Insights into the anti-angiogenic properties of phosphaplatins.

Phosphaplatins are platinum-based antitumor compounds that, unlike other clinically utilized platinum drugs (i.e. cisplatin, carboplatin, and oxaliplatin), appear to target proteins rather than DNA. Because of their unique mode of action, phosphaplatins are promising drug candidates for cisplatin-resistant cancers. In this study, we discovered that Pt(II) and Pt(IV) phosphaplatins possess diverse antitumor properties. In addition to targeting apoptosis antigen (FAS) and proapoptotic gene products as described previously, phosphaplatins also target angiogenesis. We demonstrate that phosphaplatins inhibit human umbilical vein endothelial cell (HUVEC) migration and tube formation in vitro and suppress tumor angiogenesis and growth in immunodeficient mice that were inoculated with A2780 ovarian cancer cells in vivo. To provide insight into this novel antitumor mechanism, phosphaplatin-treated HUVECs were found to exhibit lower gene expression levels of vascular endothelial growth factors (VEGFs) and the VEGFR-2 receptor compared to untreated cells. Kinase inhibition studies suggest that phosphaplatins are inhibitors of VEGFR-2. In ligand exchange experiments using both Pt atomic absorption and 31P NMR spectroscopies, we show that phosphaplatins most likely bind to VEGFR-2 through metal-ligand coordination rather than electrostatic interactions. These studies enhance our understanding of the diverse and novel mechanisms of action of the phosphaplatin antitumor agents, which could potentially be used as chemotherapeutic agents against cisplatin-resistant cancers.

Absence of Activation of DNA Repair Genes and Excellent Efficacy of Phosphaplatins against Human Ovarian Cancers: Implications To Treat Resistant Cancers.

Phosphaplatins, platinum(II) and platinum(IV) complexes coordinated to a pyrophosphate moiety, exhibit excellent antitumor activities against a variety of cancers. To determine whether phosphaplatins trigger resistance to treatment by engaging DNA damage repair genes, a yeast genome-wide fitness assay was used. Treatment of yeast cells with pyrodach-2 (D2) or pyrodach-4 (D4) revealed no particular sensitivity to nucleotide excision repair, homologous recombination repair, or postreplication repair when compared with platin control compounds. Also, TNF receptor superfamily member 6 (FAS) protein was overexpressed in phosphaplatin-treated ovarian tumor cells, and platinum colocalized with FAS protein in lipid rafts. An overactivation of sphingomyelinase (ASMase) was noted in the treated cells, indicating participation of an extrinsic apoptotic mechanism due to increased ceramide release. Our results indicate that DNA is not the target of phosphaplatins and accordingly, that phosphaplatins might not cause resistance to treatment. Activation of ASMase and FAS along with the colocalization of platinum with FAS in lipid rafts support an extrinsic apoptotic signaling mechanism that is mediated by phosphaplatins.

Decreased cytochrome c oxidase subunit VIIa in aged rat heart mitochondria: immunocytochemistry.

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24-month old (aged) rats compared to 6-month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi-quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity.

Function of mitochondrial Stat3 in cellular respiration.

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3(-/-) cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity.

Measurement of complex III activity is critical to the diagnosis of human mitochondrial disease and the study of mitochondrial pathobiology. Activity is measured as the maximal rate of antimycin A-sensitive reduction of exogenous cytochrome c by detergent-solubilized mitochondria. Complex III activity exhibited an unexpected variation based upon the commercial source of cytochrome c owing to an increase in the antimycin A-insensitive background reduction of cytochrome c and variable increases in total activity. Analysis of cytochrome c (producing a high-background) by fast protein liquid chromatography yielded a contaminant peak containing a lipid extractable component with redox spectra and mass spectroscopy fragmentation suggestive of a quinol. Measurement of inhibitor-sensitive rates are critical for the accurate and reproducible measurement of complex III activity and serve as a key quality control to screen for non-enzymatic reactions that obscure complex III activity.

Ischemic defects in the electron transport chain increase the production of reactive oxygen species from isolated rat heart mitochondria.

