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Carcinogenesis Nov 2014Cancer susceptibility varies between people, affected by genotoxic exposures, genetic makeup and physiological state. Yet, how these factors interact among each other to...
Cancer susceptibility varies between people, affected by genotoxic exposures, genetic makeup and physiological state. Yet, how these factors interact among each other to define cancer risk is largely unknown. Here, we uncover the interactive effects of genetical, environmental and physiological factors on genome rearrangements driven by homologous recombination (HR). Using FYDR mice to quantify HR-driven rearrangements in pancreas tissue, we show that DNA methylation damage (induced by methylnitrosourea) and cell proliferation (induced by thyroid hormone) each induce HR and together act synergistically to induce HR-driven rearrangements in vivo. These results imply that developmental or regenerative proliferation as well as mitogenic exposures may sensitize tissues to DNA damaging exposures. We exploited mice genetically deficient in alkyl-adenine DNA glycosylase (Aag) to analyse the relative contributions of unrepaired DNA base lesions versus intermediates formed during base excision repair (BER). Remarkably, results show that, in the pancreas, Aag is a major driver of spontaneous HR, indicating that BER intermediates (including abasic sites and single strand breaks) are more recombinogenic than the spontaneous base lesions removed by Aag. Given that mammals have about a dozen DNA glycosylases, these results point to BER as a major source of pressure on the HR pathway in vivo. Taken together, methylation damage, cell proliferation and Aag interact to define the risk of HR-driven sequence rearrangements in vivo. These data identify important sources of sequence changes in a cancer-relevant organ, and advance the effort to identify populations at high-risk for cancer.
Topics: Animals; Carcinogenesis; Cell Proliferation; DNA Damage; DNA Glycosylases; DNA Methylation; DNA Repair; Homologous Recombination; Humans; Methylnitrosourea; Mice; N-Glycosyl Hydrolases; Neoplasms
PubMed: 25155011
DOI: 10.1093/carcin/bgu177 -
BMC Cancer Aug 2017Real-time polymerase chain reaction (PCR) has become an increasingly important technique for gene expression profiling because it can provide insights into complex...
BACKGROUND
Real-time polymerase chain reaction (PCR) has become an increasingly important technique for gene expression profiling because it can provide insights into complex biological and pathological processes and be used to predict disease or treatment outcomes. Although normalized data are necessary for an accurate estimation of mRNA expression levels, several pieces of evidence suggest that the expression of so-called housekeeping genes is not stable. This study aimed to validate reference genes for the normalization of real-time PCR in an N-methyl-N-nitrosourea (MNU)-induced T-cell lymphoma mouse model.
METHODS
T-cell lymphomas were generated in p53-deficient mice by treatment with 37.5 mg/kg MNU. Thymus and spleen were identified as the primary target organs with the highest incidences of lymphomas. We analyzed the RNA expression levels of eight potential endogenous reference genes (Gapdh, Rn18s, Actb, Hprt, B2M, Rplp0, Gusb, Ctbp1). The expression stabilities of these reference genes were tested at different time points after MNU treatment using geNorm and NormFinder algorithms.
RESULTS
A total of 65% of MNU-treated mice developed T-cell lymphomas, with the spleen and thymus as the major target organs. All candidate reference genes were amplified efficiently by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Gene stability evaluation after MNU treatment and during lymphomagenesis revealed that Ctbp1 and Rplp0 were the most stably expressed genes in the thymus and spleen, respectively. RT-PCR of thymus RNA using two additional sets of primer confirmed that Ctbp1 was the most stable of all the candidate reference genes.
CONCLUSIONS
We provided suitable endogenous controls for gene expression studies in the T-cell lymphoma model.
Topics: Alcohol Oxidoreductases; Animals; DNA-Binding Proteins; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Genes, Essential; Lymphoma, T-Cell; Methylnitrosourea; Mice; Mice, Knockout; Real-Time Polymerase Chain Reaction; Ribosomal Proteins; Tumor Suppressor Protein p53
PubMed: 28807016
DOI: 10.1186/s12885-017-3536-6 -
International Journal of Cancer Sep 2018The Women's Health Initiative studies reported that the menopausal hormone therapy (MHT) regimen containing conjugated equine estrogen (CEE) and medroxyprogesterone...
