Oxidative Stress-induced DNA Damage Research Articles

Potential of Vitamin E Deficiency, Induced by Inhibition of α-Tocopherol Efflux, in Murine Malaria Infection.

Although epidemiological and experimental studies have suggested beneficial effects of vitamin E deficiency on malaria infection, it has not been clinically applicable for the treatment of malaria owing to the significant content of vitamin E in our daily food. However, since α-tocopherol transfer protein (α-TTP) has been shown to be a determinant of vitamin E level in circulation, manipulation of α-tocopherol levels by α-TTP inhibition was considered as a potential therapeutic strategy for malaria. Knockout studies in mice indicated that inhibition of α-TTP confers resistance against malaria infections in murines, accompanied by oxidative stress-induced DNA damage in the parasite, arising from vitamin E deficiency. Combination therapy with chloroquine and α-TTP inhibition significantly improved the survival rates in murines with malaria. Thus, clinical application of α-tocopherol deficiency could be possible, provided that α-tocopherol concentration in circulation is reduced. Probucol, a recently found drug, induced α-tocopherol deficiency in circulation and was effective against murine malaria. Currently, treatment of malaria relies on the artemisinin-based combination therapy (ACT); however, when mice infected with malarial parasites were treated with probucol and dihydroartemisinin, the beneficial effect of ACT was pronounced. Protective effects of vitamin E deficiency might be extended to manage other parasites in future.

Effects of Amaranthus cruentus L. on aflatoxin B1- and oxidative stress-induced DNA damage in human liver (HepG2) cells

Amaranth is presently an underutilized crop despite its high content of micronutrients/bioactive phytochemicals and its capacity to thrive in harsh environmental condition. The present study aimed at determining the health benefits of Amaranthus cruentus L. in terms of protection against DNA damage induced by the mycotoxin aflatoxin B1 (AFB1) and oxidative stress using comet assay. The antioxidant potential was further investigated using electron paramagnetic resonance spectroscopy (EPR) and an ARE/Nrf2 reporter gene assay in vitro in a human liver model using the HepG2 cell line. Ethanolic extracts from fresh leaves grown under controlled conditions were used and additionally analyzed for their phytochemical content using liquid chromatography-mass spectrometry (LC-MS). The extracts inhibited both AFB1- and oxidative stress-induced DNA damage in a concentration dependent way with a maximum effect of 57% and 81%, respectively. Oxidative stress triggered using ferrous sulfate was blocked by up to 38% (EPR); the potential to induce antioxidant enzymes using ARE/Nrf2-mediated gene expression was also confirmed. Based on these in vitro findings, further studies on the health-protecting effects of A. cruentus are encouraged to fully explore its health promoting potential and provide the scientific basis for encouraging its cultivation and consumption.

Schisandrin A prevents oxidative stress-induced DNA damage and apoptosis by attenuating ROS generation in C2C12 cells

The generation of excessive reactive oxygen species (ROS) by oxidative stress has various deleterious effects on cellular constituents. Therefore, the discovery of natural products that can inhibit the production of ROS may be effective in suppressing the onset and treatment of oxidative stress-mediated diseases. Despite the antioxidant efficacy studies on various substances in the genus Schisandra used as traditional medicine, research on the efficacy of schisandrin A, a class of active lignans, remains insufficient. This study was conducted to evaluate the protective effect of schisandrin A on DNA damage and apoptosis induced by hydrogen peroxide (H2O2) in C2C12 cells. Results showed that schisandrin A effectively attenuated H2O2-induced cytotoxicity and DNA damage, which was related to the blockage of ROS accumulation. Schisandrin A also prevented the decrease in ATP content by H2O2 and restored the H2O2-induced activation of AMP-activated protein kinase. In addition, schisandrin A prevented the release of H2O2-induced cytochrome c into the cytoplasm presumably by inhibiting the loss of mitochondrial membrane potential and the changes in the Bcl-2 family protein expression by H2O2. Furthermore, the blocking of H2O2-induced apoptosis by schisandrin A was associated with the inhibition of poly (ADP-ribose) polymerase degradation by the inactivation of caspase-3. These results suggest that schisandrin A maintains energy metabolism through the preservation of mitochondrial function while eliminating ROS generated by H2O2 in C2C12 cells. Therefore, our data indicate that schisandrin A may have a beneficial effect on the prevention and treatment of diseases associated with apoptosis induced by oxidative stress.

