Positive regulation of ERK activation and MKP-1 expression by peroxovanadium complex bpV (phen)

Lower micromolar concentrations of peroxovanadium compound potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV (phen)] stimulate RINm5F cell metabolic activity. 1 and 3 mol/L bpV (phen) induces strong and sustained activation of extracellular signal-regulated kinase (ERK). However, it seems that bpV (phen) does not effect c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In addition, bpV (phen) induces mitogen-activated protein kinase phosphatase-1 (MKP-1) expression. We found that ERK activation could be completely abolished if RINm5F cells were incubated with both bpV (phen) and PD 98059, a specific inhibitor of upstream ERK kinase MEK1. On the other hand, this combined treatment up-regulated activation of stress kinases, JNK and p38 MAPK, significantly suppressed MKP-1 expression and induced cell death. Thus, our results suggest that the mechanism underlying bpV (phen) survival-enhancing effect could be associated with induced ERK activation and MKP-1 expression.     

Molecular mechanisms involved in the antiproliferative action of protein tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate

Potassium bisperoxo(1,10-phenanthroline)oxovanadate, bpV(phen), a powerful protein phosphotyrosine phosphatase inhibitor and a potent insulinomimetic, influenced three fundamental cellular processes in HL-60 human leukemic cells: 1) inhibition of proliferation, 2) induction of differentiation and 3) apoptotic cell death. In the presence of micromolar concentrations of bpV(phen) cell number and DNA synthesis decreased progressively with time of incubation. A single treatment with bpV(phen) (3 microM) activated a differentiation program; after 6 days of incubation 82% of cells were differentiated, but differentiation started already within the first 24 h. Concentrations of 5-10 microM bpV(phen) caused the characteristic DNA ladder pattern, starting after 4.5 h. Differentiation in HL-60 cells appear to be associated with activation of extracellular signal-regulated kinase while apoptosis is connected with phosphorylation and activation of both extracellular signal-regulated kinase and c-Jun N-terminal kinase in a concentration and time-dependent manner. The antiproliferative and apoptotic action of bpV(phen) could be exploited in combination chemotherapy in leukemia.     

Peroxovanadium compounds: Biolgoical actions and mechanism of insulin-mimesis

When used alone, both vanadate and hydrogen peroxide (H₂O₂) are weakly insulin-mimetic, while in combination they are strongly synergistic due to the formation of aqueous peroxovanadium species pV_(aq). Administration of these pV_(aq) species leads to activation of the insulin receptor tyrosine kinase (IRK), autophosphorylation at tyrosine residues and inhibition of phosphotyrosine phosphatases (PTPs). We therefore undertook to synthesize a series of peroxovanadium (pV) compounds containing one or two peroxo anions, an oxo anion and an ancillary ligand in the inner co-ordination sphere of vanadium, whose properties and insulin-mimetic potencies could be assessed. These pV compounds were shown to be the most potent inhibitors of PTPs yet described. Their PTP inhibitory potency correlated with their capacity to stimulate IRK activity. Some pV compounds showed much greater potency as inhibitors of insulin receptor (IR) dephosphorylation than epidermal growth factor receptor (EGFR) dephosphorylation, implying relative specificity as PTP inhibitors. Replacement of vanadium with either molybdenum or tungsten resulted in equally potent inhibition of IR dephosphorylation. However IRK activation was reduced by greater than 80% suggesting that these compounds did not access intracellular PTPs. The insulin-like activity of these pV compounds were demonstrablein vivo. Intra venous (i.v.) administration of bpV(pic) and bpV(phen) resulted in the lowaring of plasma glucose concentrations in normal rats in a dose dependent manner. The greater potency of bpV(pic) compared to bpV(phen) was explicable, in part, by the capacity of the former but not the latter to act on skeletal muscle as well as liver. Finally administration of bpV(phen) and insulin led to a synergism, where tyrosine phosphorylation of the IR -subunit increased by 20-fold and led to the appearance of four insulin-dependentin vivo substrates. The insulin-mimetic properties of they pV compounds raises the possibility for their use as insulin replacements in the management of diabetes mellitus.     

