Carcinogen-induced DNA Damage Research Articles

Inhibition of Benzo[a]pyrene-induced DNA Adduct in Buccal Cells of Smokers by Black Raspberry Lozenges.

Using LC-MS/MS analysis we previously showed for the first time (Carcinogenesis 43:746-753, 2022) that levels of DNA damage-induced by benzo[a]pyrene (B[a]P), an oral carcinogen and tobacco smoke (TS) constituent, were significantly higher in buccal cells of smokers than those in non-smokers; these results suggest the potential contribution of B[a]P in the development of oral squamous cell carcinoma (OSCC) in humans. Treating cancers, including OSCC at late stages even with improved targeted therapies, continues to be a major challenge. Thus interception/prevention remains a preferable approach for OSCC management and control. In previous preclinical studies we and others demonstrated the protective effects of black raspberry (BRB) against carcinogen-induced DNA damage and OSCC. Thus, to translate preclinical findings we tested the hypothesis, in a Phase 0 clinical study, that BRB administration reduces DNA damage induced by B[a]P in buccal cells of smokers. After enrolling 27 smokers, baseline buccal cells were collected before the administration of BRB lozenges (5/day for 8 weeks, 1 gm BRB powder/lozenge) at baseline, at the middle and the end of BRB administration. The last samples were collected at four weeks after BRB cessation (washout period). B[a]P-induced DNA damage (BPDE-N2-dG) was evaluated by LC-MS/MS. BRB administration resulted in a significant reduction in DNA damage: 26.3% at the midpoint (p = 0.01506) compared to baseline, 36.1% at the end of BRB administration (p = 0.00355), and 16.6% after BRB cessation (p = 0.007586). Our results suggest the potential benefits of BRB as a chemopreventive agent against the development of TS-initiated OSCC.

Benzo[a]pyrene-induced up-regulation of circ_0003552 via ALKBH5-mediated m6A modification promotes DNA damage in human bronchial epithelial cells

Benzo [a]pyrene (B [a]P) is a widespread environmental chemical pollutant that has been linked to the development of various diseases. However, the specific mechanism of action remains unclear. In this study, human bronchial epithelial 16HBE and BEAS-2B cells were exposed to B [a]P at 0–32 μM to assess the DNA-damaging effects. B [a]P exposure resulted in elevated expression of γ-H2AX, a marker of DNA damage. The m6A RNA methylation assay showed that B [a]P exposure increased the extent of m6A modification and the demethylase ALKBH5 played an integral role in this process. Moreover, the results of the comet assay and Western blot analysis showed an increase in m6A modification mediated by ALKBH5 that promoted DNA damage. Furthermore, the participation of a novel circular RNA, circ_0003552, was assessed by high-throughput sequencing under the condition of high m6A modification induced by B [a]P exposure. In subsequent functional studies, an interference/overexpression system was created to confirm that circ_0003552 participated in regulation of DNA damage. Mechanistically, circ_0003552 had an m6A binding site that could regulate its generation. This study is the first to report that B [a]P upregulated circ_0003552 through m6A modification, thereby promoting DNA damage. These findings revealed that epigenetics played a key role in environmental carcinogen-induced DNA damage, and the quantitative changes it brought might provide an early biomarker for future medical studies of genetic-related diseases and a new platform for investigations of the interaction between epigenetics and genetics.

Genistein and Procyanidin B2 Reduce Carcinogen-Induced Reactive Oxygen Species and DNA Damage through the Activation of Nrf2/ARE Cell Signaling in Bronchial Epithelial Cells In Vitro.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiation therapy are currently providing the basis for cancer therapies, although both are associated with significant side effects. Thus, cancer prevention through dietary modifications has been receiving growing interest. The potential of selected flavonoids in reducing carcinogen-induced reactive oxygen species (ROS) and DNA damage through the activation of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2)/antioxidant response element (ARE) pathway was studied in vitro. Dose-dependent effects of pre-incubated flavonoids on pro-carcinogen 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKAc)-induced ROS and DNA damage in human bronchial epithelial cells were studied in comparison to non-flavonoids. The most effective flavonoids were assessed for the activation of Nrf2/ARE pathway. Genistein, procyanidin B2 (PCB2), and quercetin significantly suppressed the NNKAc-induced ROS and DNA damage. Quercetin significantly upregulated the phosphorylated protein kinase B/Akt. PCB2 significantly upregulated the activation of Nrf2 and Akt through phosphorylation. Genistein and PCB2 significantly upregulated the phospho-Nrf2 nuclear translocation and catalase activity. In summary, genistein and PCB2 reduced the NNKAc-induced ROS and DNA damage through the activation of Nrf2. Further studies are required to understand the role of dietary flavonoids on the regulation of the Nrf2/ARE pathway in relation to carcinogenesis.

