Intracellular mapping of the distribution of metals derived from the antitumor metallocenes

The intracellular distribution of transition metals in V79 Chinese hamster lung cells treated with subtoxic doses of the organometallic anticancer complexes Cp₂MCl₂, where Cp is η ⁵ -cyclopentadienyl and M is Mo, Nb, Ti, or V, has been studied by synchrotron-based X-ray fluorescence (XRF). While significantly higher concentrations of Mo and Nb were found in treated cells compared with control cells, distinct differences in the cellular distribution of each metal were observed. Analysis of thin sections of cells was consistent with some localization of Mo in the nucleus. Studies with a noncytotoxic thiol derivative of molybdocene dichloride showed an uneven distribution of Mo in the cells. For comparison, the low levels of Ti and V in cells treated with the more toxic titanocene and vanadocene complexes, respectively, resulted in metal concentrations at the detection limit of XRF. The results agree with independent chemical studies that have concluded that the biological chemistry of each of the metallocene dihalides is unique.Electronic Supplementary Material Supplementary material is available for this article at .     

Inhibition of human topoisomerase II by the antitumor metallocenes

The ability of antitumor active metallocenes Cp2MCl2, (M=Ti, V, Mo, Nb) and the biologically inactive derivative (MeCp)2TiCl2, to inhibit the relaxation of supercoiled plasmid DNA pBR322 by human topoisomerase II has been studied by gel electrophoresis. All metallocenes inhibit the enzyme with maximum inhibition observed at 2.0 mM (Cp2TiCl2), 3.0 mM (Cp2MoCl2), 0.2 mM (Cp2NbCl2), 0.25 mM (Cp2VCl2) and 2.0 mM (MeCpTiCl2). The implications for the mechanism of antitumor activity of the metallocene dihalides are discussed.     

A (1)H NMR study of the interaction of antitumor metallocenes with glutathione

The interaction of the antitumor active metallocene dihalides Cp(2)MCl(2) (M=Ti, Nb, Mo) and 1 equiv. of glutathione was studied by (1)H NMR spectroscopy at pD 2-7 in 4 mM NaCl solutions. No interaction between glutathione and titanocene dichloride was detected at pD 2, while at pD 5-7 competitive hydrolysis of the cyclopentadienyl ligands occurred. With niobocene dichloride formation of approximately 20% of an adduct was observed at pD 2 and 5, but hydrolysis of the Cp ligands in the adduct occurred over 24 h. Molybdocene dichloride formed two stable adducts at pD 6 which were tentatively assigned as a Cp(2)Mo-glutathione chelate involving coordination of the cysteine thiol and glycine carboxylate to the metal centre, and a thiol centred 1:2 Cp(2)Mo-glutathione complex. The implications for the mechanism of antitumor action of the metallocene dihalides is discussed.     

Novel benzyl-substituted molybdocene anticancer drugs

From the reaction of 6-(p-methoxyphenyl) fulvene (1a), 6-(3,4-dimethoxyphenyl) fulvene (1b) and 6-(3,4,5-trimethoxyphenyl) fulvene (1c) with LiBEt₃H, lithiated cyclopentadienide intermediates (2a-c) were synthesised. These intermediates were then transmetallated to molybdocene using MoCl₄ (synthesized in situ) to yield the benzyl-substituted molybdocenes bis-[(p-methoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3a), bis-[(3,4-dimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3b), and bis-[(3,4,5-trimethoxybenzyl)cyclopentadienyl] molybdenum (IV) dichloride (3c). The molybdocene 3a was characterised by single crystal X-ray diffraction. All three molybdocenes had their cytotoxicity investigated through MTT based preliminary in vitro testing on the human renal cell line Caki-1 in order to determine their IC50 values and compare them with the corresponding titanocene and vanadocene dichloride derivatives. Molybdocenes 3b-c were found to have the same IC50 values of 290 μM, while 3a yielded a value of 84 μM, respectively     

Constructing transferrin receptor targeted drug delivery system by using doxorubicin hydrochloride and vanadocene dichloride