Activation of the Nrf2/HO-1 Signaling Pathway Contributes to the Protective Effects of Sargassum serratifolium Extract against Oxidative Stress-Induced DNA Damage and Apoptosis in SW1353 Human Chondrocytes.

Oxidative stress in chondrocytes plays a critical role in the pathogenesis of osteoarthritis as an important cause of articular cartilage degradation. Sargassum serratifolium C. Agardh, a marine brown algae, is known to have potent antioxidant activity. Nevertheless, no study has been conducted yet on the protective efficacy against oxidative stress in chondrocytes. Therefore, the aim of the current study is to investigate the mechanism of the antioxidative effect of ethanol extract of S. serratifolium (EESS) on DNA damage and apoptosis induced by hydrogen peroxide (H2O2) in SW1353 human chondrocytes. For this purpose, SW1353 cells exposed to H2O2 in the presence or absence of EESS were applied to cell viability assay, comet assay, immunoblotting and flow cytometry analyses. Our results showed that EESS effectively attenuated H2O2-induced cytotoxicity and DNA damage associated with the inhibition of reactive oxygen species (ROS) accumulation. EESS also weakened the mitochondria membrane permeabilization by H2O2, and recovered H2O2-induced decreased expression of anti-apoptotic Bcl-2 and pro-caspase-3, and degradation of poly (ADP-ribose) polymerase. In addition, EESS increased not only expression, but also phosphorylation of nuclear factor-erythroid 2 related factor 2 (Nrf2), and promoted the expression of heme oxygenase-1 (HO-1), a critical target enzyme of Nrf2, but decreased the expression of kelch-like ECH-associated protein-1; however, the inhibition of HO-1 activity by zinc protoporphyrin abolished the antioxidant potential induced by EESS against H2O2-mediated oxidative stress. Therefore, the results of this study suggest that the antioxidant efficacy of EESS in chondrocytes is at least involved in the Nrf2/HO-1 signaling pathway-dependent mechanism.

OGG1-initiated base excision repair exacerbates oxidative stress-induced parthanatos

Oxidative stress-induced DNA damage has been well acknowledged as a major cause leading to cell death, which is etiologically linked to ischemic injury and degenerative alterations. The most common oxidation product of DNA is base lesion 8-oxo-7,8-dihydroguanine (8-oxoG), which is repaired by 8-oxoG glycosylase1 (OGG1)-initiated baseexcision repair (BER) pathway (OGG1-BER); however, the role of OGG1-BER in oxidative stress-induced cell death is poorly investigated. DNA strand breaks and apurinic/apyrimidinic (AP) sites are effective substrates to activate DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP1). Overactivation of PARP1 is associated with apoptosis-inducing factor (AIF)-mediated and caspase-independent cell death (parthanatos). We hypothesized that after an excessive oxidative insult, OGG1-BER-generated strand breaks result in hyperactivation of PARP1 and consequently cell death. To test, wild type, knockout, siRNA-depleted MEFs and neuroblastoma cells, or those expressing repair-deficient OGG1 mutants were oxidatively stressed and the role of OGG1 was examined. Results showed that OGG1-BER further increases the levels of ROS-induced DNA damage by generating repair intermediates, leading to PARP1 overactivation and cell death. Cells lacking or expressing repair-deficient OGG1 showed lower levels of DNA strand lesions, PARP1 activation, and nuclear translocation of apoptosis-inducing factor, resulting in the increased resistance to ROS-induced parthanatos. These results suggested that OGG1 guards genome integrity through either lesion repair or elimination of cells with malignant potential, to maintain the homeostasis of the host, which might depend on the magnitude of guanine oxidation.

