Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Characterization of the roles of activated charcoal and Chelex in the induction of PrfA regulon expression in complex medium.

The foodborne pathogen Listeria monocytogenes is able to survive across a wide range of intra- and extra-host environments by appropriately modulating gene expression patterns in response to different stimuli. Positive Regulatory Factor A (PrfA) is the major transcriptional regulator of virulence gene expression in L. monocytogenes. It has long been known that activated charcoal is required to induce the expression of PrfA-regulated genes in complex media, such as Brain Heart Infusion (BHI), but not in chemically defined media. In this study, we show that the expression of the PrfA-regulated hly, which encodes listeriolysin O, is induced 5- and 8-fold in L. monocytogenes cells grown in Chelex-treated BHI (Ch-BHI) and in the presence of activated charcoal (AC-BHI), respectively, relative to cells grown in BHI medium. Specifically, we show that metal ions present in BHI broth plays a role in the reduced expression of the PrfA regulon. In addition, we show that expression of hly is induced when the levels of bioavailable extra- or intercellular iron are reduced. L. monocytogenes cells grown Ch-BHI and AC-BHI media showed similar levels of resistance to the iron-activated antibiotic, streptonigrin, indicating that activated charcoal reduces the intracellular labile iron pool. Metal depletion and exogenously added glutathione contributed synergistically to PrfA-regulated gene expression since glutathione further increased hly expression in metal-depleted BHI but not in BHI medium. Analyses of transcriptional reporter fusion expression patterns revealed that genes in the PrfA regulon are differentially expressed in response to metal depletion, metal excess and exogenous glutathione. Our results suggest that metal ion abundance plays a role in modulating expression of PrfA-regulated virulence genes in L. monocytogenes.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Streptonigrin at low concentration promotes heterochromatin formation.

Heterochromatin is essential for regulating global gene transcription and protecting genome stability, and may play a role in tumor suppression. Drugs promoting heterochromatin are potential cancer therapeutics but very few are known. In order to identify drugs that can promote heterochromatin, we used a cell-based method and screened NCI drug libraries consisting of oncology drugs and natural compounds. Since heterochromatin is originally defined as intensely stained chromatin in the nucleus, we estimated heterochromatin contents of cells treated with different drugs by quantifying the fluorescence intensity of nuclei stained with Hoechst DNA dye. We used HeLa cells and screened 231 FDA-approved oncology and natural substance drugs included in two NCI drug libraries representing a variety of chemical structures. Among these drugs, streptonigrin most prominently caused an increase in Hoechst-stained nuclear fluorescence intensity. We further show that streptonigrin treated cells exhibit compacted DNA foci in the nucleus that co-localize with Heterochromatin Protein 1 alpha (HP1α), and exhibit an increase in total levels of the heterochromatin mark, H3K9me3. Interestingly, we found that streptonigrin promotes heterochromatin at a concentration as low as one nanomolar, and at this concentration there were no detectable effects on cell proliferation or viability. Finally, in line with a previous report, we found that streptonigrin inhibits STAT3 phosphorylation, raising the possibility that non-canonical STAT function may contribute to the effects of streptonigrin on heterochromatin. These results suggest that, at low concentrations, streptonigrin may primarily enhance heterochromatin formation with little toxic effects on cells, and therefore might be a good candidate for epigenetic cancer therapy.

Streptonigrin Inhibits SENP1 and Reduces the Protein Level of Hypoxia-Inducible Factor 1α (HIF1α) in Cells.

Streptonigrin (CAS no. 3930-19-6) is a natural product shown to have antitumor activities in clinical trials conducted in the 1960s-1970s. However, its use in clinical studies eventually faded, and the molecular mechanisms of streptonigrin antitumor effects remain poorly defined. Despite its lack of current clinical use, efforts on its total synthesis have continued. Here, we show that streptonigrin binds and inhibits the SUMO-specific protease SENP1. NMR studies identified that streptonigrin binds to SENP1 on the surface where SUMO binds and disrupts SENP1-SUMO1 interaction. Site-directed mutations in combination with NMR chemical shift perturbation suggest key roles of aromatic π stacking interactions in binding streptonigrin. Treatment of cells with streptonigrin resulted in increased global SUMOylation levels and reduced level of hypoxia inducible factor alpha (HIF1α). These findings inform both the design of SENP1 targeting strategy and the modification of streptonigrin to improve its efficacy for possible future clinical use.

