Synergistic DNA Damage Research Articles

Pro-Oxidant Auranofin and Glutathione-Depleting Combination Unveils Synergistic Lethality in Glioblastoma Cells with Aberrant Epidermal Growth Factor Receptor Expression.

Glioblastoma (GBM) is the most prevalent and advanced malignant primary brain tumor in adults. GBM frequently harbors epidermal growth factor receptor (EGFR) wild-type (EGFRwt) gene amplification and/or EGFRvIII activating mutation. EGFR-driven GBM relies on the thioredoxin (Trx) and/or glutathione (GSH) antioxidant systems to withstand the excessive production of reactive oxygen species (ROS). The impact of EGFRwt or EGFRvIII overexpression on the response to a Trx/GSH co-targeting strategy is unknown. In this study, we investigated Trx/GSH co-targeting in the context of EGFR overexpression in GBM. Auranofin is a thioredoxin reductase (TrxR) inhibitor, FDA-approved for rheumatoid arthritis. L-buthionine-sulfoximine (L-BSO) inhibits GSH synthesis by targeting the glutamate-cysteine ligase catalytic (GCLC) enzyme subunit. We analyzed the mechanisms of cytotoxicity of auranofin and the interaction between auranofin and L-BSO in U87MG, U87/EGFRwt, and U87/EGFRvIII GBM isogenic GBM cell lines. ROS-dependent effects were assessed using the antioxidant N-acetylsteine. We show that auranofin decreased TrxR1 activity and increased ROS. Auranofin decreased cell vitality and colony formation and increased protein polyubiquitination through ROS-dependent mechanisms, suggesting the role of ROS in auranofin-induced cytotoxicity in the three cell lines. ROS-dependent PARP-1 cleavage was associated with EGFRvIII downregulation in U87/EGFRvIII cells. Remarkably, the auranofin and L-BSO combination induced the significant depletion of intracellular GSH and synergistic cytotoxicity regardless of EGFR overexpression. Nevertheless, molecular mechanisms associated with cytotoxicity were modulated to a different extent among the three cell lines. U87/EGFRvIII exhibited the most prominent ROS increase, P-AKT(Ser-473), and AKT decrease along with drastic EGFRvIII downregulation. U87/EGFRwt and U87/EGFRvIII displayed lower basal intracellular GSH levels and synergistic ROS-dependent DNA damage compared to U87MG cells. Our study provides evidence for ROS-dependent synergistic cytotoxicity of auranofin and L-BSO combination in GBM in vitro. Unraveling the sensitivity of EGFR-overexpressing cells to auranofin alone, and synergistic auranofin and L-BSO combination, supports the rationale to repurpose this promising pro-oxidant treatment strategy in GBM.

The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation.

TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.

Chromosomal damage, gene expression and alternative transcription in human lymphocytes exposed to mixed ionizing radiation as encountered in space

Astronauts travelling in space will be exposed to mixed beams of particle radiation and photons. Exposure limits that correspond to defined cancer risk are calculated by multiplying absorbed doses by a radiation-type specific quality factor that reflects the biological effectiveness of the particle without considering possible interaction with photons. We have shown previously that alpha radiation and X-rays may interact resulting in synergistic DNA damage responses in human peripheral blood lymphocytes but the level of intra-individual variability was high. In order to assess the variability and validate the synergism, blood from two male donors was drawn at 9 time points during 3 seasons of the year and exposed to 0–2 Gy of X-rays, alpha particles or 1:1 mixture of both (half the dose each). DNA damage response was quantified by chromosomal aberrations and by mRNA levels of 3 radiation-responsive genes FDXR, CDKN1A and MDM2 measured 24 h post exposure. The quality of response in terms of differential expression of alternative transcripts was assessed by using two primer pairs per gene. A consistently higher than expected effect of mixed beams was found in both donors for chromosomal aberrations and gene expression with some seasonal variability for the latter. No synergy was detected for alternative transcription.