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

The relationship between the structures of periphery ligands and the DNA binding mode of [Ru(II)(1,10-phenanthroline)(L1L2)dipyrido[3,2-a:2',3'-c]phenazine](n+) (L1=Cl or pyridine and L2=pyridine, n=1,2)

The binding modes of the [Ru(II)(1,10-phenanthroline)(L₁L₂) dipyrido[3,2-a:2′,3′-c]phenazine]²⁺ {[Ru(phen)(py) Cl dppz]⁺ (L₁ = Cl, L₂ = pyridine) and ([Ru(phen)(py)₂dppz]²⁺ (L₁ = L₂ = pyridine)} to native DNA is compared to that of the [Ru(II)(1,10-phenanthroline)₂dipyrido[3,2-a:2′,3′-c]phenazine]²⁺ complex ([Ru(phen)₂dppz]²⁺) by various spectroscopic and hydrodynamic methods including electric absorption, linear dichroism (LD), fluorescence spectroscopy, and viscometric titration. All measured properties, including red-shift and hypochromism in the dppz absorption band, nearly perpendicular molecular plane of the dppz ligand with respect to the local DNA helix axis, prohibition of the ethidium binding, the light switch effect and binding stoichiometry, increase in the viscosity upon binding to DNA, increase in the melting temperature are in agreement with classical intercalation of dppz ligand of the [Ru(phen)₂dppz]²⁺ complex, in which both phenanthroline ligand anchored to the DNA phosphate groups by electrostatic interaction. [Ru(phen)(py)₂ dppz]²⁺ and [Ru(phen)(py) Cl dppz]⁺ complexes had one of the phenanthroline ligand replaced by either two pyridine ligands or one pyridine plus a chlorine ion. They exhibited similar protection from water molecules, interaction with DNA bases, and occupying site that is common with ethidium. The dppz ligand of these two Ru(II) complex were greatly tilted relative to the DNA helix axis, suggesting that the dppz ligand resides inside the DNA and is not perpendicular relative to the DNA helix axis. These observation suggest that anchoring the [Ru(phen)₂dppz]²⁺complex by both phenanthroline is essential for the dppz ligand to be classically intercalated between DNA base-pairs.     

Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V).

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).     

New insights in the DNA-[Cr(phen)2(dppz)] binding and photocleavage properties by the complex with an intercalating ligand

Due to the key role of DNA in cell life and pathological processes, the design of specific chemical nucleases, DNA probes and alkylating agents is an important research area for the development of new therapeutic agents and tools in Biochemistry. Hence, the interaction of small molecules with DNA has attracted in particular a great deal of attention.The aim of this study was to investigate the ability of [Cr(phen)₂(dppz)]³⁺ to associate with DNA and to characterize it as photocleavage reagent for Photodynamic Therapy (PDT).Chromium(III) complex [Cr(phen)₂(dppz)]³⁺, (dppz = dipyridophenazine, phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended π system, has been found to bind strongly to double strand oligonucleotides (ds-oligo) and plasmid DNA with intrinsic DNA binding constants, K_b, of (3.9 ± 0.3) × 10⁵ M⁻¹ and (1.1 ± 0.1) × 10⁵ M⁻¹, respectively. The binding properties to DNA were investigated by UV-visible (UV-Vis) absorption spectroscopy and electrophoretic studies. UV-Vis absorption data provide clearly that the chromium(III) complex interacts with DNA intercalatively. Competitive binding experiments show that the enhancement in the emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(phen)₂(dppz)]³⁺, indicating that the Cr(III) complex displaces EthBr from its binding site in plasmid DNA. Moreover, [Cr(phen)₂(dppz)]³⁺, non-covalently bound to DNA, promotes the photocleavage of plasmid DNA under 457 nm irradiation. We also found that the irradiated Cr(III)-plasmid DNA association is able to impair the transforming capacity of bacteria. These results provide evidence confirming the responsible and essential role of the excited state of [Cr(phen)₂(dppz)]³⁺ for damaging the DNA structure. The combination of DNA, [Cr(phen)₂(dppz)]³⁺ and light, is necessary to induce damage. In addition, assays of the photosensitization of transformed bacterial suspensions suggest that Escherichia coli may be photoinactivated by irradiation in the presence of [Cr(phen)₂(dppz)]³⁺. In sum, our results allow us to postulate the [Cr(phen)₂(dppz)]³⁺ complex as a very attractive candidate for DNA photocleavage with potential applications in Photodynamic Therapy (PDT).     

