genes involved in control of apoptosis and DNA damage repair that were significantly correlated with response to oxaliplatin. Show less
KP1019 [indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] (FFC14A) is a metal complex with promising anticancer activity. Since chemoresistance is a major obstacle in chemotherapy, this st Show more
KP1019 [indazolium trans-[tetrachlorobis(1H-indazole)ruthenate (III)] (FFC14A) is a metal complex with promising anticancer activity. Since chemoresistance is a major obstacle in chemotherapy, this study investigated the influence of several drug resistance mechanisms on the anticancer activity of KP1019. Here we demonstrate that the cytotoxic effects of KP1019 are neither substantially hampered by overexpression of the drug resistance proteins multidrug resistance-related protein 1, breast cancer resistance protein, and lung resistance protein nor the transferrin receptor and only marginally by the cellular p53 status. In contrast, P-glycoprotein overexpression weakly but significantly (up to 2-fold) reduced KP1019 activity. P-glycoprotein-related resistance was based on reduced intracellular KP1019 accumulation and reversible by known P-glycoprotein modulators. KP1019 dose dependently inhibited ATPase activity of P-glycoprotein with a K(i) of approximately 31 microM. Furthermore, it potently blocked P-glycoprotein-mediated rhodamine 123 efflux under serum-free conditions (EC(50), approximately 8 microM), however, with reduced activity at increased serum concentrations (EC(50) at 10% serum, approximately 35 microM). Moreover, P-glycoprotein-mediated daunomycin resistance could only be marginally restored by KP1019 in serum-containing medium, also indicating an influence of serum proteins on the interaction between KP1019 and P-glycoprotein. Acquired KP1019 resistance was investigated by selecting KB-3-1 cells against KP1019 for more than 1 year. Only an approximately 2-fold KP1019 resistance could be induced, which unexpectedly was not due to overexpression of P-glycoprotein or other efflux pumps. Accordingly, KP1019-resistant cells did not display reduced drug accumulation. Their unique cross-resistance pattern confirmed an ABC transporter-independent resistance phenotype. In summary, the likeliness of acquiring insensitivity to KP1019 during therapy is expected to be low, and resistance should not be based on overexpression of drug efflux transporters. Show less
The reaction of trans-[RuCl(2)(PPh(3))(3)] (Ph = C(6)H(5)) with 2-thio-1,3-pyrimidine (HTPYM) and 6-thiopurines (TPs) produced mainly crystalline solids that consist of cis,cis,trans-[Ru(PPh(3))(2)(N, Show more
The reaction of trans-[RuCl(2)(PPh(3))(3)] (Ph = C(6)H(5)) with 2-thio-1,3-pyrimidine (HTPYM) and 6-thiopurines (TPs) produced mainly crystalline solids that consist of cis,cis,trans-[Ru(PPh(3))(2)(N,S-TPYM)(2)] (1) and cis,cis,trans-[Ru(PPh(3))(2)(N(7),S-TPs)(2)]X(2) (X = Cl(-), CF(3)SO(3)(-)). In the case of TPs, other coordination isomers have never been isolated and reported. Instead, the mother liquor obtained after filtration of 1 produced red single crystals of trans,cis,cis-[Ru(PPh(3))(2)(N,S-TPYM)(2)].2H(3)O(+).2Cl(-) (2.2H(3)O(+).2Cl(-)). Selected ruthenium(II)-thiobase complexes were studied for their structural, reactivity, spectroscopic, redox, and cytotoxic properties. Single crystals of 1 contain thiopyrimidinato anions chelated to the metal center via N and S. The Ru[bond]N bonds are significantly elongated for 1 [2.122(2) and 2.167(2) A] with respect to 2 [2.063(3) A] because of the trans influence from PPh(3). The coordination pseudo-octahedron for 2 is significantly elongated at the apical sites (PPh(3) ligands). Solutions of cis,cis,trans isomers in air are stable for weeks, whereas those of 2 turn green within 24 h, in agreement with the respective redox potentials. cis,cis,trans- and trans,cis,cis-[Ru(PH(3))(2)(N,S-TPYM)(2)], as optimized through the DFT methods at the Becke3LYP level are in good agreement with experimental geometrical parameters (1 and 2), with cis,cis,trans being more stable than trans,cis,cis by 3.88 kcal. The trend is confirmed by molecular modeling based on semiempirical (ZINDO/1) and molecular mechanics (MM) methods. Cytotoxic activity measurements for cis,cis,trans-[Ru(PPh(3))(N-THZ)(N(7),S -H(2)TP)(2)]Cl(2) (4) (THZ = thiazole, H(2)TP = 6-thiopurine) and cis,cis,trans-[Ru(PPh(3))(2)(N(7),S-HTPR)2]Cl(2) (5) (HTPR = 6-thiopurine riboside) against ovarian cancer cells A2780/S gave IC(50) values of 17 +/- 1 and 29 +/- 9 microM, respectively. Furthermore, the spectral analysis of HTPYM, TPs, and their Ru(II) complexes in solution shows that intense absorptions occur in the UVA/vis region of light, whereas standard nucleobases absorb in the UVB region. Show less