Bcl-2 Interacts with Beclin 1 and Regulates Autophagy in 7, 12-Dimethylbenz[a]anthracene-Induced Hamster Buccal-Pouch Squamous-Cell Tumorigenesis.

Autophagy is a programmed cell death procedure, which has essential functions in tumorigenesis. However, its temporal expression and function under different status are yet to be determined. This study aims to investigate the temporal expression of autophagy and its possible function in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch cancer model (HBPCM). A total of 50 hamster buccal-pouch tumorigenesis models were established by painting DMBA for 4, 8, 10 and 13 weeks. The expression and subcellular localization of LC3, Beclin 1 and Bcl-2 in buccal lesions were evaluated by immunohistochemical staining and Western blotting. DNA damage was observed by immunohistochemical staining of 8-oHdG. The relationship between Beclin 1 and Bcl-2 was analyzed by immunofluorescence colocalization. The expression levels of LC3 and Beclin 1 associated with autophagy in the experimental buccal pouch of HBPCM were significantly upregulated after 4 weeks (P<0.05), but gradually downregulated after 13 weeks of HBPCM induction. By contrast, the expression level of Bcl-2 was significantly upregulated after 13 weeks. The co-localized regions of Bcl-2 and Beclin 1 peaked after 4 weeks and then decreased gradually. The DNA damage in epithelial cells increased slightly after 4 weeks, and then rapidly decreased over the next 2 months. Autophagy is motivated by a tumor suppressor that diminishes carcinogen-induced DNA damage. However, autophagy is gradually suppressed, which may be attributed to the interaction between Bcl-2 and Beclin 1. This result indicates that the promotion of autophagy may suppress malignant transformation and provide new insights on future potential treatments of HBPCM.

Tumour necrosis factor-α (TNF-α) enhances dietary carcinogen-induced DNA damage in colorectal cancer epithelial cells through activation of JNK signaling pathway

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazol [4,5-b] pyridine (PhIP) present in cooked meat are pro-carcinogens and considered to be potential risk factors for CRC. Their carcinogenic and mutagenic effects require metabolic activation primarily by cytochrome P450 1 family enzymes (CYPs); the expression of these enzymes can be modulated by aryl hydrocarbon receptor (AhR) activation and the tumour microenvironment, involving mediators of inflammation. In this study, we hypothesized that tumour necrosis factor-α (TNF-α), a key mediator of inflammation, modulates BaP- and PhIP-induced DNA damage in colon cancer epithelial cells. Importantly, we observed that TNF-α alone (0.1–100 pg/ml) induced DNA damage (micronuclei formation) in HCT-116 cells and co-treatment of TNF-α with BaP or PhIP showed higher levels of DNA damage compared to the individual single treatments. TNF-α alone or in combination with BaP or PhIP did not affect the expression levels of CYP1A1 and CYP1B1 (target genes of AhR signaling pathways). The DNA damage induced by TNF-α was elevated in p53 null HTC-116 cells compared to wild type cells, suggesting that TNF-α-induced DNA damage is suppressed by functional p53. In contrast, p53 status failed to affect BaP and PhIP induced micronucleus frequency. Furthermore, JNK and NF-κB signaling pathway were activated by TNF-α treatment but only inhibition of JNK significantly reduced TNF-α-induced DNA damage. Collectively, these findings suggest that TNF-α induced DNA damage involves JNK signaling pathway rather than AhR and NF-κB pathways in colon cancer epithelial cells.