DNA-binding and photocleavage studies of [Ru(phen)2(NMIP)]

A novel ligand 2′-(2″-nitro-3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]-phenanthroline (NMIP) and its complex [Ru(phen)₂(NMIP)]²⁺ have been synthesized and characterized by mass spectroscopy, ¹H NMR and cyclic voltammetry. Binding of the complex with calf thymus DNA (CT DNA) has been investigated by spectroscopic methods, viscosity and electrophoresis measurements. The experimental results indicate that [Ru(phen)₂(NMIP)]²⁺ binds to DNA via partial intercalative mode and the individual enantiomers of it bind to DNA in different rates. [Ru(phen)₂(NMIP)]²⁺ has also been found to promote cleavage of plasmid pBR 322 DNA from the supercoiled Form I to the open circular Form II upon irradiation.     

Binding analysis of ytterbium(III) complex containing 1,10-phenanthroline with DNA and its antimicrobial activity

To evaluate the biological preference of [Yb(phen)₂(OH₂)Cl₃](H₂O)₂ (phen is 1,10-phenanthroline) for DNA, interaction of Yb(III) complex with DNA in Tris–HCl buffer is studied by various biophysical and spectroscopic techniques which reveal that the complex binds to DNA. The results of fluorescence titration reveal that [Yb(phen)₂(OH₂)Cl₃](H₂O)₂ has strongly quenched in the presence of DNA. The binding site number n, apparent binding constant K _b, and the Stern–Volmer quenching constant K _SV are determined. ΔH ⁰, ΔS ⁰, and ΔG ⁰ are obtained based on the quenching constants and thermodynamic theory (ΔH ⁰?>?0, ΔS ⁰?>?0, and ΔG ⁰?<?0). The experimental results show that the Yb(III) complex binds to DNA by non-intercalative mode. Groove binding is the preferred mode of interaction for [Yb(phen)₂(OH₂)Cl₃](H₂O)₂ to DNA. The DNA cleavage results show that in the absence of any reducing agent, Yb(III) complex can cleave DNA. The antimicrobial screening tests are also recorded and give good results in the presence of Yb(III) complex.     

Interactions of mixed ligand ruthenium(II) complexes containing an amino acid and 1,10-phenanthroline with DNA

Mixed ligand ruthenium(II) complexes containing an amino acid (AA) and 1,10-phenanthroline (phen), i.e. [Ru(AA)(phen)₂]ⁿ⁺ (n=1,2, AA=glycine (gly), l-alanine (l-ala), l-arginine (l-arg)) have been synthesized. The interactions of these complexes and [Ru(phen)₃]²⁺ with DNA have been examined by absorption, luminescence, and circular dichroism spectroscopic methods. Absorption spectral properties revealed that [Ru(AA)(phen)₂]⁺ (AA=gly, l-ala) interacted with CT-DNA by the electrostatic binding mode. [Ru(l-arg)(phen)₂]²⁺ exhibited the greatest hypochromicity, red shift, and binding constant, indicating that this complex may partially intercalate into the base-pairs of DNA. These results were also suggested by luminescence spectroscopy. CD spectral properties have been examined to understand the detailed interactions of the ruthenium(II) complexes with artificial DNA. In the case of Δ-[Ru(l-arg)(phen)₂]²⁺, the solution on adding [poly(dG-dC)]₂ exhibited two well-defined positive peaks, which the shorter and longer wavelength peaks were assigned as originating from the major and the minor groove binding modes, respectively. Then, the solution on adding [poly(dA-dT)]₂ exhibited only one positive peak, which was assigned as a peak corresponding to the minor groove binding mode.     