XRCC4 rs6869366 polymorphism is associated with susceptibility to both nicotine dependence and/or schizophrenia

Abstract Background: Oxidative stress induced DNA damage has been assumed to contribute to the etiopathogenesis of schizophrenia (Sch). Smoking prevalence was more common in patients with Sch. The X-ray repair cross-complementation group 4 (XRCC4) gene plays an important role in the repair of DNA double-strand breaks. Objective: The purpose of this study was to investigate whether XRCC4 rs6869366 polymorphism has a relationship both in nicotine dependence (ND) and Sch+ND risk. Methods: One hundred and four patients with Sch+ND, 133 subjects with ND only and 70 healthy controls were enrolled in the study. XRCC4 rs6869366 polymorphism was analyzed using PCR-RFLP assay. Results: The frequency of XRCC4 rs6869366 GG genotype was more common in the ND and Sch+ND group than controls (p = 0.001 and p = 0.001, respectively). XRCC4 rs6869366 TT genotype was lower in both ND and Sch+ND group compared to controls (p = 0.001 and p = 0.001, respectively). Also, XRCC4 rs6869366 G allele was higher in Sch+ND group than controls (p = 0.001) while XRCC4 rs6869366 T allele was lower in ND group than healthy controls (p=0.001). XRCC4 rs6869366 GT genotype was lower in ND group than control group (p = 0.003). Discussion: These results suggested that the XRCC4 rs6869366 polymorphism G related genotype/allele was associated with susceptibility to both ND and Sch+ND in a Turkish population.

Reactive oxygen species impact on sperm DNA and its role in male infertility

Reactive oxygen species (ROS) have been extensively studied as a cause of male infertility. Excessive levels of ROS coupled with a deficiency in antioxidants can lead to oxidative stress (OS), which in turn can lead to nuclear and mitochondrial DNA damage, telomere shortening, epigenetic alterations and Y chromosomal microdeletions. In this review, we discuss how OS induces DNA damage and the types of DNA damage that can occur. We also briefly touch on the clinical consequences of OS-induced DNA damage.

Endonuclease G promotes mitochondrial genome cleavage and replication.

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.

8. Evaluation of Snow Fungus Polysaccharides on benzo[a]pyrene-induced DNA Damage

Snow fungus, Tremella fuciformis, has been demonstrated to have numerous health benefits including purported chemopreventive properties due to free radical-scavenging ability. Protective effects derived from snow fungus polysaccharides are evaluated on Chinese hamster lung fibroblasts (CCL-39) exposed to carcinogen benzo[a]pyrene known to cause free radical formation and oxidative stress to cells.
 In this experiment, it was hypothesized that the naturally occurring polysaccharides in snow fungus are able to protect against or reduce oxidative stress-induced DNA damage. Polysaccharides were isolated through an alkaline extraction and in-vitro digestion. DNA damage was measured using the single-cell gel electrophoresis comet assay after exposure to benzo[a]pyrene and polysaccharide extract to lung fibroblasts. Results were calculated using the mean and standard deviation data of tail length and area, respectively. Each damaged cell was measured and analyzed through ImageJ Editing Software. The results indicate a promising trend which depict snow fungus polysaccharides yielding lower levels of DNA damage compared to cells exposed to benzo[a]pyrene and compared to the negative control (phosphate buffered saline and Dulbecco’s cell medium). This study suggests polysaccharides from Tremella fuciformis could truly prevent cellular DNA damage by protecting against oxidative stress.

Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli.