Transferrin has shown potential in the delivery of anticancer drugs into primarily proliferating malignant cells that over-express transferrin receptors. Constructing transferrin receptor targeted drug delivery system has been widespread concerned. In this study, whether transferrin could transport noncovalent binding drugs into cancer cells has been investigated. Two representative compounds, doxorubicin hydrochloride (Dox) and vanadocene dichloride (Cp(2)VCl(2)), have been chosen to study the interactions with h-Tf and apo-Tf, and the influences in the presence of h-Tf and apo-Tf by using fluorescence spectroscopy, circular dichroism (CD) spectroscopy and MTT assay. The results have shown that both doxorubicin and Cp(2)VCl(2) could bind to h-Tf and apo-Tf but with different binding modes. The results of MTT assay demonstrate that the presence of both h-Tf and apo-Tf has enhanced the antiproliferative activity of Cp(2)VCl(2). However, the anticancer activity of the mixture of doxorubicin and h-Tf is basically the same as that of doxorubicin does. Our studies indicate that transferrin plays an important role in the transport and targeted delivery of Cp(2)VCl(2) into cancer cells.     

Binding of V(IV) to human transferrin: potential relevance to anticancer activity of vanadocene dichloride

The action mechanism of vanadocene dichloride, Cp2VCl2 (Cp=eta5-C5H5), has been investigated by interaction with human serum transferrin for its promising antitumor activities. Our results have shown that Cp2VCl2 binds to transferrin and form a new complex, and the calculated apparent association constant is 1.37 x 10(5)M(-1) from the fluorescence quenching. Simultaneously, the variation of the secondary structure of transferrin occurs, most probably due to the coordination of the amino residues of protein with VIV. It was evidenced that Cp is released free in solution after VIV binding to transferrin by 1H NMR measurements. These results have shown that Cp2VCl2 forms a complex with transferrin, which may provide a possible pathway in the transport and targeted delivery of the antitumor agent.     

Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride

The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.     

Synthesis,Ti(IV) intake by apotransferrin and cytotoxic properties of functionalized titanocene dichlorides

Functionalization of cyclopentadienyl (Cp) ligands and incorporation of these into a Ti(IV) center require careful design and selection of the appropriate synthetic routes to obtain the desired product in reasonably good yields. As part of our research efforts in the area of titanocene antitumor agents, we have revisited the synthesis of Cp rings with electron-withdrawing groups and their corresponding titanocene dichlorides, (Cp-R)₂TiCl₂ and (Cp-R)CpTiCl₂, where R is CO₂CH₃ and CO₂CH₂CH₃. These complexes were characterized by elemental analysis and ¹H and ¹³C NMR and IR spectroscopies. This report presents the first detailed synthetic route for (Cp-CO₂CH₂CH₃)CpTiCl₂ and provides an alternate route for synthesis of (Cp-R)₂TiCl₂ complexes. The ability of these complexes to deliver Ti(IV) to apotransferrin was investigated to elucidate how the functionalized Cp ligands affect the titanium intake by apotransferrin. The subject complexes transfer Ti(IV) to human apotransferrin, loading both N- and C-lobes. The antitumor activity of these complexes against HT-29 cancer colon cells was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Carboethoxy Cp functionalization results in complexes with a toxicity comparable to that of titanocene dichloride. The carbomethoxy-functionalized complexes proved to be nonactive at the time intervals studied here, regardless of their ability to donate the titanium atom to human apotransferrin.     

Titanium(IV) targets phosphoesters on nucleotides: implications for the mechanism of action of the anticancer drug titanocene dichloride

