The physiological functions of hydrogen sulfide (H 2 S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective upregulation of the H 2 S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H 2 S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H 2 S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patientderived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H 2 S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H 2 S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H 2 S as a tumor growth factor and anticancer drug target.

The purpose of the current study was to investigate the effect of the recently synthesized mitochondrially-targeted H2S donor, AP39 [10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], on bioenergetics, viability, and mitochondrial DNA integrity in bEnd.3 murine microvascular endothelial cells in vitro, under normal conditions, and during oxidative stress. Intracellular H2S was assessed by the fluorescent dye 7-azido-4-methylcoumarin. For the measurement of bioenergetic function, the XF24 Extracellular Flux Analyzer was used. Cell viability was estimated by the combination of the MTT and LDH methods. Oxidative protein modifications were measured by the Oxyblot method. Reactive oxygen species production was monitored by the MitoSOX method. Mitochondrial and nuclear DNA integrity were assayed by the Long Amplicon PCR method. Oxidative stress was induced by addition of glucose oxidase. AP39 (30 – 300 nM) to bEnd.3 cells increased intracellular H2S levels, with a preferential response in the mitochondrial regions. AP39 exerted a concentration-dependent effect on mitochondrial activity, which consisted of a stimulation of mitochondrial electron transport and cellular bioenergetic function at lower concentrations (30–100 nM) and an inhibitory effect at the higher concentration of 300 nM. Under oxidative stress conditions induced by glucose oxidase, an increase in oxidative protein modification and an enhancement in MitoSOX oxidation was noted, coupled with an inhibition of cellular bioenergetic function and a reduction in cell viability. AP39 pretreatment attenuated these responses. Glucose oxidase induced a preferential damage to the mitochondrial DNA; AP39 (100 nM) pretreatment protected against it. In conclusion, the current paper documents antioxidant and cytoprotective effects of AP39 under oxidative stress conditions, including a protection against oxidative mitochondrial DNA damage.

Background: DNA apurinic/apyrimidinic (AP) sites are toxic and mutagenic if unrepaired by AP endonucleases. Results: Structural, mutational, and computational analyses of prototypic AP endonucleases APE1 and Nfo identify surprising similarities. Conclusion: APE1 and Nfo reveal functional equivalences illuminating their catalytic reaction. Significance: A conserved catalytic geometry is specific to AP site removal despite different enzyme structures and metal ions.

Reactive oxygen species (ROS), continuously generated as byproducts of respiration, inflict more damage on the mitochondrial (mt) than on the nuclear genome because of the nonchromatinized nature and proximity to the ROS source of the mitochondrial genome. Such damage, particularly single-strand breaks (SSBs) with 5-blocking deoxyribose products generated directly or as repair intermediates for oxidized bases, is repaired via the base excision/SSB repair pathway in both nuclear and mt genomes. Here, we show that EXOG, a 5-exo/endonuclease and unique to the mitochondria unlike FEN1 or DNA2, which, like EXOG, has been implicated in the removal of the 5-blocking residue, is required for repairing endogenous SSBs in the mt genome. EXOG depletion induces persistent SSBs in the mtDNA, enhances ROS levels, and causes apoptosis in normal cells but not in mt genome-deficient rho0 cells. Thus, these data show for the first time that persistent SSBs in the mt genome alone could provide the initial trigger for apoptotic signaling in mammalian cells.A mammalian cell contains up to several thousands copies of duplex, circular 16.5-kb mt 2 genome within 80 -700 mitochondria depending on the cell type (1, 2). The mtDNA encodes essential subunits of the respiratory chain, tRNA and rRNAs, all of which are critical for maintaining oxidative phosphorylation (OXPHOS) (3). OXPHOS accounts for about 85% of oxygen consumed by the cell, and early reports estimated that under physiological conditions ϳ5% of consumed oxygen is partially reduced to ROS (4). Although recent reports indicate a much lower level of ROS production in normal cells, even a low and persistent ROS level has long term detrimental effects (5, 6). Thus, the mitochondria, the major cellular site for ROS generation, are under continuous oxidative stress that results in oxidative damage to DNA, as well as proteins and lipids (7).ROS-induced DNA damage includes multitude of mutagenic oxidized bases and single-strand breaks (SSBs) containing 3Ј-and 5Ј-blocking groups in DNA, which are generated both directly or as intermediates during BER (8, 9). Because of close proximity of the site of ROS generation and nonchromatinized state of the mt genome, the mutation rate in human mtDNA is 20 -100-fold higher relative to the nDNA (10). As summarized in recent reviews (11-13), repair of oxidized base lesions or abnormal bases is initiated with their excision by a DNA glycosylase. A monofunctional glycosylase, such as uracil-DNA glycosylase, excises U from the DNA to generate an abasic (AP) site, which is then cleaved by AP endonuclease (APE1) in mammalian cells to generate 3Ј-OH and nonligatable 5Ј-deoxyribose phosphate (dRP) residues. In the nucleus, the 5Ј-dRP could be removed by DNA polymerase ␤ (pol ␤) via its intrinsic dRP lyase activity. In the mitochondria, the DNA polymerase ␥ (pol ␥) with similar dRP lyase activity is also able to remove the dRP moiety (14). In the case of oxidized base repair by DNA glycosylases with intrinsic AP lyase activity, such as 8-oxoguanine-DNA g...