The PerR-Regulated P1B-4-Type ATPase (PmtA) Acts as a Ferrous Iron Efflux Pump in Streptococcus pyogenes.

Streptococcus pyogenes (group A Streptococcus [GAS]) is an obligate human pathogen responsible for a broad spectrum of human disease. GAS has a requirement for metal homeostasis within the human host and, as such, tightly modulates metal uptake and efflux during infection. Metal acquisition systems are required to combat metal sequestration by the host, while metal efflux systems are essential to protect against metal overload poisoning. Here, we investigated the function of PmtA (PerR-regulated metal transporter A), a P1B-4-type ATPase efflux pump, in invasive GAS M1T1 strain 5448. We reveal that PmtA functions as a ferrous iron [Fe(II)] efflux system. In the presence of high Fe(II) concentrations, the 5448ΔpmtA deletion mutant exhibited diminished growth and accumulated 5-fold-higher levels of intracellular Fe(II) than did the wild type and the complemented mutant. The 5448ΔpmtA deletion mutant also showed enhanced susceptibility to killing by the Fe-dependent antibiotic streptonigrin as well as increased sensitivity to hydrogen peroxide and superoxide. We suggest that the PerR-mediated control of Fe(II) efflux by PmtA is important for bacterial defense against oxidative stress. PmtA represents an exemplar for an Fe(II) efflux system in a host-adapted Gram-positive bacterial pathogen.

The SloR metalloregulator is involved in the Streptococcus mutans oxidative stress response.

SloR, a 25-kDa metalloregulatory protein in Streptococcus mutans modulates the expression of multiple genes, including the sloABC operon that encodes essential Mn2+ transport and genes that promote cariogenesis. In this study, we report on SloC- and SloR-deficient strains of S. mutans (GMS284 and GMS584, respectively) that demonstrate compromised survivorship compared with their UA159 wild-type progenitor and their complemented strains (GMS285 and GMS585, respectively), when challenged with streptonigrin and/or in growth competition experiments. The results of streptonigrin assays revealed significantly larger zones of inhibition for GMS584 than for either UA159 or GMS585, indicating weakened S. mutans survivorship in the absence of SloR. Competition assays revealed a compromised ability for GMS284 and GMS584 to survive peroxide challenge compared with their SloC- and SloR-proficient counterparts. These findings are consistent with a role for SloC and SloR in S. mutans aerotolerance. We also predicted differential expression of oxidative stress tolerance genes in GMS584 versus UA159 and GMS585 when grown aerobically. The results of quantitative RT-PCR experiments revealed S. mutans sod, tpx, and sloC expression that was upregulated in GMS584 compared with UA159 and GMS585, indicating that the impact of oxidative stress on S. mutans is more severe in the absence of SloR than in its presence. The results of electrophoretic mobility shift assays indicate that SloR does not bind to the sod or tpx promoter regions directly, implicating intermediaries that may arbitrate the SloR response to oxidative stress.

Antisense transcription regulates the expression of the enterohemorrhagic Escherichia coli virulence regulatory gene ler in response to the intracellular iron concentration.