Cyanidin-3-O-Glucoside-Rich Haskap Berry Administration Suppresses Carcinogen-Induced Lung Tumorigenesis in A/JCr Mice.

In our previous study, we demonstrated that cyanidin-3-O-glucoside (C3G)-rich haskap (Lonicera caerulea L.) berry extracts can attenuate the carcinogen-induced DNA damage in normal lung epithelial cells in vitro. Here, the efficacy of lyophilized powder of whole haskap berry (C3G-HB) in lowering tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, (NNK)-induced lung tumorigenesis in A/JCr mice was investigated. Three weeks after daily oral administration of C3G-HB (6 mg of C3G in 0.2 g of C3G-HB/mouse/day), lung tumors were initiated by a single intraperitoneal injection of NNK. Dietary C3G-HB supplementation was continued, and 22 weeks later, mice were euthanized. Lung tumors were visualized through positron emission tomography (PET) and magnetic resonance imaging (MRI) 19 weeks after NNK injection. Dietary supplementation of C3G-HB significantly reduced the NNK-induced lung tumor multiplicity and tumor area but did not affect tumor incidence. Immunohistochemical analysis showed reduced expression of proliferative cell nuclear antigen (PCNA) and Ki-67 in lung tissues. Therefore, C3G-HB has the potential to reduce the lung tumorigenesis, and to be used as a source for developing dietary supplements or nutraceuticals for reducing the risk of lung cancer among high-risk populations.

The Molecular Chaperone Heat Shock Protein 70 Controls Liver Cancer Initiation and Progression by Regulating Adaptive DNA Damage and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways.

Delineating the mechanisms that drive hepatic injury and hepatocellular carcinoma (HCC) progression is critical for development of novel treatments for recurrent and advanced HCC but also for the development of diagnostic and preventive strategies. Heat shock protein 70 (HSP70) acts in concert with several cochaperones and nucleotide exchange factors and plays an essential role in protein quality control that increases survival by protecting cells against environmental stressors. Specifically, the HSP70-mediated response has been implicated in the pathogenesis of cancer, but the specific mechanisms by which HSP70 may support malignant cell transformation remains to be fully elucidated. Here, we show that genetic ablation of HSP70 markedly impairs HCC initiation and progression by distinct but overlapping pathways. This includes the potentiation of the carcinogen-induced DNA damage response, at the tumor initiation stage, to increase the p53-dependent surveillance response leading to the cell cycle exit or death of genomically damaged differentiated pericentral hepatocytes, and this may also prevent their conversion into more proliferating HCC progenitor cells. Subsequently, activation of a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) negative feedback pathway diminishes oncogenic signals, thereby attenuating premalignant cell transformation and tumor progression. Modulation of HSP70 function may be a strategy for interfering with oncogenic signals driving liver cell transformation and tumor progression, thus providing an opportunity for human cancer control.

Microbial metabolites of proanthocyanidins reduce chemical carcinogen-induced DNA damage in human lung epithelial and fetal hepatic cells in vitro

Seven selected microbial metabolites of proanthocyanidins (MMP), 3-phenylpropionic, 4-hydroxyphenyl acetic, 3-(4-hydroxyphenyl) propionic, p-coumaric, benzoic acid, pyrogallol (PG), and pyrocatechol (PC) were evaluated for their ability to reduce chemical carcinogen-induced toxicity in human lung epithelial cells (BEAS-2B) and human fetal hepatic cells (WRL-68). Cells pre-treated with MMP were exposed to a known chemical carcinogen, 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to assess MMP-mediated cytoprotection and reduction of DNA damage. PG in BEAS-2B and PC in WRL-68 cells mitigated the NNKOAc-induced cytotoxicity. Pre-incubation of PG depicted significant protection against NNKOAc-induced DNA damage in BEAS-2B cells. PC in WRL-68 cells showed similar activity. To understand the mechanisms of PG- and PC-mediated DNA damage reduction, the effect on DNA damage response (DDR) proteins, cellular reactive oxygen species (ROS), total antioxidant capacity (TAC), glutathione peroxidase (GPx), and caspase activity were studied. PG and PC alter the DDR and may promote ATR-Chk1 and ATM-Chk2 pathways, respectively. Cellular oxidative stress induced by NNKOAc was mitigated by PG and PC through enhanced GPx expression and TAC. PG and PC suppressed the activation of the extrinsic apoptotic pathway (caspase 3 and 8) provoked by NNKOAc. MMP are beneficial in chemoprevention by reducing cellular DNA damage.