Abstract Reactions between the anticancer drug titanocene dichloride (Cp2TiCl2) and various nucleotides and their constituents in aqueous solution or N,N-dimethylformamide (DMF) have been investigated by 1H and 31P NMR spectroscopy and in the solid state by IR spectroscopy. In aqueous solution over the pH* (pH meter reading in D2O) range 2.3-6.5, CMP forms one new species with Ti(IV) bound only to the phosphate group. In acidic media at pH*<4.6, three species containing titanocene bound to the phosphate group of dGMP, AMP, dTMP and UMP are formed rapidly. The bases also appear to influence titanocene binding. Only one of these Ti(IV)-bound species can be detected in the pH* range of 4.6-6.5 in each case. The order of reactivity towards Cp2TiCl2(aq) at pH* ca. 3 is GMP>TMP approximately AMP > CMP. At pH* > 7.0, hydrolysis of Cp2TiCl2 predominated and little reaction with the nucleotides was observed. Binding of deoxyribose 5'-phosphate and 4-nitrophenyl phosphate to Cp2TiCl2(aq) via their phosphate groups was detected by 31P NMR spectroscopy, but no reaction between Cp2TiCl2(aq) and deoxyguanosine, 9-ethylguanine or deoxy-D-ribose was observed in aqueous solution. The nucleoside phosphodiesters 3',5'-cyclic GMP and 2',3'-cyclic CMP did not react with Cp2TiCl2(aq) in aqueous solution; however, in the less polar solvent DMF, 3',5'-cyclic GMP coordination to [Cp2Ti]2+ via its phosphodiester group was readily observed. Binding of titanocene to the phosphodiester group of the dinucleotide GpC was also observed in DMF by 31P NMR. The nucleoside triphosphates ATP and GTP reacted more extensively with Cp2TiCl2(aq) than their monophosphates; complexes with bound phosphate groups were formed in acidic media and to a lesser extent at neutral pH. Cleavage of phosphate bonds in ATP (and GTP) by Cp2TiCl2(aq) to form inorganic phosphate, AMP (or GMP) and ADP (or GDP) was observed in aqueous solutions. In addition, titanocene binding to ATP was not inhibited by Mg(II), but the ternary complex titanocene-ATP-Mg appeared to form. These reactions contrast markedly with those of the drug cisplatin, which binds predominantly to the base nitrogen atoms of nucleotides and only weakly to the phosphate groups. The high affinity of Ti(IV) for phosphate groups may be important for its biological activity.     

Host cell and EBNA-2 regulation of Epstein-Barr virus latent-cycle promoter activity in B lymphocytes.

The six latent-cycle nuclear antigens (EBNAs) of Epstein-Barr virus (EBV), whose genes share 5' leader exons and two promoters (Cp and Wp), are differentially expressed by cells of the B lineage. To examine the possibility that EBNA gene expression is regulated through selective use of Cp and Wp, we monitored the activity of promoter-chloramphenicol acetyltransferase (CAT) gene constructs transfected into EBV-positive and EBV-negative B lymphocytes and Burkitt's lymphoma cells. Wp was a much stronger promoter than Cp in EBV genome-negative B-cell lines and was used exclusively in primary B cells. When B cells were infected with transforming EBV, Cp became the stronger promoter. This switch was not observed when B cells were infected with an immortalization-deficient virus, P3HR-1, which lacks the EBNA-2 open reading frame and expresses a mutant leader protein (EBNA-LP). Cp function was transactivated when EBV-negative or P3HR-1-infected B cells were cotransfected with Cp and a 12-kb fragment of DNA (BamHI-WWYH) that spanned the P3HR-1 deletion. This activity was mapped to the EBNA-2 gene within WWYH; constructs expressing EBNA-LP did not induce Cp function, and the deletion of 405 bp from the EBNA-2 open reading frame abolished transactivation. This research demonstrates host cell and EBNA-2 regulation of latent-cycle promoter activity in B lymphocytes, a mechanism with implications for persistence of EBV-infected lymphoid cells in vivo.     

The preparation,characterisation and in vitro cytotoxicity of potentially chemotherapeutic heterobimetallic complexes containing early and late transition metals

The reactions of phosphine Ph(2)P(CH(2))(2)SO(3)Na with Cp(2)M'Cl(2) (M'=Ti, Zr) in aqueous solution give the metallophosphines, Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2) (Cp=cyclopentadienyl) and CpZr(OH)(OSO(2)(CH(2))(2)PPh(2))(2). These react with CODM"Cl(2) (M"=Pd, Pt) (COD=1,5-cyclooctadiene) in dichloromethane to give heterobimetallic complexes Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2)M"Cl(2) and CpZr(OH)(OSO(2)(CH(2))(2) PPh(2))(2)M"Cl(2) respectively. The compounds are characterised by infrared and NMR spectroscopies and elemental analysis. Electrospray mass spectra of the complexes are reported and compared to those of Cp(2)M'Cl(2) in water and dimethylsulfoxide (DMSO). For zirconocene dichloride and its product heterobimetallic complexes, the addition of ethylenediamine tetraacetic acid disodium salt (Na(2)H(2)EDTA) was found to be an effective ionisation enhancement agent for the electrospray mass spectral studies. Cytotoxicity studies for the previously reported Cl(2)Pt(PPh(2)(CH(2))(2)SO(3)H)(2).3.5H(2)O (Wedgwood et al., Inorg. Chim. Acta 290 (1999) 189), and the compounds Cp(2)Ti(OSO(2)(CH(2))(2) PPh(2))(2).1.5H(2)O and Cp(2)Ti(OSO(2)(CH(2))(2)PPh(2))(2)PtCl(2).4H(2)O reported here, have been evaluated by colony formation assay against cisplatin-sensitive and -resistant cell lines L929 and L929/R to highlight potential chemotherapeutic activity. The compound Cl(2)Pt(PPh(2)(CH(2))(2)SO(3)H)(2).3.5H(2)O overcomes cisplatin resistance.     