DNA binding and bending by dinuclear complexes comprising ruthenium polypyridyl centres linked by a bis(pyridylimine) ligand

The interaction of enantiomerically pure dinuclear complexes of the form [Ru₂(L-L)₄L¹]⁴⁺ (where L-L = 2,2^′-bipyridine (bpy) or 1,10-phenanthroline (phen) and L¹ = bis(pyridylimine) ligand ((C₅H₄N)CN(C₆H₄))₂CH₂)) with ct-DNA have been investigated by absorbance, circular dichroism, fluorescence displacement assays, thermal analysis, linear dichroism and gel electrophoresis. The complexes all bind more strongly to DNA than ethidium bromide, stabilise DNA and have a significant bending effect on DNA. The data for Δ,Δ-[Ru₂(bpy)₄L¹]⁴⁺ are consistent with it binding to DNA outside the grooves wrapping the DNA about it. By way of contrast the other complexes are groove-binders. The phen complexes provide a chemically and enantiomerically stable alternative to the DNA-coiling di-iron triple-helical cylinder previously studied. In contrast to the di-iron helicates, the phen complexes show DNA sequence effects with Δ,Δ-[Ru₂(phen)₄L¹]⁴⁺ binding preferentially to GC and Λ,Λ-[Ru₂(phen)₄L¹]⁴⁺ to AT.     

Study on Interaction of DNA from Calf Thymus with 1,10-phenanthrolinehexyldithiocarbamatopalladium(II) nitrate as Potential Antitumor Agent

Abstract

A novel palladium(II) complex has been synthesized with hexyldithiocarbamate (Hex-dtc) and 1,10-phenanthroline (phen) by the reaction of [Pd(phen)(H₂O)₂](NO₃)₂ with sodium salt of hexyldithiocarbamate and a complex of type [Pd(Hex-dtc) (phen)]NO₃ has been obtained. The complex has been characterized by elemental analysis, molar conductance, ¹H NMR, IR and electronic spectroscopic studies. The dithiocarbamate ligand acts in bidentate fashion. This water-soluble complex was screened against chronic myelogenous leukemia cell line, K562, for cytotoxic effects and showed significant antitumor activity much lower than that of cisplatin. The interaction of this complex with calf thymus DNA (ctDNA) was extensively investigated by a variety of spectroscopic techniques. Absorbance titration experiments imply the interaction of 4 Pd(II) complex molecules per 1000 nucleotides on DNA with positive cooperativity in the binding process and the complex denature the DNA at very low concentration (～14.3 μM). Fluorescence titration spectra and fluorescence Scatchard plots suggest that the Pd(II) complex intercalate in DNA. The gel chromatograms obtained from Sephadex G-25 column experiments showed that the binding of metal complex with DNA is so strong that it does not readily break. Furthermore, some thermodynamic and binding parameters found in the process of UV-Visible studies are described. They may provide specificity of the compound with ctDNA.     

Diastereomeric bactericidal effect of Ru(phenanthroline)2dipyridophenazine

Metal susceptibility assays and spot plating were used to investigate the antimicrobial activity of enantiopure [Ru(phen)₂dppz]²⁺ (phen =1,10‐phenanthroline and dppz = dipyrido[3,2‐a:2´,3´‐c]phenazine) and [μ‐bidppz(phen)₄Ru₂]⁴⁺ (bidppz =11,11´‐bis(dipyrido[3,2‐a:2´,3´‐c]phenazinyl)), on Gram‐negative Escherichia coli and Gram‐positive Bacillus subtilis as bacterial models. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) were determined for both complexes: while [μ‐bidppz(phen)₄Ru₂]⁴⁺ only showed a bactericidal effect at the highest concentrations tested, the antimicrobial activity of [Ru(phen)₂dppz]²⁺ against B. subtilis was comparable to that of tetracyline. In addition, the Δ‐enantiomer of [Ru(phen)₂dppz]²⁺ showed a 2‐fold higher bacteriostatic and bactericidal effect compared to the Λ‐enantiomer. This was in accordance with the enantiomers relative binding affinity for DNA, thus strongly indicating DNA binding as the mode of action.     