Development and validation of an HPLC-MS/MS method for the simultaneous analysis of volatile organic compound metabolites, hydroxylated polycyclic aromatic hydrocarbons, and 8-hydroxy-2′-deoxyguanosine in human urine

Humans are widely and concurrently exposed to volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs). However, few studies have reported the internal co-exposure levels of these chemicals in occupational and general populations. Specifically, the associations revealed between the urinary levels of metabolites of VOCs (mVOCs), hydroxylated PAHs (OH-PAHs), and oxidative stress biomarkers for humans remain limited. In this study, a method based on solid-phase extraction (SPE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of 22 mVOCs, 12 OH-PAHs, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in human urine samples. The method was validated with all target analyte accuracies and precisions in the range of 76 %–120 % and 1 %–14 % at three levels of spiked urine samples, respectively. The limit of detection (LOD) and limit of quantification (LOQ) of the target analytes were 0.01–0.34 ng/mL and 0.01–7.57 ng/mL, respectively. And the method was applied to measure urinary levels of target analytes from 38 petrochemical workers in Guangzhou, South China. Except for 3-hydroxy-benzo[a]pyrene, all target analytes were detected in the urine samples. The average levels were 0.05–12.6 ng/mL for individual OH-PAHs, 0.20–73620 ng/mL for individual mVOCs, and 1.00 ng/mL for 8-OHdG. Additionally, 3-hydroxy-phenanthrene, 1-hydroxy-pyrene, 6-hydroxy-chrysene, N-acetyl-S-(trichlorovinyl)-L-cysteine, 2-methylhippuric acid, thiodiacetic acid, trans, trans-Muconic acid, and N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine had statistically significant positive effects on 8-OHdG levels, while 1-hydroxy-naphthalene, 1,2-dihydroxybenzene, and hippuric acid showed a negative effect on 8-OHdG, indicating these metabolites could lead to synergistic or antagonistic oxidative DNA damage. This study provides a robust analytical method that permits a comprehensive assessment of co-exposure to PAHs and VOCs and their potential adverse health effects.

Salvia chinensis Benth Inhibits Triple-Negative Breast Cancer Progression by Inducing the DNA Damage Pathway.

ObjectiveTriple-negative breast cancer (TNBC) is distinguished by early recurrence and metastases, a high proclivity for treatment resistance, and a lack of targeted medicines, highlighting the importance of developing innovative therapeutic techniques. Salvia chinensis Benth (SCH) has been widely studied for its anticancer properties in a variety of cancers. However, its significance in TNBC treatment is rarely discussed. Our study investigated the anticancer effect of SCH on TNBC and the underlying mechanisms.MethodsFirst, we used clonogenic, cell viability, flow cytometry, and Transwell assays to assess the effect of SCH on TNBC. Bioinformatic studies, especially network pharmacology-based analysis and RNA sequencing analysis, were performed to investigate the constituents of SCH and its molecular mechanisms in the suppression of TNBC. High-performance liquid chromatography and thin-layer chromatography were used to identify two major components, quercetin and β-sitosterol. Then, we discovered the synergistic cytotoxicity of quercetin and β-sitosterol and assessed their synergistic prevention of cell migration and invasion. Breast cancer xenografts were also created using MDA-MB-231 cells to test the synergistic therapeutic impact of quercetin and β-sitosterol on TNBC in vivo. The impact on the DNA damage and repair pathways was investigated using the comet assay and Western blot analysis.ResultsOur findings showed that SCH decreased TNBC cell growth, migration, and invasion while also inducing cell death. We identified quercetin and β-sitosterol as the core active components of SCH based on a network pharmacology study. According to RNA sequencing research, the p53 signaling pathway is also regarded as a critical biological mechanism of SCH treatment. The comet assay consistently showed that SCH significantly increased DNA damage in TNBC cells. Our in vivo and in vitro data revealed that the combination of quercetin and β-sitosterol induced synergistic cytotoxicity and DNA damage in TNBC cells. In particular, SCH particularly blocked the inter-strand cross-link repair mechanism and the double-strand breach repair caused by the homologous recombination pathway, in addition to inducing DNA damage. Treatment with quercetin and β-sitosterol produced similar outcomes.ConclusionThe current study provides novel insight into the previously unknown therapeutic potential of SCH as a DNA-damaging agent in TNBC.