Association between NER Pathway Gene Polymorphisms and Wilms Tumor Risk

Nucleotide excision repair (NER) is an essential mechanism of the body to defend against exogenous carcinogen-induced DNA damage. Defects in NER may impair DNA repair capacity and, therefore, increase genome instability and cancer susceptibility. To explore genetic predispositions to Wilms tumor, we conducted a case-control study totaling 145 neuroblastoma cases and 531 healthy controls. We systematically selected 19 potentially functional SNPs in six key genes within the NER pathway (ERCC1, XPA, XPC, XPD, XPF, and XPG). The odds ratio (OR) and 95% confidence interval (CI) were calculated to measure the strength of associations. We identified significant associations between two XPD SNPs and Wilms tumor risk. The XPD rs3810366 polymorphism significantly enhanced Wilms tumor risk (dominant model: adjusted OR = 2.12, 95% CI = 1.26–3.57). Likewise, XPD rs238406 conferred a significantly increased risk for the disease (dominant model: adjusted OR = 2.30, 95% CI = 1.40–3.80; recessive model: adjusted OR = 1.64, 95% CI = 1.11–2.44). Moreover, online expression quantitative trait locus (eQTL) analysis demonstrated that these two polymorphisms significantly affected XPD gene expression in transformed fibroblast cells. Our study provides evidence of the association between the two XPD polymorphisms and Wilms tumor risk. However, these findings warrant validation in larger studies.

Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Normal Human Bronchial Epithelial Cells.

Scope Human neoplastic transformation due to DNA damage poses an increasing global healthcare concern. Maintaining genomic integrity is crucial for avoiding tumor initiation and progression. The present study aimed to investigate the efficacy of an apple flavonoid fraction (AF4) against various carcinogen-induced toxicity in normal human bronchial epithelial cells and its mechanism of DNA damage response and repair processes. Methods and Results AF4-pretreated cells were exposed to nicotine-derived nitrosamine ketones (NNK), NNK acetate (NNK-Ae), methotrexate (MTX), and cisplatin to validate cytotoxicity, total reactive oxygen species, intracellular antioxidants, DNA fragmentation, and DNA tail damage. Furthermore, phosphorylated histone (γ-H2AX) and proteins involved in DNA damage (ATM/ATR, Chk1, Chk2, and p53) and repair (DNA-PKcs and Ku80) mechanisms were evaluated by immunofluorescence and western blotting, respectively. The results revealed that AF4-pretreated cells showed lower cytotoxicity, total ROS generation, and DNA fragmentation along with consequent inhibition of DNA tail moment. An increased level of γ-H2AX and DNA damage proteins was observed in carcinogen-treated cells and that was significantly (p ≤ 0.05) inhibited in AF4-pretreated cells, in an ATR-dependent manner. AF4 pretreatment also facilitated the phosphorylation of DNA-PKcs and thus initiation of repair mechanisms. Conclusion Apple flavonoids can protect in vitro oxidative DNA damage and facilitate repair mechanisms.