On the Use of Chiral Compounds for Probing the DNA Handedness: Z to B Conversion in Poly(dGm5dC) Upon Binding of Fe(phen)3 2+ and Ru(phen)3 2+

Abstract

In order to examine whether chiral metal complexes can be used to discriminate between right- and left-handed DNA conformational states we have studied the enantioselective interactions of Fe(phen)₃ ²⁺ and Ru(phen)₃ ²⁺ (phen = 1,10-phenanthroline)with poly(dGm⁵dC) under B- and Z-form conditions. With the inversion-labile Fe(phen)₃ ²⁺, enantioselectivity leads to shifts in the diastereomeric binding equilibria. This effect, known as the “Pfeiffer effect” (1–4), is monitored as a slowly emerging circular dichroism of the solution, corresponding to a net excess of the favoured enantiomer. With Ru(phen)₃ ²⁺, which is stable to intramolecular inversion, the difference in DNA-binding strengths of the enantiomers results in an excess of the less favoured enantiomer in the bulk solution. This excess is detected in the dialysate of the DNA/metal complex solution. With both complexes we find that the Δ-enantiomer is favoured when the polynucleotide adopts the B-form, as previously shown, but also when it initially adopts the Z-form conformational state.

This observation, together with evidence from UV-circular dichroism and binding data, indicates that the binding of these metal complexes induces a Z- to B-form transition in Z- form poly(dGm⁵dC). Consequently, neither of the studied chiral DNA-binders can easily be used to discriminate the DNA handedness.     

Synthesis, structure, DNA binding and photocleavage activity of a ruthenium(II) complex with 11-(9-acridinyl)dipyrido[3,2-a:2′,3′-c]phenazine ligand

In our search for new DNA intercalating ligands, a novel bifunctional intercalator 11-(9-acridinyl)dipyrido[3,2-a:2′,3′-c]phenazine, acdppz (has two potentially effective intercalators via dipyridophenazine(dppz) and acridine which are linked together via C-C bond) and its corresponding Ru(II) polypyridyl complex [Ru(phen)₂(acdppz)]²⁺ (where phen = 1,10-phenanthroline) have been synthesized and characterized. The electrochemical behaviors of the ligand and its complex have been thoroughly examined. The structure of acdppz and [Ru(phen)₂(acdppz)]²⁺ were determined by X-ray crystallography. From the crystal structure of the complex, we found that the dppz moiety is not coplanar with the acridine ring, having a dihedral angle of 64.79 in the acdppz. The selected bond lengths and angles for the crystal structure of [Ru(phen)₂(acdppz)]²⁺ were compared to the geometry-optimized molecular structure of [Ru(phen)₂(acdppz)]²⁺ derived by Gaussian. The interaction of [Ru(phen)₂(acdppz)]²⁺ with calf-thymus (CT) DNA was investigated by absorption and viscometry titration, thermal denaturation studies. The above measurements indicated that the complex binds less strongly with the CT DNA due to the intercalation by the ruthenium bound acdppz with an intrinsic binding constant of 2.6 × 10⁵ M⁻¹. Molecular-modeling studies also support an intercalative mode of binding of the complex to the model duplex d(CGCAATTGCG)₂ possibly from the major groove with a slight preference for GC rich region. Additionally, the title complex promotes the cleavage of plasmid pBR322 DNA upon irradiation under aerobic conditions.     

[Cu(phen)2] acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)₂]²⁺, where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)₂]²⁺ acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)₂]²⁺ were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10⁻¹² to 1 × 10⁻⁶ M with a detection limit of 1.99 × 10⁻¹³ M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.     

Variation of DNA photocleavage efficiency for [(TL)2Ru(dpp)]Cl2 complexes where TL = 2,2′-bipyridine, 1,10-phenanthroline, or 4,7-diphenyl-1,10-phenanthroline