Targeted liposomes for combined delivery of artesunate and temozolomide to resistant glioblastoma

The effective treatment of glioblastoma (GBM) is a great challenge because of the blood-brain barrier (BBB) and the growing resistance to single-agent therapeutics. Targeted combined co-delivery of drugs could circumvent these challenges; however, the absence of more effective combination drug delivery strategies presents a potent barrier. Here, a unique combination ApoE-functionalized liposomal nanoplatform based on artesunate-phosphatidylcholine (ARTPC) encapsulated with temozolomide (ApoE-ARTPC@TMZ) was presented that can successfully co-deliver dual therapeutic agents to TMZ-resistant U251-TR GBM in vivo. Examination in vitro showed ART-mediated inhibition of DNA repair through the Wnt/β-catenin signaling cascade, which also improved GBM sensitivity to TMZ, resulting in enhanced synergistic DNA damage and induction of apoptosis. In assessing BBB permeation, the targeted liposomes were able to effectively traverse the BBB through low-density lipoprotein family receptors (LDLRs)-mediated transcytosis and achieved deep intracranial tumor penetration. More importantly, the targeted combination liposomes resulted in a significant decrease of U251-TR glioma burden in vivo that, in concert, substantially improved the survival of mice. Additionally, by lowering the effective dosage of TMZ, the combination liposomes reduced systemic TMZ-induced toxicity, highlighting the preclinical potential of this novel integrative strategy to deliver combination therapies to brain tumors.

Inducing Synergistic DNA Damage by TRIP13 and PARP1 Inhibitors Provides a Potential Treatment for Hepatocellular Carcinoma.

Thyroid hormone receptor interactor 13 (TRIP13), an AAA-ATPase, participates in the development of many cancers. This study explores the function of TRIP13 and synergistic effects of TRIP13 and PARP1 inhibitors in hepatocellular carcinoma (HCC). The dose-dependent effects of TRIP13 and PARP1 inhibitors on HCC cells proliferation or migration were investigated by the CCK-8 and Transwell assays. Using siRNA or lentivirus to knock down TRIP13, we tested HCC cell and tumor growth in vitro and in vivo. The DNA damage caused by TRIP13 and PARP1 inhibitors was measured by the phosphorylation of H2AX, one of the DNA damage biomarkers. The phosphorylation of H2AX was increased after treatment with DCZ0415 or TRIP13 knockdown. Combining DCZ0415 with PARP1 inhibitor, Olaparib induced synergistic anti-HCC activity. We also found that the overexpression of TRIP13 is significantly associated with early recurrent HCC and poor survival. Up-regulation of TRIP13 in HCC was regulated by transcription factor SP1. In conclusion, our study demonstrated that DCZ0415 targeting TRIP13 impaired non-homologous end-joining repair to inhibit HCC progression and had a synergistic effect with PARP1 inhibitor Olaparib in HCC, suggesting a potential treatment of HCC.

Oxidative Stress-Dependent Synergistic Antiproliferation, Apoptosis, and DNA Damage of Ultraviolet-C and Coral-Derived Sinularin Combined Treatment for Oral Cancer Cells.

Simple SummaryCombined treatments with low side effects enhance anticancer applications. This study focusses on validating the potential synergistic antiproliferation of the combined treatment of ultraviolet-C and the coral-derived compound sinularin (UVC/sinularin) in oral cancer cells. This study confirms that UVC/sinularin synergistically and selectively inhibits oral cancer cell proliferation with low cytotoxicity on normal oral cells. The mechanisms involve the enhanced cellular and mitochondrial oxidative stress that cause apoptosis, DNA damage, and mitochondrial dysfunction in oral cancer cells.Combined treatment is increasingly used to improve cancer therapy. Non-ionizing radiation ultraviolet-C (UVC) and sinularin, a coral Sinularia flexibilis-derived cembranolide, were separately reported to provide an antiproliferation function to some kinds of cancer cells. However, an antiproliferation function using the combined treatment of UVC/sinularin has not been investigated as yet. This study aimed to examine the combined antiproliferation function and explore the combination of UVC/sinularin in oral cancer cells compared to normal oral cells. Regarding cell viability, UVC/sinularin displays the synergistic and selective killing of two oral cancer cell lines, but remains non-effective for normal oral cell lines compared to treatments in terms of MTS and ATP assays. In tests using the flow cytometry, luminescence, and Western blotting methods, UVC/sinularin-treated oral cancer cells exhibited higher reactive oxygen species production, mitochondrial superoxide generation, mitochondrial membrane potential destruction, annexin V, pan-caspase, caspase 3/7, and cleaved-poly (ADP-ribose) polymerase expressions than that in normal oral cells. Accordingly, oxidative stress and apoptosis are highly induced in a combined UVC/sinularin treatment. Moreover, UVC/sinularin treatment provides higher G2/M arrest and γH2AX/8-hydroxyl-2′deoxyguanosine-detected DNA damages in oral cancer cells than in the separate treatments. A pretreatment can revert all of these changes of UVC/sinularin treatment with the antioxidant N-acetylcysteine. Taken together, UVC/sinularin acting upon oral cancer cells exhibits a synergistic and selective antiproliferation ability involving oxidative stress-dependent apoptosis and cellular DNA damage with low toxic side effects on normal oral cells.