Heteroaryl substituted titanocenes as potential anti-cancer drugs

From the reaction of Super Hydride (LiBEt(3)H) with 6-(furyl)fulvene (1a), 6-(thiophenyl)fulvene (1b) or 6-(N-methyl-pyrrole)fulvene (1c) the corresponding lithium cyclopentadienide intermediates (2a-c) were obtained. These intermediates were reacted with titanium tetrachloride and bis-[(furyl-2-cyclopentadienylmethane)] titanium(IV) dichloride (3a) and bis-[(thiophenyl-2-cyclopentadienylmethane)] titanium(IV) dichloride (3b) and bis-[(N-methylpyrrole-2-cyclopentadienylmethane)] titanium(IV) dichloride (3c) were obtained and subsequently characterised by X-ray crystallography. When titanocenes 3a-c were tested against pig kidney (LLC-PK) cells inhibitory concentrations (IC(50)) of 1.6x10(-4)M, 1.5x10(-4)M and 9.1x10(-4)M, respectively, were observed. These values represent improved cytotoxicity against LLC-PK, when compared to their corresponding ansa substituted analogues and also in comparison to unsubstituted titanocene dichloride.     

Ceruloplasmin is an endogenous protectant against kainate neurotoxicity

To determine the role of ceruloplasmin (Cp) in epileptic seizures, we used a kainate (KA) seizure animal model and examined hippocampal samples from epileptic patients. Treatment with KA resulted in a time-dependent decrease in Cp protein expression in the hippocampus of rats. Cp-positive cells were colocalized with neurons or reactive astrocytes in KA-treated rats and epileptic patient samples. KA-induced seizures, initial oxidative stress (i.e., hydroxyl radical formation, lipid peroxidation, protein oxidation, and synaptosomal reactive oxygen species), altered iron status (increasing Fe²⁺ accumulation and L-ferritin-positive reactive microglial cells and decreasing H-ferritin-positive neurons), and impaired glutathione homeostasis and neurodegeneration (i.e., Fluoro-Nissl and Fluoro-Jade B staining analyses) were more pronounced in Cp antisense oligonucleotide (ASO)- than in Cp sense oligonucleotide-treated rats. Consistently, Cp ASO facilitated KA-induced lactate dehydrogenase (LDH) release, Fe²⁺ accumulation, and glutathione loss in neuron-rich and mixed cultures. However, Cp ASO did not alter KA-induced LDH release or Fe²⁺ accumulation in the astroglial culture, but did facilitate impairment in glutathione homeostasis in the same culture. Importantly, treatment with human Cp protein resulted in a significant attenuation against these neurotoxicities induced by Cp ASO. Our results suggest that Cp-mediated neuroprotection occurs via the inhibition of seizure-associated oxidative damage (including impairment in glutathione homeostasis), Fe²⁺ accumulation, and alterations in ferritin immunoreactivity. Moreover, interactive modulation between neurons and glia was found to be important for Cp upregulation in the attenuation of epileptic damage in both animals and humans.     

Methoxy-phenyl substituted ansa-titanocenes as potential anti-cancer drugs derived from fulvenes and titanium dichloride

Starting from 6-(4'-methoxyphenyl)fulvene (1a), 6-(2',4',6'-trimethoxyphenyl)fulvene (1b), or 6-(3',5'-dimethoxyphenyl)fulvene (1c), [1,2-di(cyclopentadienyl)-1,2-di(4'-methoxyphenyl)-ethanediyl] titanium dichloride (2a), [1,2-di(cyclopentadienyl)-1,2-bis(2',4',6'-trimethoxyphenyl)-ethanediyl] titanium dichloride (2b), and [1,2-di(cyclopentadienyl)-1,2-bis(3',5'-dimethoxyphenyl)-ethanediyl] titanium dichloride (2c) were synthesised. When titanocenes 2a-c were tested against pig kidney carcinoma cells (LLC-PK) inhibitory concentrations (IC50) of 2.8 x 10(-4), 3.6 x 10(-4) and 2.1 x 10(-4) M, respectively, were observed.     

