Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

Induction of DNA damage by free radicals generated either by organic or inorganic arsenic (AsIII, MMAIII, and DMAIII) in cultures of B and T lymphocytes

The aim of this work is based in the premise that inorganic arsenic (As^III) and trivalentmethylated metabolites monomethylarsonous (MMA^III) and dimethylarsinous (DMA^III) participate in DNA damage through the generation of reactive oxygen species (ROS). We have utilized two lymphoblastic lines, Raji (B cells) and Jurkat (T cells), which were treated with the trivalent arsenic species (dose: 0–100 μM) and analyzed by two assays (comet assay and flow cytometry) in the determination of DNA damage and ROS effects in vivo. The results showed that the damage to the DNA and the generation of ROS are different in both cellular lines with respect to the dose of organic arsenic, and the order of damage is MMA^III>DMA^III>As^III. This fact suggests that the DMA^III is not always the more cytotoxic intermediary xenobiotic, as has already been reported in another study.     

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Abstract

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Combined effects of 872 MHz radiofrequency radiation and ferrous chloride on reactive oxygen species production and DNA damage in human SH‐SY5Y neuroblastoma cells

The aim of the present study was to investigate possible cooperative effects of radiofrequency (RF) radiation and ferrous chloride (FeCl₂) on reactive oxygen species (ROS) production and DNA damage. In order to test intracellular ROS production as a possible underlying mechanism of DNA damage, we applied the fluorescent probe DCFH‐DA. Integrity of DNA was quantified by alkaline comet assay. The exposures to 872 MHz RF radiation were conducted at a specific absorption rate (SAR) of 5 W/kg using continuous waves (CW) or a modulated signal similar to that used in Global System for Mobile Communications (GSM) phones. Four groups were included: (1) Sham exposure (control), (2) RF radiation, (3) Chemical treatment, (4) Chemical treatment, and RF radiation. In the ROS production experiments, human neuroblastoma (SH‐SY5Y) cells were exposed to RF radiation and 10 µg/ml FeCl₂ for 1 h. In the comet assay experiments, the exposure time was 3 h and an additional chemical (0.015% diethyl maleate) was used to make DNA damage level observable. The chemical treatments resulted in statistically significant responses, but no effects from either CW or modulated RF radiation were observed on ROS production, DNA damage or cell viability. Bioelectromagnetics 31:417–424, 2010. © 2010 Wiley‐Liss, Inc.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

AGEs induces apoptosis and autophagy via reactive oxygen species in human periodontal ligament cells

The apoptosis of human periodontal ligament cells (HPDLCs) may be an important factor of the negative effect of advanced glycation end products (AGEs) on the periodontal tissue of diabetic patients. However, the pathways or potential effects of apoptosis in AGEs-treated HPDLCs have not been fully elucidated. Autophagy is closely related to apoptosis. Herein, we investigated the potential mechanism of apoptosis and autophagy in HPDLCs treated with AGEs via an in vitro model. We found that AGEs-treated HPDLCs showed a time- and concentration-dependent reduction in the cell survival rate. The mitochondrial-dependent apoptosis was induced in AGEs-treated HPDLCs, as confirmed by the mitochondrial membrane potential depolarization, decreased Bcl-2 expression, increased Bax expression, and increased caspase-3 and PARP cleavage. Autophagy was also induced in AGEs-treated HPDLCs, as indicated by the conversion of LC3-II/LC3-I and the presence of autophagosomes. Interestingly, our study results suggested that apoptosis and autophagy were related to reactive oxygen species (ROS) production. In addition, AGEs-induced autophagy acted as a latent factor in decreasing the generation of ROS in HPDLCs and protecting against the AGEs-induced apoptosis. In summary, our study shows that ROS are essential in AGEs-induced HPDLCs apoptosis and autophagy, which may be a molecular mechanism for the repairment of ROS-induced damage in HPDLCs treated with AGEs to promote cell survival. The present study might provide new insights into the therapeutic targeting of HPDLCs autophagy, which could be an additional strategy for periodontitis in patients with diabetes mellitus.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa)

The effect of aluminium (Al) on seedlings of two rice cultivars, Pusa Basmati and Vikas was investigated after different hours of exposure to 80 mol/L of external Al supply. With increasing time of exposure, the growing seedlings readily absorbed Al and its localization was greater in roots than shoots. Prolonged exposure to Al intensified lipid peroxidation, changed the activities of SOD and peroxidase and caused DNA damage. However, differential responses were observed between the seedlings of two rice cultivars under Al stress. A close inverse relationship existed between decreased root growth and increased Al accumulation, lipid peroxidation, SOD, peroxidase activities and DNA damage. The results demonstrate that roots are the major sites of Al localization and accumulation of Al promoted oxygen free radicals mediated peroxidation of membranes as evidenced by increased MDA levels and the activities of SOD and peroxidase. Our results for the first time showed that Al can cause DNA damage in rice.     