Reduction-Responsive Chemo-Capsule-Based Prodrug Nanogel for Synergistic Treatment of Tumor Chemotherapy.

Chemotherapy is currently the most universal therapeutics to tumor treatment; however, limited curative effect and undesirable drug resistance effect are the two major clinical bottlenecks. Herein, we develop a two-in-one cross-linking strategy to prepare a stimuli-responsive prodrug nanogel by virtue of delivering a combination of chemotherapeutic drugs of 10-hydroxy camptothecin and doxorubicin for ameliorating the deficiencies of chemotherapy and amplifying the cancer therapeutic efficiency. The obtained prodrug nanogel has both high drug loading capacity and suitable nanoscale size, which are beneficial to the cell uptake and tumor penetration. Moreover, the chemotherapeutic drugs are released from the prodrug nanogel in response to the reductive tumor microenvironment, enhancing tumor growth inhibition in vitro and in vivo by the synergistic DNA damage. Based on these results, the unique prodrug nanogel would be a promising candidate for satisfactory tumor treatment-based chemotherapy by a simple but efficient strategy.

Redox Signaling in the Endoplasmic Reticulum Regulates Endothelial Surface N-glycoforms during Inflammation

Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.

Combined Treatment with Low Cytotoxic Ethyl Acetate Nepenthes Extract and Ultraviolet-C Improves Antiproliferation to Oral Cancer Cells via Oxidative Stress.

Ultraviolet-C (UVC) irradiation provides an alternative radiotherapy to X-ray. UVC sensitizer from natural products may improve radiotherapy at low cytotoxic side effects. The aim of this study is to assess the regulation for oral cancer cell proliferation by a combined treatment of UVC and our previously reported anti-oral cancer natural product (ethyl acetate extract of Nepenthes adrianii × clipeata; EANA). The detailed possible UVC sensitizing mechanisms of EANA such as effects on cell proliferation, cell cycle, apoptosis, and DNA damage are investigated individually and in combination using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay, flow cytometry, and western blotting at low dose conditions. In a 24 h MTS assay, the low dose EANA (5 μg/mL) and low dose UVC (12 J/m2) individually show 80% and combinedly 57% cell proliferation in oral cancer Ca9-22 cells; but no cytotoxicity to normal oral HGF-1 cells. Mechanistically, low dose EANA and low dose UVC individually induce apoptosis (subG1 accumulation, pancaspase activation, and caspases 3, 8, 9), oxidative stress (reactive oxygen species, mitochondrial superoxide, and mitochondrial membrane potential depletion), and DNA damage (γH2AX and 8-hydroxy-2′-deoxyguanosine). Moreover, the combined treatment (UVC/EANA) synergistically induces these changes. Combined low dose treatment-induced antiproliferation, apoptosis, oxidative stress, and DNA damage were suppressed by the ROS scavenger N-acetylcysteine. In conclusion, UVC/EANA shows synergistic antiproliferation, oxidative stress, apoptosis, and DNA damage to oral cancer cells in an oxidative stress-dependent manner. With the selective killing properties of low dose EANA and low dose UVC, EANA provides a novel UVC sensitizing agent to improve the anti-oral cancer therapy.