Differential response of interleukin-2 production to chronic copper supplementation in healthy humans

BACKGROUND: Copper (Cu) is an essential trace element for many biological processes including maintenance of both innate and acquired branches of immunity. OBJECTIVE: To measure the effect of copper supplementation on IL-2 and TNF-alpha production in subjects with lower and higher ceuloplasmin (Cp) values within normal range. DESIGN: Healthy adults (17 men and 16 women) with normal-low (low Cp) and normal-high Cp (high Cp) values were supplemented with 10 mg Cu/day (as CuSO(4)) during 2 months. METHOD: Before and after supplementation blood mononuclear cells were incubated in the absence or presence of phytohaemagglutinin or lipopolysaccharide for induction of IL-2 and TNF-alpha, respectively. The secretion of cytokines was measured by ELISA. Cu supplementation did not modify classical biochemical markers of Cu status. RESULTS: After supplementation, a significant increase in IL-2 production was found only in subjects with normal-low plasma Cp. Before and after Cu supplementation geometric mean and range +/- 1 SEM values were 1,566 (1,287-1,905) and 2,514 (2,159-2,927) pg/mL, respectively (two-way ANOVA for repeated measures: Cp level p < 0.001; time = NS; interaction Cp level and time p < 0.05). We did not observe changes in TNF-alpha production after Cu supplementation. CONCLUSIONS: Cu supplementation increased secretion of IL-2 and not TNF-alpha, which suggests an activation of proliferative but not inflammatory cytokines. These results support hypothesis that IL-2 may be a good indicator to identify a subgroup of individuals (polymorphism) who differs in Cu metabolism.     

Studies in humans on the mechanism of potent spermicidal and apoptosis-inducing activities of vanadocene complexes

We previously demonstrated that bis-cyclopentadienyl (Cp) complexes of vanadium(IV) (vanadocenes) are potent spermicidal and apoptosis-inducing agents. To gain further insight into the structure-function relationships controlling these two properties of vanadocenes, we have synthesized analogues in which the bis-Cp rings were substituted with one or five electron-donating methyl groups. The three complexes included vanadocene dichloride (VDC), bis(methylcyclopentadienyl) vanadium dichloride (VMDC), and bis(pentamethylcyclopentadienyl) vanadium dichloride (VPMDC). The concentration-dependent effect of these vanadocenes on sperm-immobilizing activity (SIA), mitochondrial membrane potential (DeltaPsim), axonemal dynein ATPase activity, and tyrosine phosphorylation of global and axoneme-specific sperm proteins was assessed by computer-assisted sperm analysis, flow cytometry, colorimetry, and immunoblotting, respectively. Apoptosis-inducing ability was quantitated by the two-color flow cytometric terminal dideoxynucleotidyl transferase-based assay that labels 3'-hydroxyl ends of fragmented DNA. All three vanadocenes induced rapid sperm immobilization (T(1/2) < 15 sec). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC by 10-fold. The EC(50) values (50% inhibitory concentration) for VDC, VMDC, and VPMDC were 7.5 microM, 4.3 microM, and 0.7 microM, respectively. Whereas SIA of vanadocenes was apparent at low micromolar concentrations, the apoptosis-inducing property was evident only at higher micromolar concentrations. The concentrations of VDC, VMDC, and VPMDC required for 50% apoptosis were 49 microM, 67 microM, and 153 microM, and for 50% reduction in sperm DeltaPsim were 435 microM, 173 microM, and 124 microM, respectively. Spermicidal activity of vanadocenes was not dependent on the inhibition of ATPase or tyrosine phosphorylation of global and sperm axonemal proteins. Due to the ability of these vanadocene complexes to rapidly generate hydroxyl radicals in the presence of oxidant, our findings provide unprecedented evidence for a novel mechanism of action for spermicidal vanadocenes. The differential concentration-dependent spermicidal and apoptosis-inducing properties of vanadocenes gives them particular utility as a new class of vaginal contraceptives.