Nikos Katsaros

Cisplatin‐Induced Apoptosis in HL‐60 Human Promyelocytic Leukemia Cells

Abstract: The anti‐apoptotic molecule BCL2 delays cell‐cycle entry from quiescence. We have recently cloned a new member of the BCL2 family of apoptosis‐related genes, BCL2L12. In the present study, the expression of BCL2 and BCL2L12 genes during cisplatin‐induced apoptosis in HL‐60 leukemic cells was investigated. The kinetics of apoptosis induction and cell toxicity were evaluated by DNA laddering and the MTT method, respectively. BCL2 and BCL2L12 expression was analyzed by RT‐PCR using gene‐specific primers. The ratio of apoptotic cells increased with increasing concentrations of cisplatin and exposure time of cell culture to the drug. Gradual, time‐dependent downregulation of BCL2 gene was observed during cisplatin treatment. Up‐regulation of BCL2L12 was observed 3 h (no DNA fragmentation) and 6 h (initiation of the DNA fragmentation) after treatment with cisplatin, followed by a decrease of its expression after 12‐h continuous treatment with the drug. It is known that the main anti‐carcinogenic effect of cisplatin is due to the induction of cell apoptosis. Present results indicate that downregulation of the BCL2 and upregulation of the BCL2L12 gene may be the underlying mechanisms.

mRNA expression analysis of a variety of apoptosis-related genes, including the novel gene of the BCL2-family, BCL2L12, in HL-60 leukemia cells after treatment with carboplatin and doxorubicin

Abstract Apoptosis is a type of programmed cell death involved in many crucial biological processes. It represents the basic mechanism for the action of chemotherapeutic agents, such as doxorubicin and carboplatin. Both are able to cause cell death through the induction of apoptosis in the human leukemic cell line HL-60. We investigated the possible alterations in the expression of apoptosis-related genes, including the novel BCL2L12 gene, which was recently cloned in our group. The kinetics of apoptosis induction and cell toxicity was investigated by DNA laddering and by the MTT method, respectively. Total RNA was extracted and cDNA was prepared by reverse transcription. BCL2, BAX, FAS, caspase-9, caspase-3 and BCL2L12 were amplified by PCR. Overexpression of FAS, BCL2L12 and caspase-3 was observed after treatment of HL-60 cells for 3 or 6 h with carboplatin, while their expression was decreased after a 12-h treatment, demonstrating that these genes may take part in the early stages of apoptosis. Overexpression of the same genes was also observed after 6 h of treatment with doxorubicin (concomitantly with DNA laddering). In the case of carboplatin-induced apoptosis we detected down-regulation of BAX, BCL2 and caspase-9, whereas in the case of doxorubicin, BAX and BCL2 remained at control levels and caspase-9 was increased.

Transition metal orotic acid complexes

Complexes of orotic acid with transition metals (Cu2+, Mn2+, VO2+, Zn2+, Hg2+, Cd2+, Fe3+, Cr3+, and Ag+) have been prepared and characterized by elemental, conductivity, magnetic measurements, i.r., n.m.r. and diffuse reflectance spectra. The ligand, in its monoanion form, coordinates through the carboxylic group to the metal. The Co2+ complex was also isolated under alkaline conditions and studied.

Organotin(IV) complexes with 2-(2′-pyridyl)quinoxaline (L): The crystal structure of the [SnEt2Cl2L]·0.5 benzene

Abstract Complexes of 2-(2′-pyridyl)quinoxaline (L) with R2SnCl2 (R=Me, Et, Bun) have been synthesized and characterized using IR, far-IR, 119Sn Mossbauer, 1H and 13C spectroscopies. The X-ray crystal structure of Et2SnCl2L shows a bidentate chelating behaviour of L, which is observed in all the diorganotin compounds presented. Interaction of the ligand L with SnCl4 resulted in the formation of a salt with the formula [(LH)2]2+[SnCl6]2−. Solution studies of the complexes R2SnCl2L (R=Me, Et, Bun) revealed partial dissociation of the ligand in chloroform.

Interaction of cis- and trans-RuCl2(DMSO)4 with the nucleotides GpA, d(GpA), ApG, d(ApG) and d(CCTGGTCC): high-field NMR characterization of the reaction products

Abstract Both cis- and trans-RuCl2(DMSO)4 (cis-Ru and trans-Ru) react with ApG, GpA, d(ApG) and d(GpA) to yield products with bifunctional metal coordination of the bases. For each dinucleotide one major product and several minor species are formed. This is in contrast to previous results on analogous reactions between trans-Ru and d(GpG) where a substantial amount of an intermediate species was found. The rates of reaction between dinucleotides and cis-Ru are approximately 20-fold slower than for trans-Ru. The compounds formed with the two isomers exhibit identical proton NMR spectra, suggesting the same coordination mode for ruthenium in the final product. The two purine bases are coordinated to ruthenium through N7 in a head-to-head conformation with the glycosidic angles being in the anti range. Coupling constants indicate a relatively pure 3′-endo conformation for the 5′-sugar and mainly 2′-endo for the 3′-sugar. The similar bifunctional binding mode of cis- and trans-Ru(II) with dinucleotides as evident from the NMR spectra are in contrast to the different mode of interaction suggested earlier for cis- and trans-Ru complexes with DNA. trans-Ru interacts with the deoxyoctanucleotide d(CCTGGTCC), giving two main products during the first 2 h of incubation time. Four H8 guanine resonances are shifted downfield, characteristic of N7 metal coordination. The products are not analyzed in detail, but it is suggested that the structures may be described as two chiral G(N7/N7) chelates.