Enteric pathogens, such as enterohemorrhagic E. coli (EHEC) O157:H7, encounter varying concentrations of iron during their life cycle. In the gastrointestinal tract, the amount of available free iron is limited because of absorption by host factors. EHEC and other enteric pathogens have developed sophisticated iron-responsive systems to utilize limited iron resources, and these systems are primarily regulated by the Fur repressor protein. The iron concentration could be a signal that controls gene expression in the intestines. In this study, we explored the role of iron in LEE (locus for enterocyte effacement) virulence gene expression in EHEC. In contrast to the expression of Fur-regulated genes, the expression of LEE genes was greatly reduced in fur mutants irrespective of the iron concentration. The expression of the ler gene, the LEE-encoded master regulator, was affected at a post-transcription step by fur mutation. Further analysis showed that the loss of Fur affected the translation of the ler gene by increasing the intracellular concentration of free iron, and the transcription of the antisense strand was necessary for regulation. The results indicate that LEE gene expression is closely linked to the control of intracellular free iron homeostasis.

Characterization of a ferric uptake regulator (Fur)-mutant of the cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT5344.

The Fur protein is the main sensor of cellular iron status in bacteria. In the present study, we inactivated the fur gene of Pseudomonas pseudoalcaligenes CECT5344 and characterized the resulting mutant. Our findings provide experimental evidence that, cyanide generates an intracellular signal equivalent to that triggered by iron deprivation, as witnessed by the induction of prrF and fiuA (ferrichrome receptor) expression in the presence of cyanide. The fur mutant also displayed slow growth, especially in minimal culture medium, increased sensitivity to cyanide in LB medium and as expected, resistance to manganese ions. Moreover, the mutant exhibited enhanced iron accumulation and increased sensitivity to streptonigrin, as well as to some inducers of oxidative stress, such as paraquat and menadione, yet it remained resistant to hydrogen peroxide. Surprisingly, neither the wild type strain nor the fur mutant strain produced siderophores that could be detected using the universal CAS-agar method.

Natural compounds' activity against cancer stem-like or fast-cycling melanoma cells.

BACKGROUND: Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. METHODS: We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. FINDINGS: Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. CONCLUSION: Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

Insights into the mechanism of streptonigrin-induced protein arginine deiminase inactivation.

Protein citrullination is just one of more than 200 known PTMs. This modification, catalyzed by the protein arginine deiminases (PADs 1-4 and PAD6 in humans), converts the positively charged guanidinium group of an arginine residue into a neutral ureido-group. Given the strong links between dysregulated PAD activity and human disease, we initiated a program to develop PAD inhibitors as potential therapeutics for these and other diseases in which the PADs are thought to play a role. Streptonigrin which possesses both anti-tumor and anti-bacterial activity was later identified as a highly potent PAD4 inhibitor. In an effort to understand why streptonigrin is such a potent and selective PAD4 inhibitor, we explored its structure-activity relationships by examining the inhibitory effects of several analogues that mimic the A, B, C, and/or D rings of streptonigrin. We report the identification of the 7-amino-quinoline-5,8-dione core of streptonigrin as a highly potent pharmacophore that acts as a pan-PAD inhibitor.

The ESX-3 secretion system is necessary for iron and zinc homeostasis in Mycobacterium tuberculosis.

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.

Analysis of the activities of RAD54, a SWI2/SNF2 protein, using a specific small-molecule inhibitor.

RAD54, an important homologous recombination protein, is a member of the SWI2/SNF2 family of ATPase-dependent DNA translocases. In vitro, RAD54 stimulates RAD51-mediated DNA strand exchange and promotes branch migration of Holliday junctions. It is thought that an ATPase-dependent DNA translocation is required for both of these RAD54 activities. Here we identified, by high-throughput screening, a specific RAD54 inhibitor, streptonigrin (SN), and used it to investigate the mechanisms of RAD54 activities. We found that SN specifically targets the RAD54 ATPase, but not DNA binding, through direct interaction with RAD54 and generation of reactive oxygen species. Consistent with the dependence of branch migration (BM) on the ATPase-dependent DNA translocation of RAD54, SN inhibited RAD54 BM. Surprisingly, the ability of RAD54 to stimulate RAD51 DNA strand exchange was not significantly affected by SN, indicating a relatively smaller role of RAD54 DNA translocation in this process. Thus, the use of SN enabled us to identify important differences in the effect of the RAD54 ATPase and DNA translocation on two major activities of RAD54, BM of Holliday junctions and stimulation of DNA pairing.