The mitochondrial genome is highly susceptible to damage by reactive oxygen species (ROS) generated endogenously as a byproduct of respiration. ROS-induced DNA lesions, including oxidized bases, abasic (AP) sites, and oxidized AP sites, cause DNA strand breaks and are repaired via the base excision repair (BER) pathway in both the nucleus and mitochondria. Repair of damaged bases and AP sites involving 1-nucleotide incorporation, named single nucleotide (SN)-BER, was observed with mitochondrial and nuclear extracts. During SN-BER, the 5-phosphodeoxyribose (dRP) moiety, generated by AP-endonuclease (APE1), is removed by the lyase activity of DNA polymerase ␥ (pol ␥) and polymerase ␤ in the mitochondria and nucleus, respectively. However, the repair of oxidized deoxyribose fragments at the 5 terminus after strand break would require 5-exo/endonuclease activity that is provided by the flap endonuclease (FEN-1) in the nucleus, resulting in multinucleotide repair patch (long patch (LP)-BER). Here we show the presence of a 5-exo/endonuclease in the mitochondrial extracts of mouse and human cells that is involved in the repair of a lyase-resistant AP site analog via multinucleotide incorporation, upstream and downstream to the lesion site. We conclude that LP-BER also occurs in the mitochondria requiring the 5-exo/endonuclease and pol ␥ with 3-exonuclease activity. Although a FEN-1 antibody cross-reacting species was detected in the mitochondria, it was absent in the LP-BER-proficient APE1 immunocomplex isolated from the mitochondrial extract that contains APE1, pol ␥, and DNA ligase 3. The LP-BER activity was marginally affected in FEN-1-depleted mitochondrial extracts, further supporting the involvement of an unidentified 5-exo/endonuclease in mitochondrial LP-BER.The mammalian mitochondrion contains 5-15 copies of the circular 16-kb mitochondrial (mt) 2 genome, and each mammalian cell thus may contain a thousand or more copies of the mt genome (1). MtDNA, encoding 13 subunits of the electron transport chain and containing genes for ribosomal RNAs and tRNAs (2), is extremely susceptible to oxidative damage, presumably because of the lack of protective histones and proximity to reactive oxygen species (ROS), which are endogenously generated by the electron transport complexes (3, 4). Such damage includes several dozen oxidized bases, abasic (AP) sites, and oxidation products of AP sites leading to DNA strand breaks (5). Endogenous mutations in mtDNA, likely to arise from these lesions, were shown to be considerably higher than in the nuclear genome (6). Approximately 10,000 AP sites were estimated to be generated per nuclear genome per day (7). Analysis of the release of 5-methylene-2-furanone, the product of ␤-, ␥-elimination of 2-deoxyribonolactone, an oxidized AP site, causing DNA strand breakage, suggests that this ribonolactone could account for 70% of the total sugar damage in DNA (8, 9). The oxidized AP sites, whose level is likely to be high especially in the mtDNA, should block replication and transcription a...

Therapeutic manipulation of the gasotransmitter hydrogen sulfide (H2S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H2S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H2S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H2S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H2S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance.

Allergic airway inflammation is characterized by increased expression of pro-inflammatory mediators, inflammatory cell infiltration, mucus hypersecretion, and airway hyperresponsiveness, in parallel with oxidative DNA base and strand damage, whose etiological role is not understood. Our goal was to establish the role of 8-oxoguanine (8-oxoG), a common oxidatively damaged base, and its repair by 8-oxoguanine DNA glycosylase 1 (Ogg1) in allergic airway inflammatory processes. Airway inflammation was induced by intranasally administered ragweed (Ambrosia artemisiifolia) pollen grain extract (RWPE) in sensitized BALB/c mice. We utilized siRNA technology to deplete Ogg1 from airway epithelium; 8-oxoG and DNA strand break levels were quantified by Comet assays. Inflammatory cell infiltration and epithelial methaplasia were determined histologically, mucus and cytokines levels biochemically and enhanced pause was used as the main index of airway hyperresponsiveness. Decreased Ogg1 expression and thereby 8-oxoG repair in the airway epithelium conveyed a lower inflammatory response after RWPE challenge of sensitized mice, as determined by expression of Th2 cytokines, eosinophilia, epithelial methaplasia, and airway hyperresponsiveness. In contrast, 8-oxoG repair in Ogg1-proficient airway epithelium was coupled to an increase in DNA single-strand break (SSB) levels and exacerbation of allergen challenge-dependent inflammation. Decreased expression of the Nei-like glycosylases Neil1 and Neil2 that preferentially excise ring-opened purines and 5-hydroxyuracil, respectively, did not alter the above parameters of allergic immune responses to RWPE. These results show that DNA SSBs formed during Ogg1-mediated repair of 8-oxoG augment antigen-driven allergic immune responses. A transient modulation of OGG1 expression/activity in airway epithelial cells could have clinical benefits.

Mitochondrial dysfunction is observed in many diseases. Targeting H2S generation to mitochondria may be cytoprotective.