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Premalignant and malignant changes of skin in a patient with oculocutaneous albinism: multiple actinic keratosis and squamous cell carcinoma

<p class="abstract"><span lang="EN-IN">Pigmentation of skin is a feature which is governed by multiple factors including the number of melanocytes, their metabolic activity of the melanocytes, the melanogenic activity of the melanosomes and lastly the morphology and differentiation of the melanosomes “Squamous cell carcinoma” (SCC) of sun-exposed skin is the most frequently observed malignancy among Albinos. It is ultimately due to lack of Eumelanin which guards against both the sunlight as well as oxidative stress-induced DNA damage. A 41 year old albino male patient presented with multiple asymptomatic raised skin lesions of different morphology and dimensions over scalp, neck, behind right ear and back with duration of 7 years. The initial lesion developed as a small papule over scalp which gradually progressed to present size and later appeared before the right ear, neck and lastly over the back. On examination, the initial lesion over scalp was verrucous plaque with adherent crusting, while other lesions were indurated, non-tender ulcers with irregular margins and punched out edges. There are few whitish raised scaly papules and plaques over the back. Routine investigations were within normal limits. Histopathology findings from the neck revealed squamous epithelium with tumour cells infiltrating the underlying stroma. Tumour cells showed pleomorphism, increased N:C ratio, hyperchromatic nuclei, prominent nucleoli and keratin pearls. Histopathology findings from the scaly lesion over the back revealed mild hyperkeratosis with dysplasia of the basal keratinocytes and prominent solar elastosis in the superficial dermis. Based on clinical and histopathogical findings, diagnosis of multiple SCC with actinic keratosis was made. Patient was referred to oncosurgeon for further management. Early detection and prompt treatment of the disease is required to reduce the spread to other parts of the body along with photo-protection all throughout life</span><span lang="EN-IN">. </span></p>

Effects of Psychological Stress from a National License Examination on the Urine 8-Hydroxy-Deoxyguanosine Levels in Young Female Students, Taking into Account the Menstrual Cycle

Objective: Urine 8-hydroxy-deoxyguanosine (U8-OHdG) was a marker of oxidative stress-induced DNA damage; it was increased by psychological stress. Since the menstrual cycle may confound or modify the association between psychological stress and U8-OHdG. The aim of this study was to verify the effect of psychological stress from a national license examination on levels of U8-OHdG, which is a biomarker of oxidative stress. And the effects of women’s menstrual cycles, which should be considered in mental and physical assessments, on U8-OHdG, were evaluated. Methods: The subjects were 18 female university students at a medical university in whom Self-rating Depression Scale (SDS) scores, State-Trait Anxiety Inventory (STAI) scores, and U8-OHdG levels were measured in 3 phases of the menstrual cycle, the follicular phase, ovulatory phase, and luteal phase. The mean values were taken for the group during a time of classroom learning. The same measurements were also made one week before and the day after a national license examination and the measurements were compared among the three periods. Results and Conclusion: State anxiety and U8-OHdG levels were significantly higher in those with a week before the national license examination than in those with classroom lecture (State anxiety: p = 0.002, U8-OHdG levels: p = 0.007). The menstrual cycle did not show a significant correlation with U8-OHdG levels. This study demonstrated that levels of the oxidative stress biomarker U8-OHdG are not affected by changes in the menstrual cycle. It also showed that U8-OHdG levels increased with the psychological stress of a national license examination.

Epigenetic alterations of a novel antioxidant gene SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) occurs with the highest frequency in China, especially in the high-risk Northern Chinese. Recent studies have reported that SLC22A3 is significantly downregulated in non-tumor (NT) esophageal tissues from familial ESCC patients compared with those from sporadic ESCC. However, the mechanism of how SLC22A3 regulates familial ESCC remains unknown. In this study, post hoc genome-wide association studies (GWAS) in 496 cases with a family history of upper gastrointestinal tract cancers and 1056 controls were performed and the results revealed that SLC22A3 is a novel susceptibility gene for familial ESCC. Reduced expression of SLC22A3 in NT esophageal tissues from familial ESCC patients significantly correlates with its promoter hypermethylation. Moreover, case-control study of Chinese descendants from different risk areas of China revealed that the methylation of the SLC22A3 gene in peripheral blood leukocyte (PBL) DNA samples could be a risk factor for developing ESCC in this high-risk population. Functional studies showed that SLC22A3 is a novel antioxidant gene, and deregulation of SLC22A3 facilitates heat stress-induced oxidative DNA damage and formation of γ-H2AX foci in normal esophageal epithelial cells. Collectively, we show that epigenetic alterations of SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

Fish oil omega-3 polyunsaturated fatty acids attenuate oxidative stress-induced DNA damage in vascular endothelial cells.