Cardiac ischemia decreases complex III activity, cytochrome c content, and respiration through cytochrome oxidase in subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The reversible blockade of electron transport with amobarbital during ischemia protects mitochondrial respiration and decreases myocardial injury during reperfusion. These findings support that mitochondrial damage occurs during ischemia and contributes to myocardial injury during reperfusion. The current study addressed whether ischemic damage to the electron transport chain (ETC) increased the net production of reactive oxygen species (ROS) from mitochondria. SSM and IFM were isolated from 6-mo-old Fisher 344 rat hearts following 25 min global ischemia or following 40 min of perfusion alone as controls. H(2)O(2) release from SSM and IFM was measured using the amplex red assay. With glutamate as a complex I substrate, the net production of H(2)O(2) was increased by 178 +/- 14% and 179 +/- 17% in SSM and IFM (n = 9), respectively, following ischemia compared with controls (n = 8). With succinate as substrate in the presence of rotenone, H(2)O(2) increased by 272 +/- 22% and 171 +/- 21% in SSM and IFM, respectively, after ischemia. Inhibitors of electron transport were used to assess maximal ROS production. Inhibition of complex I with rotenone increased H(2)O(2) production by 179 +/- 24% and 155 +/- 14% in SSM and IFM, respectively, following ischemia. Ischemia also increased the antimycin A-stimulated production of H(2)O(2) from complex III. Thus ischemic damage to the ETC increased both the capacity and the net production of H(2)O(2) from complex I and complex III and sets the stage for an increase in ROS production during reperfusion as a mechanism of cardiac injury.

Ischemic preconditioning does not protect via blockade of electron transport.

Ischemic preconditioning (IPC) before sustained ischemia decreases myocardial infarct size mediated in part via protection of cardiac mitochondria. Reversible blockade of electron transport at complex I immediately before sustained ischemia also preserves mitochondrial respiration and decreases infarct size. We proposed that IPC would attenuate electron transport from complex I as a potential effector mechanism of cardioprotection. Isolated, Langendorff-perfused rat hearts underwent IPC (3 cycles of 5-min 37 degrees C global ischemia and 5-min reperfusion) or were perfused for 40 min without ischemia as controls. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated. IPC did not decrease ADP-stimulated respiration measured in intact mitochondria using substrates that donate reducing equivalents to complex I. Maximally expressed complex I activity measured as rotenone-sensitive NADH:ubiquinone oxidoreductase in detergent-solubilized mitochondria was also unaffected by IPC. Thus the protection of IPC does not occur as a consequence of a partial decrease in complex I activity leading to a decrease in integrated respiration through complex I. IPC and blockade of electron transport both converge on mitochondria as effectors of cardioprotection; however, each modulates mitochondrial metabolism during ischemia by different mechanisms to achieve cardioprotection.

Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion.

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.

Reversal of mitochondrial defects before ischemia protects the aged heart.

Myocardial injury is increased in the aged heart during ischemia and reperfusion. Aging decreases oxidative metabolism in interfibrillar mitochondria (IFM) located between the myofibrils. We asked whether reversal of aging defects in IFM before ischemia would decrease injury in the aged heart following ischemia and reperfusion. Treatment with acetylcarnitine (AcCN) increases the activity of cytochrome oxidase in the aged heart. Aged (24 months) and adult (6 months) Fischer 344 rats were treated with AcCN (300 mg/kg i.p. 3 h before excision of the heart) or served as controls. AcCN restored oxidative phosphorylation and the activity of complexes III and IV in IFM from aged hearts to rates present in adults. Isolated hearts underwent 25 min global ischemia and 30 min reperfusion without additional treatment. Contractile recovery during reperfusion improved in hearts from AcCN-treated aged rats compared to aged controls and were similar to adults in recovery. AcCN-treated aged hearts sustained less damage, indicated by decreased lactate dehydrogenase (LDH) release during reperfusion. AcCN treatment did not alter functional recovery or LDH release in adults. Restoration of mitochondrial function in the aged heart before ischemia was accompanied by enhanced contractile recovery and decreased tissue injury following ischemia and reperfusion.

Blockade of electron transport during ischemia protects cardiac mitochondria.