Gap junctional communication promotes apoptosis in a connexin-type-dependent manner.

Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to α-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca(2+) signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca(2+) concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca(2+) or a Ca(2+)-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca(2+) increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells.

[Microbial model of Halobacterium salinarum for screening inhibitors of sterol biosynthesis].

A highly effective and simple microbial test system for screening inhibitors of sterol biosynthesis (ISB) is described. The system is based on cultivation of the bacterial strain Halobacterium salinarum (former Halobacterium halobium), that possesses mevalonate pathway of sterol biosynthesis and is much similar in the biosynthesis to cholesterol formation in humans. In the H. salinarum test system the ISB were found as compounds that inhibited the test culture growth. Mevalonate which is one of the crucial intermediates of sterol biosynthesis dismissed the inhibitory effect of many microbial metabolites thus being evident of their action at the early stages of the sterol biosynthesis, including the HMG-CoA reductase stage. The H. salinarum test system was developed as a micromethod and could be easily mechanized by miniaturization of the microbiological procedures, cultivation in sterile 96-well plates and using automatic micropipettes and dispensers. The H. salinarum test system was effective in testing crude extracts of the culture broths and advantageous at early stage of screening. The use of the H. salinarum test system was shown possible for screening antitumor antibiotics.

Borrelia burgdorferi, a pathogen that lacks iron, encodes manganese-dependent superoxide dismutase essential for resistance to streptonigrin.

Borrelia burgdorferi, the causative agent of Lyme disease, exists in nature through a complex life cycle involving ticks of the Ixodes genus and mammalian hosts. During its life cycle, B. burgdorferi experiences fluctuations in oxygen tension and may encounter reactive oxygen species (ROS). The key metalloenzyme to degrade ROS in B. burgdorferi is SodA. Although previous work suggests that B. burgdorferi SodA is an iron-dependent superoxide dismutase (SOD), later work demonstrates that B. burgdorferi is unable to transport iron and contains an extremely low intracellular concentration of iron. Consequently, the metal cofactor for SodA has been postulated to be manganese. However, experimental evidence to support this hypothesis remains lacking. In this study, we provide biochemical and genetic data showing that SodA is a manganese-dependent enzyme. First, B. burgdorferi contained SOD activity that is resistant to H(2)O(2) and NaCN, characteristics associated with Mn-SODs. Second, the addition of manganese to the Chelex-treated BSK-II enhanced SodA expression. Third, disruption of the manganese transporter gene bmtA, which significantly lowers the intracellular manganese, greatly reduced SOD activity and SodA expression, suggesting that manganese regulates the level of SodA. In addition, we show that B. burgdorferi is resistant to streptonigrin, a metal-dependent redox cycling compound that produces ROS, and that SodA plays a protective role against the streptonigrin. Taken together, our data demonstrate the Lyme disease spirochete encodes a manganese-dependent SOD that contributes to B. burgdorferi defense against intracellular superoxide.

Streptonigrin inhibits ß-Catenin/Tcf signaling and shows cytotoxicity in ß-catenin-activated cells.