Pyrazole and imidazole complexes of vanadium(II) chloride

Complexes of vanadium(II) chloride with pyrazole and imidazole of the type VCl2(pyrazole)4 and VCl2 (imidazole)6 were prepared. The complexes are unstable in air and substitution labile in solution. The electronic spectra of their methanolic solutions suggest octahedral symmetry. The participation of methanol in the first coordination sphere appears to be a function of the concentration of the ligand in solution. The values of Dq and B are calculated. The far infrared spectrum of the pyrazole complex in the crystalline state indicates a trans configuration. Magnetic measurements give values for the magnetic moments lower than the spin-only value.

Biological Activity of Some Cobalt(II) andMolybdenum(VI) Complexes: in vitro Cytotoxicity

Cytotoxicity and cell growth inhibition studies were performed for five distinct cobalt(ll) [Co2(acac)tpmc](ClO4)3, [Co2(dibzac)tpmc](ClO4)3, [Co2(hfac)tpmc](CIO4)2, [Co2(tmhd)tpmc](CIO4)3 and [Co2(ox)tpmc](CIO4)2.3H20 and five molybdenum(Vl) complexes, [MoO2(pipdtc)2], [MoO2(morphdtc)], [MoO2(timdtc)2], [MoO2(pzdtc)2] and [MoO2(N-Mepzdtc)2]. The former were tested in two leukemia cell lines: chronic myelogenic leukemia (K562) and human promyelocytic cell line (U937). They showed to have relatively high toxicity in K562 cells and a relatively low cytotoxicity in U937 cells, as assessed by both MTT and Trypan Blue assays. The five molybdenum complexes were tested in human promyelotic U937 cell line and they showed to have high toxicity.

Synthesis, spectroscopic and magnetic resonance studies of mercury(II) and methylmercury(II) complexes of azathioprine, a biologically active mercaptopurine derivative

Synthetic and spectroscopic studies of the Hg(II) and MeHg(II) complexes of azathioprine (AZA), a biologically active 6-mercaptopurine derivative, were undertaken. The altered coordination behavior of AZA with respect to the parent mercaptopurine, with sulfur no longer being the primary donor atom, was confirmed. As concluded by the 1H NMR, 13C NMR, and IR spectroscopic data, Hg(II) binds to the N(9) position of deprotonated AZA, while in the MeHg(II) compound, coordination occurs through the N(3) and N(9) positions of the purine ring. The values of the coupling constants 2J (199Hg-1H), 1J(199Hg-13C) for the MeHg(II) compound further support complexation via nitrogen atoms of the purine. Elemental analyses confirmed the compounds to be Hg(AZA)2 (1) and [(MeHg)2(AZA)](NO3) (2); conductivity measurement values show that 1 is a nonelectrolyte and 2 is a 1:1 electrolyte. Furthermore, the FAB-MS of the compounds confirms direct binding of the metal to the ligand, and in the case of the MeHg(II) compound, the successive loss of one and two MeHg(II) moieties can be clearly observed.

Interaction of Uranyl Ions with Daunorubicin and Adriamycin

The interaction of the uranyl(VI) ion (UO22+) with two anthracyclines, adriamycin (Adr) and daunorubicin (Dnr), has been studied by absorption, circular dichroism, fluorescence and NMR spectroscopy. We have shown that both drugs can form two complexes with UO22+, a process strongly dependent on the dielectric constant of the solvent, the metal-to-ligand ratio (R), and the pH. The first complex (I) involves coordination of the metal ion to the [C(11)−O−, C(12)=O] chelating site, while the second complex (II) involves coordination to the [C(6)−O−, C(5)=O] site. In aqueous solutions at pH 6.5, the formation of complex I takes place with two drug molecules per UO22+ ion. At higher pH or R values, complex II forms, and probably has a polymeric structure with one uranyl ion bridging two drug chromophores through both the keto-phenolate sites. In methanolic solutions the simultaneous formation of both complexes I and II is demonstrated by both CD and NMR spectroscopy. Dissociation of the complexes occurs upon interaction of the drug complexes with native DNA, with the free anthracyclines intercalating between the base pairs of the nucleotide.

The solution structure of the Ga(III)-bleomycin A2 complex resolved by NMR and molecular modeling; interaction with d(CCAGGCCTGG)

The solution structure of the GaIII-bleomycin A2 complex (GaBLM) has been determined using 2D NMR methods in combination with molecular dynamics calculations. Complete assignment of the amide and amine protons, observation of 80 NOEs and measurement of 15 3JH-H coupling constants provided us with a well-defined structure using a restrained simulated annealing protocol. On the basis of distance and dihedral angle constraints agreement, along with potential energy considerations, the favored model is a five-coordinate complex with the primary amine of β-aminoalanine holding the axial position of a distorted tetragonal pyramid. The disaccharide moiety of GaBLM is not a ligand, sharing the same side of the equatorial plane with the axial amine ligand. Titration of the self-complementary oligonucleotide d(CCAGGCCTGG) with GaBLM results in the formation of only one 1:1 complex in slow exchange on the NMR time scale. Our data indicate that the bithiazole moiety intercalates between the C6•G15 and C7•G14 base pairs, in a similar mode to that reported by earlier studies. Structural implications and comparisons to other metallo-bleomycins are discussed.