The Women's Health Initiative studies reported that the menopausal hormone therapy (MHT) regimen containing conjugated equine estrogen (CEE) and medroxyprogesterone acetate increased, whereas CEE alone reduced breast cancer incidence. These observations suggest the possibility that CEE might exert unique actions on breast and also suggest the need to eliminate the progestogen from MHT regimens. A MHT regimen called a tissue selective estrogen complex (TSEC), containing CEE plus bazedoxifene (BZA), to avoid the need for a progestogen, was developed and FDA approved. Our study addressed two questions regarding this TSEC: (i) whether CEE exert effects on breast cancer which differ from those of estradiol (E ) and (ii) whether BZA antagonize the effects of E and CEE on breast cancer? Two rodent models (NMU and ACI) were used to compare the effect of CEE with E on mammary tumor formation, proliferation and apoptosis. In both the NMU and ACI models, E significantly increased tumor incidence and multiplicity whereas in striking contrast CEE did not, even though the estrogenic effects of CEE and E on uterine weight were identical. Mechanistically E blocked whereas CEE stimulated apoptosis (cleaved caspase-3) in ACI animals and only E stimulated proliferation (Ki67). BZA exerted highly potent anti-estrogenic effects on tumors by completely blocking palpable tumor formation. These data suggest that the CEE/BZA TSEC may be a safer, breast-antagonistic, MHT agent for women and might have potential to prevent breast cancer while relieving menopausal symptoms.
Topics: Animals; Apoptosis; Carcinogens; Cell Proliferation; Drug Therapy, Combination; Estrogen Replacement Therapy; Estrogens; Estrogens, Conjugated (USP); Female; Indoles; Mammary Neoplasms, Animal; Methylnitrosourea; Progestins; Rats; Rats, Inbred ACI; Rats, Sprague-Dawley; Selective Estrogen Receptor Modulators
PubMed: 29577272
DOI: 10.1002/ijc.31401 -
Journal of Toxicologic Pathology Apr 2022The retina consists of several layers, and drugs can affect the retina and choroid separately. Therefore, investigating the target layers of toxicity can provide useful...
The retina consists of several layers, and drugs can affect the retina and choroid separately. Therefore, investigating the target layers of toxicity can provide useful information pertaining to its modes of action. Herein, we compared gene expression profiles obtained via microarray analyses using samples of target layers collected via laser capture microdissection and samples of the whole globe of the eye of rats treated with -methyl--nitrosourea. Pathway analyses suggested changes in the different pathways between the laser capture microdissection samples and the whole globe samples. Consistent with the histological distribution of glial cells, upregulation of several inflammation-related pathways was noted only in the whole globe samples. Individual gene expression analyses revealed several gene expression changes in the laser capture microdissection samples, such as caspase- and glycolysis-related gene expression changes, which is similar to previous reports regarding -methyl--nitrosourea-treated animals; however, caspase- and glycolysis-related gene expressions did not change or changed unexpectedly in the whole globe samples. Analyses of the laser capture microdissection samples revealed new potential candidate genes involved in the modes of action of -methyl--nitrosourea-induced retinal toxicity. Collectively, our results suggest that specific retinal layers, which may be targeted by specific toxins, are beneficial in identifying genes responsible for drug-induced ocular toxicity.
PubMed: 35516843
DOI: 10.1293/tox.2021-0064 -
Breast Cancer Research : BCR Jun 2018Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or...
BACKGROUND
Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases.
METHODS
Here, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study.
RESULTS
Metformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism.
CONCLUSIONS
These findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.
Topics: Animals; Aromatase; Breast; Breast Neoplasms; Disease Progression; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Neoplasms, Animal; Metformin; Methylnitrosourea; Ovariectomy; Postmenopause; Rats; Stromal Cells; Tumor Microenvironment
PubMed: 29898754
DOI: 10.1186/s13058-018-0974-2 -
Journal of the National Cancer Institute Jun 2017A nontoxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study was undertaken to...
BACKGROUND
A nontoxic chemopreventive intervention efficacious against different subtypes of breast cancer is still a clinically unmet need. The present study was undertaken to determine the efficacy of an Ayurvedic medicine phytochemical (Withaferin A, [WA]) for chemoprevention of breast cancer and to elucidate its mode of action.
METHODS
Chemopreventive efficacy of WA (4 and 8 mg/kg body weight) was determined using a rat model of breast cancer induced by N-methyl-N-nitrosourea (MNU; n = 14 for control group, n = 15 for 4 mg/kg group, and n = 18 for 8 mg/kg group). The mechanisms underlying breast cancer chemoprevention by WA were elucidated by immunoblotting, biochemical assays, immunohistochemistry, and cytokine profiling using plasma and tumors from the MNU-rat (n = 8-12 for control group, n = 7-11 for 4 mg/kg group, and n = 8-12 for 8 mg/kg group) and/or mouse mammary tumor virus-neu (MMTV-neu) models (n = 4-11 for control group and n = 4-21 for 4 mg/kg group). Inhibitory effect of WA on exit from mitosis and leptin-induced oncogenic signaling was determined using MCF-7 and/or MDA-MB-231 cells. All statistical tests were two-sided.
