Jack C. Yalowich

Mechanisms of cardiolipin oxidation by cytochrome c : Relevance to Pro- and antiapoptotic functions of etoposide

Execution of apoptotic program in mitochondria is associated with accumulation of cardiolipin peroxidation products required for the release of proapoptotic factors into the cytosol. This suggests that lipid antioxidants capable of inhibiting cardiolipin peroxidation may act as antiapoptotic agents. Etoposide, a widely used antitumor drug and a topoisomerase II inhibitor, is a prototypical inducer of apoptosis and, at the same time, an effective lipid radical scavenger and lipid antioxidant. Here, we demonstrate that cardiolipin oxidation during apoptosis is realized not via a random cardiolipin peroxidation mechanism but rather proceeds as a result of peroxidase reaction in a tight cytochrome c/cardiolipin complex that restrains interactions of etoposide with radical intermediates generated in the course of the reaction. Using low-temperature and ambient-temperature electron paramagnetic resonance spectroscopy of H2O2-induced protein-derived (tyrosyl) radicals and etoposide phenoxyl radicals, respectively, we established that cardiolipin peroxidation and etoposide oxidation by cytochrome c/cardiolipin complex takes place predominantly on protein-derived radicals of cytochrome c. We further show that etoposide can inhibit cytochrome c-catalyzed oxidation of cardiolipin competing with it as a peroxidase substrate. Peroxidase reaction of cytochrome c/cardiolipin complexes causes cross-linking and oligomerization of cytochrome c. With nonoxidizable tetraoleoyl-cardiolipin, the cross-linking occurs via dityrosine formation, whereas bifunctional lipid oxidation products generated from tetralinoleoyl-cardiolipin participate in the production of high molecular weight protein aggregates. Protein aggregation is effectively inhibited by etoposide. The inhibition of cardiolipin peroxidation by etoposide, however, is realized at far higher concentrations than those at which it induces apoptotic cell death. Thus, oxidation of cardiolipin by the cytochrome c/cardiolipin peroxidase complex, which is essential for apoptosis, is not inhibited by proapoptotic concentrations of the drug.

The effect of dexrazoxane (ICRF-187) on doxorubicin- and daunorubicin-mediated growth inhibition of Chinese hamster ovary cells.

Dexrazoxane (ICRF-187) is clinically used to reduce doxorubicin-induced cardiotoxicity. Because dexrazoxane, doxorubicin and daunorubicin all act on DNA topoisomerase II, a study was undertaken to see what effect dexrazoxane had on the growth inhibitory effects of doxorubicin and daunorubicin towards Chinese hamster ovary cells. Dexrazoxane exhibited significant antagonism of doxorubicin- and daunorubicin-mediated growth inhibition when the cells were preincubated with dexrazoxane before the anthracycline was added. Continuous exposure of cells to either anthracycline and low concentrations of dexrazoxane resulted in additive growth inhibitory effects at low anthracycline concentrations, and no effect at higher anthracycline concentrations.

c-Abl kinase regulates curcumin-induced cell death through activation of c-Jun N-terminal kinase.

Curcumin, a natural phenolic compound found in turmeric (Curcuma longa) exhibits anticancer properties, attributed to its antiproliferative and apoptosis-inducing activity. The ubiquitously expressed nonreceptor tyrosine kinase c-Abl regulates stress responses induced by oxidative agents such as ionizing radiation and H2O2. In this study, we show that c-Abl is an important component of the cell death response activated by curcumin and that Abl mediates this response partly through activation of c-Jun N-terminal kinase (JNK). Therefore, inhibition of Abl by STI571 [imatinib (Gleevec)] treatment or down-regulation of Abl expression through Abl-specific short-hairpin RNA (shRNA) diminished cell death induction and JNK activation. Highlighting the interdependent nature of the Abl and JNK signaling in the curcumin-induced cell death response, a JNK inhibitor [anthra(1,9-cd)pyrazol-6(2H)-one-1,9-pyrazoloanthrone (SP600125)] caused very little cell death inhibition in STI571-pretreated cells and in Abl shRNA-expressing cells. Moreover, treatment with Abl and JNK inhibitor alone or together caused similar levels of cell death inhibition. Although p53 induction in response to curcumin treatment is dependent on Abl, we found that Abl→p53 signaling is not necessary for curcumin-induced cell death. Taken together, the results demonstrate the differential roles played by Abl→p53 and Abl→JNK signaling events in modulating the cell death response to curcumin.

Thiol-modulated mechanisms of the cytotoxicity of thimerosal and inhibition of DNA topoisomerase II alpha.