Mechanism of synergistic DNA damage induced by caffeic acid phenethyl ester (CAPE) and Cu(II): Competitive binding between CAPE and DNA with Cu(II)/Cu(I)

Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.

P-224 - Mechanism of the synergistic DNA damage and unusual hydroxyl radical production by the non-enzymatic activation of the anti-tuberculosis drug isoniazid by Mn(III)

Isoniazid (INH) has been used as a frontline anti-tuberculosis drug, which can be activated to produce reactive radical species by enzymes and metal ions. However, the underlying activation mechanism of INH by Mn(III) remains unclear. Here we found that INH/Mn(III) together induced synergistic oxidative DNA damage (8-oxo-dG, DSBs), which could be inhibited by the typical •OH scavengers, and to our surprise, also inhibited by SOD, but not by catalase. •OH was found to be produced by INH/Mn(III) by both ESR secondary spin-trapping and fluorescent methods, which was also inhibited by SOD, but not by catalase. We further found that Mn(III) was reduced to Mn(II) by INH, while INH was oxidized initially to form the characteristic N-centered isoniazidyl radical, which decompose to produce the C-centered isonicotinic acyl radical. During this redox process, O2•- was produced. More interestingly, we found that •OH can be produced by Mn(II) and O2•- only when INH was simultaneously present. Together, we proposed that the synergistic DNA damage induced by INH/Mn(III) was due to the unusual O2•--dependent and H2O2-independent production of •OH via forming reactive MnOOH intermediate. This study provided new insight on the non-enzymatic activation of INH by Mn(III).

85 - Site-specific Production of Hydroxyl Radicals and Synergistic DNA Damage Induced by the Non-enzymatic Activation of the Anti-tuberculosis Drug Isoniazid by Cu(II)

Isoniazid (INH) has been recognized as a frontline anti-tuberculosis drug against Mycobacterium tuberculosis (Mtb), which is readily activated by Mtb catalase peroxidase enzyme to produce the reactive isonicotinic acyl radical intermediate responsible for its anti-tuberculosis activity. However, it is not clear whether INH can be activated non-enzymatically by Cu(II). Here we found that INH and Cu(II) together could induce synergistic DNA damage, including DNA strand breakage and 8-oxodG formation, while neither of them alone has any effect. DNA damage induced by INH/Cu(II) could be inhibited by Cu(I)-specific chelator and catalase, but not by SOD and the typical •OH radical scavengers. Interestingly, •OH, •H, C- and N-centered radicals were found to be produced during Cu(II)-catalyzed oxidation of INH by ESR spin-trapping method. The C-centered radical was further unequivocally identified by ESR and ESI-Q-TOF-MS as isonicotinic acyl radical, which can react with nicotinamide coenzyme NADH to form the critical isonicotinic acyl-NAD adducts. Low temperature ESR studies showed that Cu(II) was reduced to Cu(I) by INH. We proposed that the synergistic DNA damage induced by INH/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. This study provided a new insight on non-enzymatic activation of INH by Cu(II), which may have important biological implications for future research on INH.

English

Considering the vast use of sweeteners by the Brazilian population and the constant need for toxicological studies of food additives, this study aimed to evaluate the cytotoxic and mutagenic potentials of the sodium saccharin and/or sodium cyclamate sweeteners in plant (Allium cepa) and animal (Mus musculus) test systems based on concentrations permitted by the Brazilian laws. In A. cepa, both the sweeteners individually and their combinations concentration and exposure time (ET: 48, 72 and 168 h) dependently exerted cytotoxic and mutagenic effects. Similarly, an increased in micronuclei formation was also observed by the sweeteners in peripheral blood cells in mice. More toxic effects were observed with the combination doses at 168 ET. There may be a synergistic effect and DNA damage with an increasing concentration and ET. In conclusion, the concentrations considered safe by the Brazilian laws had significant cytotoxic and mutagenic activity on the eukaryotic cells. Key words: Allium cepa, Mus musculus, sweeteners, toxicity.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.

Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading.

Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.