Subsarcolemmal mitochondria sustain progressive damage during myocardial ischemia. Ischemia decreases the content of the mitochondrial phospholipid cardiolipin accompanied by a decrease in cytochrome c content and a diminished rate of oxidation through cytochrome oxidase. We propose that during ischemia mitochondria produce reactive oxygen species at sites in the electron transport chain proximal to cytochrome oxidase that contribute to the ischemic damage. Isolated, perfused rabbit hearts were treated with rotenone, an irreversible inhibitor of complex I in the proximal electron transport chain, immediately before ischemia. Rotenone pretreatment preserved the contents of cardiolipin and cytochrome c measured after 45 min of ischemia. The rate of oxidation through cytochrome oxidase also was improved in rotenone-treated hearts. Inhibition of the electron transport chain during ischemia lessens damage to mitochondria. Rotenone treatment of isolated subsarcolemmal mitochondria decreased the production of reactive oxygen species during the oxidation of complex I substrates. Thus, the limitation of electron flow during ischemia preserves cardiolipin content, cytochrome c content, and the rate of oxidation through cytochrome oxidase. The mitochondrial electron transport chain contributes to ischemic mitochondrial damage that in turn augments myocyte injury during subsequent reperfusion.

Production of reactive oxygen species by mitochondria: central role of complex III.

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.

Aging defect at the QO site of complex III augments oxyradical production in rat heart interfibrillar mitochondria.

Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart.

DNA oxidation by peroxo-chromium(v) species: oxidation of guanosine to guanidinohydantoin.

Reactions of peroxo-chromium(v) complexes with DNA afforded mainly guanine oxidation yielding, a four-electron oxidation product, guanidinohydantoin, and exhibited extensive base labile strand scission.

Interfibrillar cardiac mitochondrial comples III defects in the aging rat heart.

We used the Fischer 344 rat as a model for aging effects on the heart. Cardiac interfibrillar mitochondria (IFM), located between the myofibrils, exhibit a decrease in protein yield and oxidative phosphorylation through complex III and IV in elderly (24 months) compared to adult controls (6 months). In contrast, subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remained unchanged. The activity of electron transport complex III decreased only in the IFM with aging. Complex III and IV require an inner mitochondrial membrane lipid, cardiolipin for maximal activity. However, the content and composition of cardiolipin was unchanged in the IFM from aging hearts. We observed electron leakage in complex III at the myxothiazol site in the aging IFM accompanied by increased superoxide production. The aging heart sustains greater injury during ischemia and reperfusion compared to adult hearts. We propose that ischemic damage combines with aging defects in complex III to increase oxidative injury in aging hearts. Ischemia damaged complex III in both SSM and IFM from adult and aging hearts via impairment of the iron-sulfur subunit without the loss of the apoprotein. Thus, at the onset of reperfusion, complex III in IFM contains two defects in electron flow, which are likely to prime complex III for enhanced oxidant production during reperfusion, leading to increased damage in aging hearts.

Preservation of cardiolipin content during aging in rat heart interfibrillar mitochondria.

Aging selectively decreases the rate of oxidative phosphorylation in the interfibrillar population of cardiac mitochondria (IFM) located between the myofibers. In contrast, subsarcolemmal mitochondria (SSM), located below the plasma membrane, remain unaffected. IFM from elderly (24-month-old) Fischer 344 rats have a decreased specific activity of complexes III and IV. Complexes III and IV require an inner mitochondrial membrane lipid environment enriched in the oxidatively sensitive phospholipid cardiolipin for maximal activity. We asked if aging decreases the content or alters the composition of cardiolipin as a potential mechanism of the aging defect in IFM. The content and composition of mitochondrial phospholipids were measured in SSM and IFM from adult and aging rat hearts. Aging did not alter the content of mitochondrial phospholipids, including cardiolipin, in either population of mitochondria. The composition of cardiolipin based on characterization of both acyl group and the individual molecular species of cardiolipin was also unaltered by aging. Lipid-mediated oxidative modification of complex III subunits was not detected, making cardiolipin-derived oxidative damage to complex III unlikely. Thus, alterations in cardiolipin are not the mechanism for the aging defect in IFM in Fischer 344 rats.