Thimerosal is an organic mercury compound that is widely used as a preservative in vaccines and other solution formulations. The use of thimerosal has caused concern about its ability to cause neurological abnormalities due to mercury accumulation during a normal schedule of childhood vaccinations. While the chemistry and the biological effects of methylmercury have been well-studied, those of thimerosal have not. Thimerosal reacted rapidly with cysteine, GSH, human serum albumin, and single-stranded DNA to form ethylmercury adducts that were detectable by mass spectrometry. These results indicated that thimerosal would be quickly metabolized in vivo because of its reactions with protein and nonprotein thiols. Thimerosal also potently inhibited the decatenation activity of DNA topoisomerase II alpha, likely through reaction with critical free cysteine thiol groups. Thimerosal, however, did not act as a topoisomerase II poison and the lack of cross-resistance with a K562 cell line with a decreased level of topoisomerase II alpha (K/VP.5 cells) suggested that inhibition of topoisomerase II alpha was not a significant mechanism for the inhibition of cell growth. Depletion of intracellular GSH with buthionine sulfoximine treatment greatly increased the K562 cell growth inhibitory effects of thimerosal, which showed that intracellular glutathione had a major role in protecting cells from thimerosal. Pretreatment of thimerosal with glutathione did not, however, change its K562 cell growth inhibitory effects, a result consistent with the rapid exchange of the ethylmercury adduct among various thiol-containing cellular reactants. Thimerosal-induced single and double strand breaks in K562 cells were consistent with a rapid induction of apoptosis. In conclusion, these studies have elucidated some of the chemistry and biological activities of the interaction of thimerosal with topoisomerase II alpha and protein and nonprotein thiols and with DNA.

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the β isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme–DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIβ.

The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα

The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-57, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of assays were used to determine whether Dp44mT and triapine target topoisomerase IIα. Neither of these compounds inhibited topoisomerase IIα decatenation or induced cleavage of pBR322 DNA in the presence of enzyme. In cells, Dp44mT did not stabilize topoisomerase IIα covalent binding to DNA using an immunoblot band depletion assay, an ICE (immunodetection of complexes of enzyme-to-DNA) assay, and a protein-DNA covalent complex forming assay. Dp44mT did not display cross resistance to etoposide resistant K562 cells containing reduced topoisomerase IIα levels. Synchronized Dp44mT-treated CHO cells did not display a G2/M cell cycle block expected of a topoisomerase II inhibitor. A COMPARE analysis of Dp44mT using the NCI 60-cell line data indicated that inhibition of cell growth was poorly correlated with DNA topoisomerase IIα mRNA levels. In summary, we found no support for the conclusion that Dp44mT inhibits cell growth through the targeting of topoisomerase IIα. Since clinical trials of triapine are underway, it will be important to better understand the intracellular targeting and mechanisms of action of the thiosemicarbazones to support forward development of these agents and newer analogs.

Potent Cytotoxic Arylnaphthalene Lignan Lactones from Phyllanthus poilanei

Two new (1 and 2) and four known arylnaphthalene lignan lactones (3–6) were isolated from different plant parts of Phyllanthus poilanei collected in Vietnam, with two further known analogues (7 and 8) being prepared from phyllanthusmin C (4). The structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods, and the structure of phyllanthusmin D (1) was confirmed by single-crystal X-ray diffraction analysis. Several of these arylnaphthalene lignan lactones were cytotoxic toward HT-29 human colon cancer cells, with compounds 1 and 7-O-[(2,3,4-tri-O-acetyl)-α-l-arabinopyranosyl)]diphyllin (7) found to be the most potent, exhibiting IC50 values of 170 and 110 nM, respectively. Compound 1 showed activity when tested in an in vivo hollow fiber assay using HT-29 cells implanted in immunodeficient NCr nu/nu mice. Mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3, but it did not inhibit DNA topoisomerase IIα activity.

Cadmium is a Catalytic Inhibitor of DNA Topoisomerase II

Cadmium (Cd(2+)) is a highly toxic and carcinogenic metal that is an environmental and occupational hazard. DNA topoisomerase II is an essential nuclear enzyme and its inhibition can result in the formation of genotoxic and recombinogenic DNA double strand breaks. In this study we showed that cadmium chloride strongly inhibited the DNA decatenation activity of human topoisomerase IIα in the low micromolar concentration range and that its inhibitory effects were reduced by glutathione. Because the activity of topoisomerase II is strongly inhibited by thiol-reactive compounds this result suggested that cadmium may be binding to critical topoisomerase II cysteine thiols. Cadmium, however, did not stabilize DNA-topoisomerase II covalent complexes, as measured by the lack of formation of DNA double strand breaks. Hence, it is not likely to be a topoisomerase II poison. Consistent with the idea that cadmium cytotoxicity may be modulated by glutathione levels, buthionine sulfoximine pretreatment to decrease glutathione levels resulted in a greatly increased cadmium-induced cytotoxicity in K562 cells. The results of this study suggest that cadmium may exert some of its cell growth inhibitory, and possibly its toxicity and carcinogenicity, by inhibiting topoisomerase IIα through reaction with critical cysteine thiols.

The Mismatch Repair System Modulates Curcumin Sensitivity through Induction of DNA Strand Breaks and Activation of G2-M Checkpoint

The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of γH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G2-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function. Mol Cancer Ther; 9(3); 558–68