48 - Non-Enzymatic Radical Activation of the Potent Anti-Tubercular Isoniazid by Cu(II)

Isoniazid (INH) is one of the most potent drugs available for tuberculosis treatment. As a pro-drug it requires activation by catalase/peroxidase to produce the reactive isonicotinic acyl radical intermediate responsible for its anti-tuberculosis activity, However, it is not clear whether INH can be activated non-enzymatically by Cu(II). Here we found that INH and Cu(II) together could induce synergistic DNA damage (strand breakage and 8-oxodG formation), while neither of them alone has any effect. DNA damage by INH/Cu(II) could be inhibited by Cu(I)-specific chelator and catalase, but not by SOD and the typical ●OH radical scavengers. Interestingly, ESR spin-trapping method showed that ●OH, ●H and a C-centered radical were produced during Cu(II)-catalyzed oxidation of INH. We proposed that the synergistic DNA damage induced by INH/Cu(II) might be due to the synergistic and site-specific production of ●OH near the binding site of copper and DNA. The C-centered radical was further unequivocally identified by ESR and HPLC-MS as isonicotinic acyl radical, which can react with nicotinamide coenzyme NADH to form the critical isonicotinic acyl-NAD adduct. The adduct can effectively inhibit in vitro activity of the enoyl-acyl carrier protein reductase InhA, an INH target in the biosynthetic pathway of mycolic acids. This study provided a new insight on non-enzymatic activation of INH by Cu(II) to produce the reactive isonicotinic acyl radical, ●OH and ●H, which may have important biological implications for future research on INH.

44 - Mechanism of Synergistic DNA Damage Induced by the Hydroquinone Metabolite of Brominated Phenolic Environmental Pollutants and Cu(II): Formation of DNA-Cu Complex and Site-Specific Production of Hydroxyl Radicals

2,6-dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants and was also found as one of disinfection byproducts in drinking water. Here we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage and 8-oxodG formation. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent and catalase, but not by SOD, nor by the typical hydroxyl radical (●OH) scavengers such as DMSO. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, CD, cyclic voltammetry and O2 consumption methods; and the highly reactive ●OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent and ESR spin-trapping methods. We propose that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of ●OH near the binding site of Cu and DNA.

PTH-336 Effects of lower doses of dca on radiosensitisation of colorectal cancer cells in vitro

Introduction Neoadjuvant radiotherapy confers a well-established benefit in locoregional recurrence for patients with locally invasive rectal cancer. However, only around 50% experience significant downstaging of their tumour. Radiation-associated toxicity renders dose escalation unachievable. Dichloroacetate (DCA) is a generic drug shown to modify tumour metabolism reversing the Warburg effect and inducing apoptosis and may potentially enhance the effect of radiation. This study aimed to investigate the effects of DCA at lower doses in combination with radiotherapy on the survival of CRC cell lines, and any possible synergistic DNA damage. Method CRC cells (LoVo) and control cells (HEK 293) treated with DCA (2,5,10 mM) and irradiation (0,4,8 Gy). Influences of the combination treatment on cell survival were analysed using MTT assays and clonogenic assays and the induction of DNA damage by quantifying gamma-H2AX foci. Statistical analyses were performed using GraphPad, differences between DCA-treated and vehicle control groups were assessed using the Mann-Whitney U test. Results DCA (5 mM) significantly reduced the survival of CRC cells in combination with 4 Gy and 8 Gy (P = 0.0244, 0.0078, respectively). DCA (10 mM) significantly reduced survival of CRC cells in in combination with 4 Gy and 8 Gy (P DCA (5 mM) significantly increased the mean number of DNA double-strand breaks (DSB) per CRC cell in combination with 4 Gy and 8 Gy (P = 0.0041, 0.023, respectively). In unirradiated CRC cells, all the DCA doses (2, 5, 10 mM) significantly increased the mean number of DSB’s per CRC cell (P Conclusion Lower doses of DCA may have an additive killing effect in combination with radiation in CRC cells. A similar picture of sensitivity was seen in normal cell line demonstrating that DCA is not completely harmless. Increasing doses of DCA associated with increased levels of DSB’s in CRC cells and it may be a potential mechanism behind DCA. Further research is warranted exploring the mechanism behind the effects of DCA and as a potential adjunct in the management of rectal cancers. Disclosure of interest None Declared.