ObjectiveOmega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), likely prevent cardiovascular disease, however their mechanisms remain unclear. Recently, the role of DNA damage in atherogenesis has been receiving considerable attention. Here, we investigated the effects of EPA and DHA on DNA damage in vascular endothelial cells to clarify their antiatherogenic mechanisms.Methods and resultsWe determined the effect of EPA and DHA on H2O2-induced DNA damage response in human aortic endothelial cells. Immunofluorescence staining showed that γ-H2AX foci formation, a prominent marker of DNA damage, was significantly reduced in the cells treated with EPA and DHA (by 47% and 48%, respectively). H2O2-induced activation of ATM, a major kinase orchestrating DNA damage response, was significantly reduced with EPA and DHA treatment (by 31% and 33%, respectively). These results indicated EPA and DHA attenuated DNA damage independently of the DNA damage response. Thus the effects of EPA and DHA on a source of DNA damage were examined. EPA and DHA significantly reduced intracellular reactive oxygen species under both basal condition and H2O2 stimulation. In addition, the mRNA levels of antioxidant molecules, such as heme oxygenase-1, thioredoxin reductase 1, ferritin light chain, ferritin heavy chain and manganese superoxide dismutase, were significantly increased with EPA and DHA. Silencing nuclear factor erythroid 2-related factor 2 (NRF2) remarkably abrogated the increases in mRNA levels of antioxidant molecules and the decrease in intracellular reactive oxygen species. Furthermore, EPA and DHA significantly reduced H2O2-induced senescence-associated β-galactosidase activity in the cells (by 31% and 22%, respectively), which was revoked by NRF2 silencing.ConclusionsOur results suggested that EPA and DHA attenuate oxidative stress-induced DNA damage in vascular endothelial cells through upregulation of NRF2-mediated antioxidant response. Therefore omega-3 fatty acids likely help prevent cardiovascular disease, at least in part, by their genome protective properties.

Oxidative stress and dietary phytochemicals: Role in cancer chemoprevention and treatment

Several epidemiological observations have shown an inverse relation between consumption of plant-based foods, rich in phytochemicals, and incidence of cancer. Phytochemicals, secondary plant metabolites, via their antioxidant property play a key role in cancer chemoprevention by suppressing oxidative stress-induced DNA damage. In addition, they modulate several oxidative stress-mediated signaling pathways through their anti-oxidant effects, and ultimately protect cells from undergoing molecular changes that trigger carcinogenesis. In several instances, however, the pro-oxidant property of these phytochemicals has been observed with respect to cancer treatment. Further, in vitro and in vivo studies show that several phytochemicals potentiate the efficacy of chemotherapeutic agents by exacerbating oxidative stress in cancer cells. Therefore, we reviewed multiple studies investigating the role of dietary phytochemicals such as, curcumin (turmeric), epigallocatechin gallate (EGCG; green tea), resveratrol (grapes), phenethyl isothiocyanate (PEITC), sulforaphane (cruciferous vegetables), hesperidin, quercetin and 2′-hydroxyflavanone (2HF; citrus fruits) in regulating oxidative stress and associated signaling pathways in the context of cancer chemoprevention and treatment.

A novel mtDNA repair fusion protein attenuates maladaptive remodeling and preserves cardiac function in heart failure.