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l -cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.     

Endometriotic cyst fluid induces reactive oxygen species (ROS) in human immortalized epithelial cells derived from ovarian endometrioma

Objectives: Endometriotic cyst fluid (ECF) contains a large amount of reactive oxygen species (ROS), and endometriotic cysts are exposed to strong oxidative stress, which may cause malignant transformation. In this study, ROS production by ECF was clinically analysed.

Methods: Human immortalized epithelial cells derived from ovarian endometrioma (EMosis-CC/TERT 1) were treated with ECF. In addition, ROS production in EMosis-CC/TERT 1 was measured, and its clinical significance was analysed.

Results: A total of 38 ECF samples were obtained from patients diagnosed with endometriotic cysts. In EMosis-CC/TERT1, significantly higher levels of ROS were induced by ECF than by the vehicle control and ferric nitrilotriacetate. There were no significant differences in ROS production by laterality and preoperative serum CA125 values. There were several patients whose cyst sizes were approximately 5?cm and had relatively high ROS production. Production of ROS by ECF was relatively higher in patients older than 40 years of age than in those younger than 40.

Discussion: Our study revealed that ROS are highly produced by ECF in EMosis-CC/TERT1 cells; therefore, exposure to ECF induced strong oxidative stress. Development of a therapeutic strategy to reduce ROS production might be useful for preventing malignant transformation of endometriotic cysts.     

Lipocalin‐2 enhances angiogenesis in rat brain endothelial cells via reactive oxygen species and iron‐dependent mechanisms

Inflammation is a key part of central nervous system pathophysiology. However, inflammatory factors are now thought to have both beneficial and deleterious effects. Here, we examine the hypothesis that lipocalin‐2 (LCN2), an inflammatory molecule that can be up‐regulated in the distressed central nervous system, may enhance angiogenesis in brain endothelial cells. Adding LCN2 (0.5–2.0 μg/mL) to RBE (Rat brain endothelial cells). 4 rat brain endothelial cells significantly increased matrigel tube formation and scratch migration, and also elevated levels of iron and reactive oxygen species. Co‐treatment with a radical scavenger (U83836E), a Nox inhibitor (apocynin) and an iron chelating agent (deferiprone) significantly dampened the ability of LCN2 to enhance tube formation and scratch migration in brain endothelial cells. These findings provide in vitro proof of the concept that LCN2 can promote angiogenesis via iron‐ and reactive oxygen species‐related pathways, and support the idea that LCN2 may contribute to the neurovascular recovery aspects of inflammation.

     

Comparison of the effect of raw and blanched‐frozen broccoli on DNA damage in colonocytes

Consumption of cruciferous vegetables may protect against colorectal cancer. Cruciferous vegetables are rich in a number of bioactive constituents including polyphenols, vitamins and glucosinolates. Before consumption, cruciferous vegetables often undergo some form of processing that reduces their content of bioactive constituents and may determine whether they exert protective effects. The aim of this study was to compare the ability of raw and blanched‐frozen broccoli to protect colonocytes against DNA damage, improve antioxidant status and induce xenobiotic metabolizing enzymes (XME). Fifteen Landrace × Large White male pigs were divided into five age‐matched and weight‐matched sets (79 days, SD 3, and 34·7 kg, SD 3·9, respectively). Each set consisted of siblings to minimize genetic variation. Within each set, pigs received a cereal‐based diet, unsupplemented (control) or supplemented with 600 g day^?1 of raw or blanched‐frozen broccoli for 12 days. The consumption of raw broccoli caused a significant 27% increase in DNA damage in colonocytes (p = 0·03) relative to the control diet, whereas blanched‐frozen broccoli had no significant effect. Both broccoli diets had no significant effect on plasma antioxidant status or hepatic and colonic XME. This study is the first to report that the consumption of raw broccoli can damage DNA in porcine colonocytes. Copyright © 2015 John Wiley & Sons, Ltd.     

Role of reactive oxygen species (ROS) in apoptosis induction

Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.