Acidic cellular microenvironment modifies carcinogen-induced DNA damage and repair

Chronic inflammation creates an acidic microenvironment, which plays an important role in cancer development. To investigate how low pH changes the cellular response to the carcinogen benzo[a]pyrene (B[a]P), we incubated human pulmonary epithelial cells (A549 and BEAS-2B) with nontoxic doses of B[a]P using culturing media of various pH’s (extracellular pH (pHe) of 7.8, 7.0, 6.5, 6.0 and 5.5) for 6, 24 and 48 h. In most incubations (pHe 7.0–6.5), the pH in the medium returned to the physiological pH 7.8 after 48 h, but at the lowest pH (pHe < 6.0), this recovery was incomplete. Similar changes were observed for the intracellular pH (pHi). We observed that acidic conditions delayed B[a]P metabolism and at t = 48 h, and the concentration of unmetabolized extracellular B[a]P and B[a]P-7,8-diol was significantly higher in acidic samples than under normal physiological conditions (pHe 7.8) for both cell lines. Cytochrome P450 (CYP1A1/CYP1B1) expression and its activity (ethoxyresorufin-O-deethylase activity) were repressed at low pHe after 6 and 24 h, but were significantly higher at t = 48 h. In addition, a DNA repair assay showed that the incision activity was ~80% inhibited for 6 h at low pHe and concomitant exposure to B[a]P. However, at t = 48 h, the incision activity recovered to more than 100% of the initial activity observed at neutral pHe. After 48 h, higher B[a]P-DNA adduct levels and γ-H2AX foci were observed at low pH samples than at pHe 7.8. In conclusion, acidic pH delayed the metabolism of B[a]P and inhibited DNA repair, ultimately leading to increased B[a]P-induced DNA damage.

Xeroderma pigmentosum, complementation group D expression in H1299 lung cancer cells following benzo[a]pyrene exposure as well as in head and neck cancer patients

ABSTRACTDNA repair genes play critical roles in response to carcinogen-induced and anticancer therapy-induced DNA damage. Benzo[a]pyrene (BaP), the most carcinogenic polycyclic aromatic hydrocarbon (PAH), is classified as a group 1 carcinogen by International Agency for Research on Cancer. The aims of this study were to (1) evaluate the effects of BaP on DNA repair activity and expression of DNA repair genes in vitro and (2) examine the role of xeroderma pigmentosum, complementation group D (XPD) mRNA expression in human head and neck cancers. Host cell reactivation assay showed that BaP inhibited nucleotide excision repair in H1299 lung cancer cells. DNA repair through the non-homologous end-joining pathway was not affected by BaP. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) and Western blot demonstrated that XPD was downregulated by BaP treatment. BaP exposure did not apparently affect expression of another 11 DNA repair genes. BaP treatment increased the DNA damage marker γ-H2AX and ultraviolet (UV) sensitivity, supporting an impairment of DNA repair in BaP-treated cells. XPD expression was also examined by quantitative RT-PCR in 68 head and neck cancers, and a lower XPD mRNA level was found in smokers’ cancer specimens. Importantly, reduced XPD expression was correlated with patient 5-year overall survival rate (35 vs. 56%) and was an independent prognostic factor (hazard ratio: 2.27). Data demonstrated that XPD downregulation was correlated with BaP exposure and human head and neck cancer survival.

Interleukin-6 promotes dietary carcinogen-induced DNA damage in colorectal cancer cells

Pro-inflammatory cytokine interleukin-6 promotes dietary carcinogen-mediated DNA damage in 2D and 3D cultured cells by inducingCYP1B1expression through miR27b downregulation.

Mangiferin activates the Nrf2-ARE pathway and reduces etoposide-induced DNA damage in human umbilical cord mononuclear blood cells

Context: Mangiferin (2-C-β-d-gluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) is a well-known natural antioxidant distributed in various plants of the Anacardiaceae and Gentianaceae families. Mangiferin can inhibit carcinogen-induced lung or colon tumor formation in experimental animals. However, the molecular mechanisms of its chemopreventive activity remain unexplored.Objective: This study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-ARE signaling in hematopoietic cells.Materials and methods: Mononuclear cells (MNCs) were isolated from human umbilical cord blood (hUCB). DNA damage was evaluated by comet and micronucleus assays. The expression of Nrf2 and NQO1 was examined by immunofluorescence and western blotting. An electrophoretic mobility shift assay (EMSA) was used to detect the binding activity of Nrf2 with NQO1-ARE sequences.Results: We found that mangiferin treatment significantly reduced DNA damage in etoposide-treated MNCs, which was verified by decreased olive tail moment (OTM) and micronucleus (MN) frequency. Mangiferin treatment significantly promoted Nrf2 translocation into the nucleus and increased nuclear Nrf2 expression. Moreover, NQO1, an Nrf2 signaling target, was significantly upregulated by mangiferin treatment, and the binding activity of Nrf2 with NQO1-ARE sequences was elevated after mangiferin treatment.Discussion and conclusion: Mangiferin activated Nrf2 signaling, upregulated NQO1 expression, and significantly reduced etoposide-induced DNA damage. Thus, mangiferin is a potential cytoprotective agent for hematopoietic cells.