Oxidative stress results in mtDNA damage and contributes to myocardial cell death. mtDNA repair enzymes are crucial for mtDNA repair and cell survival. We investigated a novel, mitochondria-targeted fusion protein (Exscien1-III) containing endonuclease III in myocardial ischemia-reperfusion injury and transverse aortic constriction (TAC)-induced heart failure. Male C57/BL6J mice (10-12 wk) were subjected to 45 min of myocardial ischemia and either 24 h or 4 wk of reperfusion. Exscien1-III (4 mg/kg ip) or vehicle was administered at the time of reperfusion. Male C57/BL6J mice were subjected to TAC, and Exscien1-III (4 mg/kg i.p) or vehicle was administered daily starting at 3 wk post-TAC and continued for 12 wk. Echocardiography was performed to assess left ventricular (LV) structure and function. Exscien1-III reduced myocardial infarct size ( P < 0.01) at 24 h of reperfusion and preserved LV ejection fraction at 4 wk postmyocardial ischemia. Exscien1-III attenuated TAC-induced LV dilation and dysfunction at 6-12 wk post-TAC ( P < 0.05). Exscien1-III reduced ( P < 0.05) cardiac hypertrophy and maladaptive remodeling after TAC. Assessment of cardiac mitochondria showed that Exscien1-III localized to mitochondria and increased mitochondrial antioxidant and reduced apoptotic markers. In conclusion, our results indicate that administration of Exscien1-III provides significant protection against myocardial ischemia and preserves myocardial structure and LV performance in the setting of heart failure. NEW & NOTEWORTHY Oxidative stress-induced mitochondrial DNA damage is a prominent feature in the pathogenesis of cardiovascular diseases. In the present study, we demonstrate the efficacy of a novel, mitochondria-targeted fusion protein that traffics endonuclease III specifically for mitochondrial DNA repair in two well-characterized murine models of cardiac injury and failure.

Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress

Melatonin has antioxidant and scavenger effects in the cellular antioxidant system. This research investigated the protective effects and underlying mechanisms of melatonin action in porcine somatic cell nuclear transfer (SCNT) embryos. The results suggested that the developmental competence of porcine SCNT embryos was considerably enhanced after melatonin treatment. In addition, melatonin attenuated the increase in reactive oxygen species levels induced by oxidative stress, the decrease in glutathione levels, and the mitochondrial dysfunction. Importantly, melatonin inhibited phospho-histone H2A.X (γH2A.X) expression and comet tail formation, suggesting that γH2A.X prevents oxidative stress-induced DNA damage. The expression of genes involved in homologous recombination and non-homologous end-joining pathways for the repair of double-stranded breaks (DSB) was reduced upon melatonin treatment in porcine SCNT embryos at day 5 of development under oxidative stress condition. These results indicated that melatonin promoted porcine SCNT embryo development by preventing oxidative stress-induced DNA damage via quenching of free radical formation. Our results revealed a previously unrecognized regulatory effect of melatonin in response to oxidative stress and DNA damage. This evidence provides a novel mechanism for the improvement in SCNT embryo development associated with exposure to melatonin.

CYT-Rx20 Inhibits Cervical Cancer Cell Growth and Migration Through Oxidative Stress-Induced DNA Damage, Cell Apoptosis, and Epithelial-to-Mesenchymal Transition Inhibition.

The β-nitrostyrene family has been reported to possess anticancer properties. However, the anticancer activity of β-nitrostyrenes on cervical cancer cells and the underlying mechanisms involved remain unexplored. In this study, a β-nitrostyrene derivative CYT-Rx20 (3'-hydroxy-4'-methoxy-β-methyl-β-nitrostyrene) was synthesized, and its anticancer activity on cervical cancer cells and the mechanisms involved were investigated. The effect of CYT-Rx20 on human cervical cancer cell growth was evaluated using cell viability assay. Reactive oxygen species (ROS) generation and annexin V staining were detected by flow cytometry. The protein expression levels of cleaved caspase-3, cleaved caspase-9, cleaved poly (ADPribose) polymerase, γH2AX, β-catenin, Vimentin, and Twist were measured by Western blotting. DNA double-strand breaks were determined by γ-H2AX foci formation and neutral comet assay. Migration assay was used to determine cancer cell migration. Nude mice xenograft was used to investigate the antitumor effects of CYT-Rx20 in vivo. CYT-Rx20 induced cytotoxicity in cervical cancer cells by promoting cell apoptosis via ROS generation and DNA damage. CYT-Rx20-induced cell apoptosis, ROS generation, and DNA damage were reversed by thiol antioxidants. In addition, CYT-Rx20 inhibited cervical cancer cell migration by regulating the expression of epithelial-to-mesenchymal transition markers. In nude mice, CYT-Rx20 inhibited cervical tumor growth accompanied by increased expression of DNA damage marker γH2AX and decreased expression of mesenchymal markers β-catenin and Twist. CYT-Rx20 inhibits cervical cancer cells in vitro and in vivo and has the potential to be further developed into an anti-cervical cancer drug clinically.