BACKGROUND: Activation of ß-catenin/T-cell factor (Tcf) signaling plays a role in human carcinogenesis. This suggests a possibility that the ß-catenin/Tcf signaling activated by the accumulation of ß-catenin in the nucleus is related to some type of human carcinogenesis. Therefore, if ß-catenin's transcriptional activity can be markedly down-regulated, tumor growth will be suppressed in ß-catenin activated types of cancer. METHODS: To investigate the activation or suppression of ß-catenin/Tcf transcription, we established a transiently transfected cell line with a constitutively active ß-catenin mutant gene whose product is not degraded. This cell line was also co-transfected with luciferase reporter gene constructs containing either an optimized (TOPflash) or mutant (FOPflash) Tcf-binding element. RESULTS: We identified the inhibitory effect of streptonigrin against ß-catenin/Tcf signaling in ß-catenin activated cells. Streptonigrin inhibited the transcriptional activity of ß-catenin/Tcf in SW480 cells and HEK293 cells transiently transfected with a constitutively active mutant ß-catenin gene. The growth inhibitory effect of streptonigrin was more evident in ß-catenin-activated cancer cells than in non-activated cancer cells. The electrophoresis mobility shift assay showed that the binding of Tcf complexes with their specific DNA-binding sites was suppressed by streptonigrin. CONCLUSION: Streptonigrin is a negative regulator of ß-catenin/Tcf signaling, and their inhibitory mechanism is related to the proliferation inhibitory effect on ß-catenin-activated cancer cells. GENERAL SIGNIFICANCE: This report reveals a molecular mechanism underlying the anti-tumor effect of streptonigrin from the perspective ß-catenin/Tcf signaling. Given its function in inhibiting ß-catenin/Tcf signaling, streptonigrin may be of interest as a leading compound for chemotherapeutic agent against ß-catenin-activated tumorigenesis.

A fluopol-ABPP HTS assay to identify PAD inhibitors.

Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator.

Relationship between heterochromatic interstitial telomeric sequences and chromosome damage induced by the radiomimetic compound streptonigrin in Chinese hamster ovary cells.

The relationship between (heterochromatic) interstitial telomeric sequences (ITSs) and the chromosome damage induced by the radiomimetic compound streptonigrin (SN) was investigated in Chinese hamster ovary (CHO) cells by using PNA- and Q-FISH techniques with a pantelomeric probe. CHO cells were exposed to increasing concentrations of SN and chromosomal aberrations were analyzed in the first mitosis after treatment. Cytogenetic analysis revealed that 16.9% and 11.7% of the total aberrations induced by SN in cells harvested 18 h and 3 h after treatment, respectively, exhibited one or more FISH-detectable telomeric signals. Although there was a significant induction by SN of chromosome breaks at centromeric regions containing ITSs, about 70% of the chromosome breaks exhibiting telomeric signals observed in SN-treated cells occurred outside the centromeric regions of chromosomes. This observation, along with the finding of entirely labeled acentric fragments in both untreated and SN-treated cells show that, although this antibiotic induces breakage at centromeric regions containing ITSs, these chromosome regions are not the preferential target for the clastogenic action of SN. In addition, our results show that heterochromatic ITSs are involved more than expected in the formation of chromatid breaks and exchanges induced by SN, and that these sequences are not preferentially involved in the formation of dicentrics, multicentrics, centric rings, chromosome breaks, acentric fragments and chromatid deletions induced by this antibiotic. These findings indicate that the involvement of heterochromatic ITSs in the chromosome damage induced by SN is not random. Moreover, our results show that SN induces telomeric repeats translocations, although this effect depends on the concentration of the drug, and that this antibiotic increases the size of ITSs, this latter effect not being related to the chromosomal sensitivity of the exposed cells to this compound. The mechanism by which SN induces amplification of heterochromatic ITSs remains to be elucidated.

Specific IKKbeta inhibitor IV blocks Streptonigrin-induced NF-kappaB activity and potentiates its cytotoxic effect on cancer cells.

Many anticancer agents activate NF-kappaB, which plays an important role in the survival of cancer cells. Inhibition of NF-kappaB activity may therefore potentiate the efficacy of anticancer agents. We found that a previously used anticancer agent Streptonigrin (SN) was also a potent NF-kappaB inducer. Using a specific IKKbeta inhibitor IV (Podolin et al., J Pharmacol Exp Ther 2005; 312: 373-381), we revealed that the activation of NF-kappaB was mediated through DNA damage-induced activation of IKK complex. Furthermore, we demonstrated that SN-induced DNA damage was unrelated to reactive oxygen species but to the hydroquinone form of SN converted by the NAD(P)H:quinine oxidoreductase (NQO1). The study suggests that the combination of SN with IKK inhibitor may improve efficacy over the use of single agent.