Body mass index and lung cancer risk: results from the ICARE study, a large, population-based case–control study

The association between body mass index (BMI) and lung cancer is still disputed because of possible residual confounding by smoking and preclinical weight loss in case-control studies. We examined this association using data from the multicenter ICARE study in France, a large, population-based case-control study. A total of 2,625 incident lung cancer cases and 3,381 controls were included. Weight was collected at interview, 2 years before the interview, and at age 30. Lifetime smoking exposure was calculated using the comprehensive smoking index (CSI). Adjusted odds ratios (aORs) and 95 % confidence intervals were estimated by unconditional logistic regression and controlled for age, area, education, CSI, occupational exposure, previous chronic bronchitis, and parental history of lung cancer. We also examined the role of weight change. Analyses were stratified by smoking status and sex. When compared with that of men with normal BMI 2 years before the interview, lung cancer aORs (95 % CI) among men with BMIs of <18.5, 25-29.9, 30-32.4, and ≥32.5 kg/m(2) were 2.7 (95 % CI 1.2-6.2), 0.9 (95 % CI 0.7-1.1), 0.8 (95 % CI 0.6-1.1), and 0.8 (95 % CI 0.6-1.0), respectively (p(trend) = 0.02). Results were more pronounced among current smokers and were similar in men and women. Weight gain over time was associated with a significant decreased risk of lung cancer. We found an inverse dose-dependent association between lung cancer risk and BMI 2 years prior to interview in current smokers. IMPACT STATEMENT: BMI might be an individual factor impacting the risk of lung cancer related to smoking's carcinogen-induced DNA damage.

Abstract 5446: Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation, DNA damage, and chronically-induced breast cell carcinogenesis

Abstract Long-term exposure to low doses of environmental carcinogens contributes to sporadic human breast cancers. Epidemiologic and experimental studies indicate that green tea catechins (GTCs) may interfere with breast cancer development. We have been developing a chronically-induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous, human breast epithelial MCF10A cells to physiologically-achievable pico-molar concentrations of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), to progressively induce cellular acquisition of cancer-associated properties, as measurable endpoints. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppressing cellular carcinogenesis. Here, we report a two-step strategy that initially used short-term endpoints that were transiently-induced by a single exposure to NNK and B[a]P. These short-term endpoints were used as targets to detect the ability of individual GTC components to block carcinogenesis. We subsequently used long-term endpoints that were constantly-induced by cumulative exposures to carcinogens as targets to verify and compare the activity of individual GTC components in suppressing carcinogenesis. We detected that a single exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to ERK pathway activation and induction of cell proliferation and chromosomal DNA damage. GTC components, at non-cytotoxic levels, were able to suppress chronically-induced cellular carcinogenesis by blocking carcinogen-induced ROS elevation, ERK activation, cell proliferation, and DNA damage in each exposure cycle. Our model may help accelerate the identification of agents to prevent carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5446. doi:1538-7445.AM2012-5446

Fish oil suppresses angiogenesis, reduces cell proliferation and DNA damage in rat mammary carcinogenesis

