A novel cisplatin mediated apoptosis pathway is associated with acid sphingomyelinase and FAS proapoptotic protein activation in ovarian cancer.

Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin.

Mitochondrial dysfunction in cardiac disease: ischemia--reperfusion, aging, and heart failure.

Mitochondria contribute to cardiac dysfunction and myocyte injury via a loss of metabolic capacity and by the production and release of toxic products. This article discusses aspects of mitochondrial structure and metabolism that are pertinent to the role of mitochondria in cardiac disease. Generalized mechanisms of mitochondrial-derived myocyte injury are also discussed, as are the strengths and weaknesses of experimental models used to study the contribution of mitochondria to cardiac injury. Finally, the involvement of mitochondria in the pathogenesis of specific cardiac disease states (ischemia, reperfusion, aging, ischemic preconditioning, and cardiomyopathy) is addressed.

Aging decreases electron transport complex III activity in heart interfibrillar mitochondria by alteration of the cytochrome c binding site.

Aging alters cardiac physiology and structure and enhances damage during ischemia and reperfusion. Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located among the myofibers, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Aging decreased the rate of oxidative phosphorylation using durohydroquinone, an electron donor to complex III, in IFM only. Complex III activity was decreased in IFM, but not SSM. Aging did not alter the content of catalytic centers of complex III (cytochromes b and c(1)and iron-sulfur protein). Complex III activity measured at physiologic ionic strength in IFM from aging hearts was decreased by 49% compared to IFM from adults, whereas activity measured at low ionic strength was unchanged, localizing the aging defect to the cytochrome c binding site of complex III. Subunits VIII and X of the cytochrome c binding site were present in complex III with the aging defect, indicating that loss of subunits did not occur. Study of aging damage to complex III will help clarify the contribution of altered electron transport in IFM to increased oxidant production during aging, formation of the aging cardiac phenotype, and the relationship of aging defects to increased damage following ischemia.

Oxidative damage of DNA by chromium(V) complexes: relative importance of base versus sugar oxidation.

Chromium(V)-mediated oxidative damage of deoxy-ribonucleic acids was investigated at neutral pH in aqueous solution by utilizing bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) (I) and bis(hydroxyethyl)-amino-tris(hydroxymethyl)methane)oxochromate(V) (II). Single-stranded and double-stranded (ds) calf thymus and human placenta DNA, as well as two oligomers, 5'-GATCTAGTAGGAGGACAAATAGTGTTTG-3' and 5'-GATCCAAGCAAACACTATTTGTCCTCCTACTA-3', were reacted with the chromium(V) complexes. Most products were separated and characterized by chroma-tographic and spectroscopic methods. Polyacrylamide gel electrophoresis experiments reveal more damage at G sites in comparison to other bases. Three primary oxidation products, 5-methylene-2-furanone (5-MF), furfural and 8-oxo-2'-deoxyguanosine, were characterized. A minor product, which appears to be thymine propenal, was also observed. The dsDNA produces more furfural than furanone. The formation of these two products resulted from hydrogen ion or hydride transfer from C1' and C5' positions of the ribose to the oxo-chromium(V) center. Since no enhancements of these products (except propenal) were observed in the presence of oxygen, mechanisms pertaining to the participation of activated oxygen species may be ruled out. The oxidation of the G base is most likely associated with an oxygen atom transfer from the oxo-metallates to the double bond between C8 and N7 of the purine ring. The formation of the propenal may be associated with an oxygen-activated species, since a marginal enhancement of this product was observed in the presence of oxygen. The formation of furfural in higher abundance over 5-MF for dsDNA was attributed to the ease of hydrogen ion (or hydride transfer) from the C5' compared to C1' position of the ribose within a Cr(V)-DNA intermediate in which the metal center is bound to the phosphate diester moiety.

Mechanisms of DNA damage by chromium(V) carcinogens.

Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0. The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety. The primary oxidation product was characterized as 5-methylene-2-furanone. Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three. Orthophosphate and 3'-phosphates were also detected in this reaction. Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites. This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well. DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety. Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations. It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed. Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.

Electron paramagnetic resonance, kinetics of formation and decomposition studies of (bis(hydroxyethyl)amino-tris(hydroxymethyl)-methane)oxochromate(V): a model chromium(V) complex for DNA damage studies.

A new chromium complex, (bis(hydroxyethyl)amino-tris(hydroxymethyl)methane)oxochromate(V), has been characterized by epr spectroscopy. The chromium(V) complex was formed by the ligand displacement reaction of bis(2-ethyl-2-hydroxybutanato) oxochromate(V) with bis(hydroxyethyl)amino-tris(hydroxy-methyl)methane (BT). Both epr and kinetic data indicate that the reaction proceeds through a chromium(V) intermediate. Kinetics of formation of the intermediate exhibit a rate saturation at higher [BT] (> 30 mM) indicating a rate law constituting an equilibrium between the parent Cr(V) complex and the bis-tris ligand followed by a pure first order process. The g-value of the intermediate is consistent with a Cr(V) complex in which the BT is coordinated in a bidentate fashion replacing a coordinated hydroxy butanoic acid ligand, affording a mixed ligand complex. The equilibrium step (K = 36 M-1) consists of monodentate coordination by the BT ligand and the limiting first order rate constant (1.9 x 10(-2) s-1) manifests the rate of chelation by the polydentate ligand. The intermediate is converted to the product upon further chelation through the complete displacement of the remaining 2-ethyl-2-hydroxy butanoic acid by a first order process (k = 0.023 s-1). The epr data support a pair of products that are in rapid equilibrium. In these products, BT functions either as a tetra or a penta-dentate ligand coordinating through four or five alkoxy sites. The enthalpy and entropy of activations related to the two chelation steps were found to be 32 +/- 2 kJ/mol and -(1.7 +/- 0.2) x 10(2) J/mol K for the intermediate, and 36 +/- 1 kJ/mol and -(1.5 +/- 0.2) x 10(2) J/mol K for the product. Our data support an associative mechanism for the chelation steps. The Cr(V)-BT product is more stable than the parent complex. The second order disproportionation rate constant for the Cr(V)-BT complex was evaluated to be 0.1 M-1 s-1 compared to 8.0 M-1 s-1 for the parent complex. This is the first example of a chromium(V) complex with a non-macrocyclic ligand coordinating through oxygen donor atoms which is stable in aqueous solution at neutral pH over a long period of time.