New water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes, all derivatives of the highly cytotoxic alpha-[Ru(azpy)(2)Cl(2)] (alpha denoting the coordinating pairs Cl, N(py), and N(azo) as cis, Show more
New water-soluble bis(2-phenylazopyridine)ruthenium(II) complexes, all derivatives of the highly cytotoxic alpha-[Ru(azpy)(2)Cl(2)] (alpha denoting the coordinating pairs Cl, N(py), and N(azo) as cis, trans, cis, respectively) have been developed. The compounds 1,1-cyclobutanedicarboxylatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(cbdca-O,O')] (1), oxalatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(ox)] (2), and malonatobis(2-phenylazopyridine)ruthenium(II), alpha-[Ru(azpy)(2)(mal)] (3), have been synthesized and fully characterized. X-ray analyses of 1 and 2 are reported, and compound 1 is the first example in which the cbdca ligand is coordinated to a ruthenium center. The cytotoxicity of this series of water-soluble bis(2-phenylazopyridine) complexes has been determined in A2780 human ovarian carcinoma and A2780cisR, the corresponding cisplatin-resistant cell line. For comparison reasons, the cytotoxicity of the complexes alpha-[Ru(azpy)(2)Cl(2)], alpha-[Ru(azpy)(2)(NO(3))(2)], beta-[Ru(azpy)(2)Cl(2)] (beta indicating the coordinating pairs Cl, N(py), and N(azo) as cis, cis, cis, respectively), and beta-[Ru(azpy)(2)(NO(3))(2)] have been determined in this cell line. All the bis(2-phenylazopyridine)ruthenium(II) compounds display a promising cytotoxicity in the A2780 cell line (IC(50) = 0.9-10 microM), with an activity comparable to that of cisplatin and even higher than the activity of carboplatin. Interestingly, the IC(50) values of this series of ruthenium compounds (except the beta isomeric compounds) are similar in the cisplatin-resistant A2780cisR cell line compared to the normal cell line A2780, suggesting that the activity of these compounds might not be influenced by the multifactorial resistance mechanism that affect platinum anticancer agents. Show less
Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen Show more
Ruthenium complexes offer the potential of reduced toxicity, a novel mechanism of action, non-cross resistance and a different spectrum of activity compared to platinum containing compounds. Thirteen novel ruthenium(II) organometallic arene complexes have been evaluated for activity (in vitro and in vivo) in models of human ovarian cancer, and cross-resistance profiles established in cisplatin and multi-drug-resistant variants. A broad range of IC50 values was obtained (0.5 to >100 microM) in A2780 parental cells with two compounds (RM175 and HC29) equipotent to carboplatin (6 microM), and the most active compound (HC11) equipotent to cisplatin (0.6 microM). Stable bi-dentate chelating ligands (ethylenediamine), a more hydrophobic arene ligand (tetrahydroanthracene) and a single ligand exchange centre (chloride) were associated with increased activity. None of the six active ruthenium(II) compounds were cross-resistant in the A2780cis cell line, demonstrated to be 10-fold resistant to cisplatin/carboplatin by a mechanism involving, at least in part, silencing of MLH1 protein expression via methylation. Varying degrees of cross-resistance were observed in the P-170 glycoprotein overexpressing multi-drug-resistant cell line 2780AD that could be reversed by co-treatment with verapamil. In vivo activity was established with RM175 in the A2780 xenograft together with non-cross-resistance in the A2780cis xenograft and a lack of activity in the 2780AD xenograft. High activity coupled to non cross-resistance in cisplatin resistant models merit further development of this novel group of anticancer compounds. Show less