Summary Background & aims The current study was designed to evaluate the effect of dietary fish oil and corn oil on carcinogen-induced DNA damage, cell proliferation and angiogenesis in female Sprague-Dawley rats. Methods Rats were treated with 7,12-dimethylbenz(α)anthracene (DMBA) (0.5 mg/0.2 ml corn oil/100 g body weight) by a tail vein injection. Rats were fed either fish oil or corn oil (0.5 ml/day/rat) by oral gavage. DNA Damage, proliferating cell nuclear antigen labeling index (PCNA-LI) and expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) of the mammary tissue were included in this study. Results Tissue levels of both 8-hydroxy-2′deoxyguanosine (8-OHdG), a marker of DNA damage and DNA repair enzyme (PARP-1) were significantly reduced ( P = 0.00000006) in the fish oil-treated group, and increased ( P = 0.00000006) in the corn oil-treated group when compared to carcinogen counterpart. Furthermore, PCNA-LI, VEGF expressions and the MVD values were lower in fish oil-treated group ( P = 0.000078; P = 0.000032; P = 0.000000005 respectively) and higher ( P = 0.000027; P = 0.00004; P = 0.0000000003 respectively) in corn oil-treated group when compared to carcinogen control. Conclusions Our results indicate that fish oil protects against carcinogen-induced DNA damage, cell proliferation and angiogenesis.

Mangiferin Reduced Etoposide-Induced DNA Damage in Mononuclear Human Umbilical Cord Blood Cells Via Activating Nrf2-Mediated Signaling

Abstract 4626 Introduction:Mangiferin is a natural antioxidant predominantly distributed in Mangifera indica L (Mango). Mangiferin also inhibits carcinogen-induced lung cancer or colon cancer (Rajendran et al, 2008; Yoshimi et al, 2001). However, the molecular mechanism of its chemopreventive activity remains incompletely understood. Moreover, whether mangiferin can prevent leukemia remains unexplored. Nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling is the promising target for cellular protection and chemoprevention. According to the lately suggested mechanisms, Nrf2 protein localized predominantly in the nucleus of various cells. The activation of Nrf2 is most likely a result of its stabilization, leading to its accumulation in the nucleus. Nrf2 controls the transcriptional activation of its downstream target genes including Phase II detoxifying enzymes such as NAD(P)Hquinone oxidoreductase 1 (NQO1). Up-regulation of these Phase II detoxifying enzymes contributes to inhibiting carcinogenesis. The present study aimed to investigate the effects of mangiferin on chemical carcinogen-induced DNA damage and Nrf2-mediated signaling in hematopoietic cells. Methods:Mononuclear human umbilical cord blood (MNC hUCB) cells were incubated with or without mangiferin prior to etoposide, a well-known chemical carcinogen for therapy-related leukemia. DNA damage in MNC hUCB cells was evaluated by comet assay and micronucleus assay. In order to explore whether mangiferin activated Nrf2-mediated signaling, MNC hUCB cells were treated with mangiferin and then the nucleus accumulation of Nrf2 was examined by confocal microscopy and western blotting. The regulation of mangiferin on the expression of NQO1 was investigated by western blotting. Results:i) Mangiferin reduced etoposide-induced DNA damage in MNC hUCB cells. Etoposide induced DNA damage in MNC hUCB cells in a dose-dependent manner. However, etoposide-induced DNA damage significantly alleviated when the cells were pre-incubated with 50 μ mol/L mangiferin for 4h. For example, the olive tail moment value significantly enhanced to 26.3-fold of control when treating with 100 μ g/ml etoposide (p=0.001), but mangiferin pre-treatment reduced the olive tail moment value to 13.4-fold of control which was only 50.8% of the single etoposide treatment group (p=0.003) in comet assay. Micronucleus frequency also significantly increased in a dose-dependent manner after etoposide treatment. Nevertheless pre-incubation with mangiferin significantly inhibited the formation of etoposide-induced micronucleus.ii) Mangiferin induced the nuclear accumulation of Nrf2 in MNC hUCB cells. In the present study, it was also observed that Nrf2 protein mainly located in nuclear area in non-treated cells under confocal microscope. The fluorescence intensity in nucleus enhanced after mangiferin treatment in a dose-dependent and time-dependent manner. Meanwhile, the western blotting results revealed that the nuclear level of Nrf2 protein significantly increased following mangiferin treatment as well. These results showed that mangiferin induced the nuclear accumulation of Nrf2 which indicated activation of Nrf2-mediated signaling.iii) The western blotting results demonstrated that mangiferin treatment increased the expression of NQO1, a downstream target gene of Nrf2 pathway, in MNC hUCB cells. Conclusion:Mangiferin protects hematopoietic cells from chemotherapeutic agent via activating Nrf2-mediated signaling. This newly identified Nrf2 activator can be a potential chemopreventive agent against therapy-related leukemia which deserves further study. Disclosures:No relevant conflicts of interest to declare.