Amifostine does not protect thyroid cancer cells in DNA damaging in vitro models.

BackgroundAmifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells.MethodsWe investigated the effects of the active form of amifostine (WR-1065) on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643) and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM), by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL) and by γ-radiation (6 Gy). DNA damage, cell viability and apoptosis were examined.ResultsWe demonstrated the selective action of WR-1065 (0.1 mM), which prevented oxidative stress–induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h) was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis.ConclusionsOur data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.

Abstract 436: Folate cycle represents a new metabolic vulnerability for hepatocellular carcinoma treatment

Abstract Hepatocellular carcinoma (HCC), primary liver cancer, ranks the third most lethal cancer worldwide due to late symptom presentation and lack of promising curative therapy. Metabolic reprogramming has been recognized as a major and new hallmark of cancer in recent years. Better understanding of its underpinning molecular mechanisms favoring cancer growth will be crucial for the development of effective HCC therapeutics. The folate cycle fuels metabolic processes and the production of metabolites essential to cell growth and tumorigenesis maintenance. Through the shuttling of a single carbon unit by a folate derivative through the tetrahydrofolate (THF) backbone in the cytoplasmic and mitochondrial compartments, metabolites like NADPH - a major cellular antioxidant for redox balance, s-adenosyl methionine (SAM) - precursor of DNA and histone methylation, and pyrimidine and purine - the building blocks of DNA are produced. We found folate to be indispensable for HCC cell growth. Furthermore, methylene-THF dehydrogenase 1-like (MTHFD1L), a key enzyme facilitating the folate cycle from the mitochondria, was found to be significantly up-regulated in HCC with association to poorer clinical features for patients. Genetic inhibition of MTHFD1L by knockdown and knockout by shRNA and CRISPR-Cas9 systems, respectively, blocked NADPH production. Rapid elevation in oxidative stress induced DNA damage and cell cycle delay; ultimately inhibiting HCC proliferation. Binding of the transcription factor NRF2, a potent protector of oxidative stress, and MTHFD1L was confirmed by ChIP assay. NRF2 over-expression using the CRISPR-activating system in HCC cells further highlighted the dependent relationship between NRF2 and MTHFD1L. Metabolomics analysis showed that MTHFD1L knockdown caused a disruption to the folate cycle and accumulation of serine. Surprisingly, MTHFD1L knockdown did not reduce the levels of SAM and nucleotides. Knockdown of MTHFD1L in HCC cells significantly inhibited primary liver tumor growth and lung metastasis in orthotopic liver implantation model. Therapeutically, the administration of methotrexate, an anti-folate agent, sensitized HCC cells towards Sorafenib treatment both in vitro and vivo. The folate cycle represents a metabolic vulnerability and attractive therapeutic target for HCC. Inhibition of MTHFD1L disrupts the folate cycle and sensitizes HCC cells towards its convention treatment agent, Sorafenib in various HCC mouse models. Our investigation unravels a metabolic vulnerability in cancer which contributes to better understanding and is beneficial for the development of precise inhibitors specifically targeting associated pathways. Citation Format: Derek Lee, Iris Ming-Jing Xu, David Kung-Chun Chiu, Robin Kit-Ho Lai, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Folate cycle represents a new metabolic vulnerability for hepatocellular carcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 436. doi:10.1158/1538-7445.AM2017-436