The complexes [(bpy)₂Ru(dpp)]Cl₂, [(phen)₂Ru(dpp)]Cl₂, and [(Ph₂phen)₂Ru(dpp)]Cl₂ (where dpp = 2,3-bis(2-pyridyl)pyrazine, bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline, Ph₂phen = 4,7-diphenyl-1,10-phenanthroline) have been investigated and found to photocleave DNA via an oxygen-mediated pathway. These light absorbing complexes possess intense metal-to-ligand charge transfer (MLCT) transitions in the visible region of the spectrum. The [(TL)₂Ru(dpp)]²⁺ systems populate ³MLCT states after visible light excitation, giving rise to emissions in aqueous solution centered at 692, 690, and 698 nm for TL = bpy, phen, and Ph₂phen respectively. The ³MLCT states and emissions are quenched by O₂, producing a reactive oxygen species. These complexes photocleave DNA with varying efficiencies, [(Ph₂phen)₂Ru(dpp)]²⁺ > [(phen)₂Ru(dpp)]²⁺ > [(bpy)₂Ru(dpp)]²⁺. The presence of the polyazine bridging ligand will allow these chromophores to be incorporated into larger supramolecular assemblies.     

Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex

Copper-1,10-phenanthroline (phen) complex [Cu(phen)₂] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)₂. HepG2 cells were more sensitive to Cu(phen)₂ with an IC50 of 4.03 μM than other three kinds of cell lines. After treated by Cu(phen)₂, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder’s occurence and Annexin V-positive increased cells indicated that Cu(phen)₂ induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)₂ treatment resulted in significant G₂/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)₂-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-x_L and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca²⁺ was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)₂ induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.     

Bisperoxovanadium complex promotes dopamine exocytosis in PC12 cells.

The effects of the peroxovanadium complex potassium bisperoxo(1,10-phenanthroline)-oxovanadate (bpV[phen]) have been studied on dopamine (DA) exocytosis in PC12 cells. Bisperoxo(1,10-phenanthroline)-oxovanadate does not elicit dopamine secretion in PC12 cells. However, treatment of PC12 cells with 30 microM bpV[phen] for 20 min significantly enhances the secretion induced by the Ca(2+)-ionophore A23187. The effects appear to be irreversible, and strikingly different from the transient and suppressing effects of orthovanadate, which, like bpV[phen], is also a protein tyrosine phosphatase inhibitor. Contrastingly, the short-lived peroxovanadates, formed in situ by the addition of hydrogen peroxide and orthovanadate, are relatively ineffective. The Ca(2+) chelating agent EGTA abolishes bpV[phen]-enhanced dopamine release. The extracellular-regulated protein kinases (ERK) and synaptophysin, proteins implicated in exocytosis, are both tyrosine-phosphorylated by bpV[phen] in a dose- and time-dependent manner, with a maximal effect at 30 microM. Pre-treatment of cells with PD98059 significantly reduced dopamine release (P<0.05). These results suggest that this peroxovanadium complex enhances dopamine exocytosis, at least in part, by ERK-mediated signaling pathway and synaptophysin-associated phosphatase(s).     

Determination of the intracellular targets and in vitro activity of a new class of chemical nuclease complexes derived from antibiotics of the quinolone family

 Alteration of bacterial DNA structure and/or associated functions in vivo by [Cu(phen)(nal)]⁺, a metal complex of the type [Cu(phen)(antib)]⁺ (where antib is a quinolone or a fluoroquinolone), was demonstrated by the induction of a recA-lacZ fusion integrated at the amyE locus of a recombinant Bacillus subtilis strain. Using the same approach, nalidixic acid alone was shown to induce 14% of the β-galactosidase levels induced by [Cu(phen)(nal)]⁺; on the other hand none of the other components, i.e. copper, phenanthroline or the complex [Cu(phen)₂]²⁺ activated significantly the recA-directed β-galactosidase activity, suggesting that the intact structure of the complex is required to reach maximum levels of induction. Results of in vitro experiments demonstrated that under reductive conditions [Cu(phen)(nal)]⁺ behaves as a powerful nuclease capable of degrading plasmid DNA; this activity was stronger than that of the chemical nuclease copper phenanthroline [Cu(phen)₂]²⁺. The nuclease activity putatively occurred by a mechanism involving hydroxyl radicals since the reaction was partially inhibited by catalase. These results support the hypothesis that the mechanism of action of quinolones could be mediated by a transition metal ion such as copper. Received: 30 September 1997 / Accepted: 30 January 1998