Green tea catechin intervention of reactive oxygen species-mediated ERK pathway activation and chronically induced breast cell carcinogenesis

Long-term exposure to low doses of environmental carcinogens contributes to sporadic human breast cancers. Epidemiologic and experimental studies indicate that green tea catechins (GTCs) may intervene with breast cancer development. We have been developing a chronically induced breast cell carcinogenesis model wherein we repeatedly expose non-cancerous, human breast epithelial MCF10A cells to bioachievable picomolar concentrations of environmental carcinogens, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), to progressively induce cellular acquisition of cancer-associated properties, as measurable end points. The model is then used as a target to identify non-cytotoxic preventive agents effective in suppression of cellular carcinogenesis. Here, we demonstrate, for the first time, a two-step strategy that initially used end points that were transiently induced by short-term exposure to NNK and B[a]P as targets to detect GTCs capable of blocking the acquisition of cancer-associated properties and subsequently used end points constantly induced by long-term exposure to carcinogens as targets to verify GTCs capable of suppressing carcinogenesis. We detected that short-term exposure to NNK and B[a]P resulted in elevation of reactive oxygen species (ROS), leading to Raf-independent extracellular signal-regulated kinase (ERK) pathway activation and subsequent induction of cell proliferation and DNA damage. These GTCs, at non-cytotoxic levels, were able to suppress chronically induced cellular carcinogenesis by blocking carcinogen-induced ROS elevation, ERK activation, cell proliferation and DNA damage in each exposure cycle. Our model may help accelerate the identification of preventive agents to intervene in carcinogenesis induced by long-term exposure to environmental carcinogens, thereby safely and effectively reducing the health risk of sporadic breast cancer.

The impact of EGFR stimulation and inhibition on BPDE induced DNA fragmentation in oral/oropharyngeal mucosa in vitro

Still, the vast majority of head and neck squamous cell carcinoma (HNSCC) can be linked to the "traditional" risk factors tobacco smoke and alcohol consumption. These tumors are believed to be the results of multiple years of carcinogenic impact on upper aerodigestive tract mucosa. The frequent observation, that one patient suffers from several synchronous cancers, multiple local recurrences, and second primary tumors led to the concept of field cancerization, first introduced by Slaughter and colleagues in 1953. As underlying molecular events, genetic instability, loss of heterozygosity, amplification, deletion, up- and down-regulation of oncogenes and/or tumor suppressor genes were revealed. One of the best studied oncogenic features of head and neck carcinogenesis are high expression levels of epidermal growth factor receptor (EGFR). Enhanced expression of the receptor was detected in histologically normal mucosa from HNSCC patients and increasing levels during the progress from hyperplasia to dysplastic lesion and invasive carcinoma were demonstrated. Whereas nearly all of our knowledge about EGFR biology in HNSCC is based on preclinical and clinical studies investigating receptor inhibitors, little is known about cause and function of EGFR in premalignant mucosa. In this study we show, that EGFR stimulation significantly decreases carcinogen induced DNA damage in normal mucosa from HNSCC patients and that this effect is completely abrogated adding an anti-EGFR antibody before stimulation, while there was no effect in non-tumor controls. The effect of EGFR inhibition was contrary. In non-tumor controls, blocking the receptor with an antibody significantly decreased DNA damage, whereas in cases no effect was seen. Our results indicate an important role of the receptor during chemical carcinogenesis. On the basis of this study we suppose, that increasing EGFR levels during head and neck carcinogenesis can be interpreted as a physiological response to permanent carcinogen impact on the mucosa.