Kinetic analysis of the cis-diamminedichloroplatinum(II)--cysteine reaction: implications to the extent of platinum--DNA binding.

The reaction between cis-diamminedichloroplatinum(II) (cis-DDP) and L-cysteine was examined at neutral pH at 37 degrees C. The reaction proceeds through a Pt(NH3)2 (cys)Cl intermediate which undergoes parallel reactions with a second molecule of cysteine to form a bis(cysteine) complex, Pt(NH3)2(cys)2 and with the starting platinum complex to form a cysteine-bridged dinuclear complex. In the presence of excess cysteine, the product is predominantly the bis(cysteine) complex. The intermediate is formed by the direct reaction of the platinum complex with cysteine with a bimolecular rate constant 2.2 +/- 0.2 x 10(-2) M-1.s-1 at 37 degrees C as well as through a rapid reaction with the mono aqua-platinum complex. The rate constant for the formation of the dimer was evaluated to be 0.24 +/- 0.4 M-1.s-1, an order of magnitude higher than that for the mononuclear complex formation. The intermediate reacts with a second cysteine molecule with a bimolecular rate constant, 5.6 +/- 0.4 x 10(-2) M-1.s-1. The rate constant for the equation of Pt(NH3)2(cys)Cl was evaluated to be 1.8 +/- 0.2 10(-4) s-1. The Pt-195 chemical shifts for the mono(cysteine), bis(cysteine), and cysteine bridged dimer were found to be -3308, -3705, and -3104 ppm. The bis(cysteine) complex at neutral pH undergoes slow reaction (t1/2 approximately equal to four days) to form a secondary product, presumably Pt(NH3)(cys)2, in which one cysteine acts a bidentate chelating agent. In acidic solution, with equimolar concentrations of cysteine and diaqua-platinum complex, the reaction predominantly yielded a cysteine bridged dimeric complex. When cysteine concentration was increased fourfold over the platinum complex, the bis(cysteine) chelate with complete removal of coordinated ammonia appeared as the dominant product. The platinum-195 chemical shift for this chelate was found to be -3290 ppm. Considering the abundance of thiols in amino acids/peptides and replication enzymes in the cellular milieu, it remains to be seen how platinum complexes react with DNA. Direct platination to replication enzymes as a possible mechanism for antineoplactic activity is yet to be ruled out.

Mechanisms of formation and decomposition of hypervalent chromium metabolites in the glutathione-chromium (VI) reaction.

A long-lived chromium(IV) intermediate is generated during the reaction between Cr(VI) and glutathione in glycine below pH 3. The intermediate reacts with the tripeptide to produce Cr(III) and oxidized glutathione. A dynamic magnetic susceptibility measurement based on a nuclear magnetic resonance method yielded a 2.8 microB magnetic movement for the chromium(IV) species. The intermediate is formed by parallel third-order and second-order processes. The third-order process (k = 5.9 x 10(2) M-2 s-1) involves first-order participation by each of the oxidant, reductant, and hydrogen ions. A hydrogen ion independent pathway leads to a sluggish second-order process (k = 0.11 M-1 s-1) that is first order with respect to reduced glutathione [GSH] and [Cr(VI)]. Chromium(IV) species is reduced to Cr(III) by a second-order process (k = 0.13 M-1 s-1) that is first order in each of [Cr(IV)] and [GSH] and does not depend on [H+]. At pH 3.4, a chromium(V) species was detected as a minor intermediate as well. In the pH range 6.5-7.5, three dominant chromium(V) intermediates were detected. The existence of Cr(IV) in low pH offers an opportunity to examine the mechanism of DNA damage by this rare oxidation state.

Inhibition of immunopurified DNA polymerase-alpha from PA-3 prostate tumor cells by platinum (II) antitumor drugs.

Cisplatin (cis-diamminedichloroplatinum(II); cis-DDP) is used as an effective drug for treatment of a variety of cancers, such as carcinomas of bladder, ovarian, and testicular origin. Immunopurified DNA polymerase-alpha from rat prostate tumor PA-3 cells was inhibited (50%) in the presence of cis-DDP (165 microM) and PtCl2(en) (cis-dichloroethylenediamine platinum (II); DEDAP) (75 microM) and remained uninhibited in the presence of trans-DDP. Immunopurified DNA polymerase-alpha was preincubated with cis-DDP and separated from unreacted cis-DDP by gel filtration chromatography. The platinated DNA polymerase-alpha was unable to initiate the DNA chain extension reaction. N-ethylmaleimide (1 mM), a thiol group modifier, also inhibited (95%) the DNA polymerase-alpha catalyzed reaction in vitro. Possible disruption of a zinc-finger motif of the DNA polymerase-alpha polypeptide chain by replacement of zinc is suggested.