RESULTS
Incidence, multiplicity, and burden of breast cancer in rats were decreased by WA administration. For example, the tumor weight in the 8 mg/kg group was lower by about 68% compared with controls (8 mg/kg vs control, mean = 2.76 vs 8.59, difference = -5.83, 95% confidence interval of difference = -9.89 to -1.76, P = .004). Mitotic arrest and apoptosis induction were some common determinants of breast cancer chemoprevention by WA in the MNU-rat and MMTV-neu models. Cytokine profiling showed suppression of plasma leptin levels by WA in rats. WA inhibited leptin-induced oncogenic signaling in cultured breast cancer cells.
CONCLUSIONS
WA is a promising chemopreventative phytochemical with the ability to inhibit at least two different subtypes of breast cancer.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Acetyl Coenzyme A; Aldehyde Dehydrogenase 1 Family; Animals; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cytokines; Deoxyguanosine; Electron Transport Complex III; Female; Forkhead Transcription Factors; Humans; Ki-67 Antigen; Lactic Acid; Leptin; MCF-7 Cells; Malates; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Methylnitrosourea; Mice; Mitosis; Mitotic Index; Rats; Receptors, Estrogen; Retinal Dehydrogenase; Retroviridae Infections; Signal Transduction; Tumor Burden; Tumor Virus Infections; Withanolides
PubMed: 28040797
DOI: 10.1093/jnci/djw293 -
Scientific Reports Jun 2021This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising...
This study aimed to investigate the impact of chronic low-level exposure to chemical carcinogens with different modes of action on the cellular response to ionising radiation. Human lymphoblastoid GM1899A cells were cultured in the presence of 4-nitroquinoline N-oxide (4NQO), N-nitroso-N-methylurea (MNU) and hydrogen peroxide (HO) for up to 6 months at the highest non-(geno)toxic concentration identified in pilot experiments. Acute challenge doses of 1 Gy X-rays were given and chromosome damage (dicentrics, acentric fragments, micronuclei, chromatid gaps/breaks) was scored. Chronic exposure to 20 ng/ml 4NQO, 0.25 μg/ml MNU or 10 μM HO hardly induced dicentrics and did not significantly alter the yield of X-ray-induced dicentrics. Significant levels of acentric fragments were induced by all chemicals, which did not change during long-term exposure. Fragment data in combined treatment samples compared to single treatments were consistent with an additive effect of chemical and radiation exposure. Low level exposure to 4NQO induced micronuclei, the yields of which did not change throughout the 6 month exposure period. As for fragments, micronuclei yields for combined treatments were consistent with an additive effect of chemical and radiation. These results suggest that cellular radiation responses are not affected by long-term low-level chemical exposure.
Topics: 4-Nitroquinoline-1-oxide; Cell Line; Cell Survival; Chromosomes; Humans; Hydrogen Peroxide; Lymphocytes; Methylnitrosourea; Micronucleus Tests; Radiation Dosage; Radiation Tolerance; Time Factors
PubMed: 34135387
DOI: 10.1038/s41598-021-91957-y -
International Journal of Experimental... Dec 2014Previous studies in the field of cancer research have suggested a possible role for statins in the reduction of risk in certain malignancies. The purpose of these...
Previous studies in the field of cancer research have suggested a possible role for statins in the reduction of risk in certain malignancies. The purpose of these studies was to examine the chemopreventive effects of pravastatin alone and in combination with pineal hormone melatonin in the N-methyl-N-nitrosourea-induced mammary carcinogenesis model. Pravastatin was given orally (1 00 mg/kg) and melatonin was added to the water (20 μg/ml). Chemoprevention began seven days prior to carcinogen administration and subsequently continued for 15 weeks until autopsy. At autopsy, mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. Parameters of experimental carcinogenesis, mechanism of action (biomarkers of apoptosis, angiogenesis and proliferation) and side effects after long-term treatment in animals were assessed. Pravastatin alone suppressed tumour frequency by 20.5% and average tumour volume by 15% compared with controls. Combined administration of the drugs decreased tumour frequency by 69% and lengthened tumour latency by nine days compared with control animals. The ration between high and low grade carcinomas was apparently reduced in both treated groups. The analysis of carcinoma cells showed significant expression increase in caspase-3 and caspase-7 after pravastatin treatment; however, combined treatment even more pronounced increase in the expression of both caspases. Regarding VEGFR-2 expression, a small effect in carcinomas of both treated groups was found. In plasma metabolism evaluation, pravastatin alone significantly decreased levels of glucose and triacylglycerols. Our results suggest a mild anti-neoplastic effect of pravastatin in this rat mammary gland carcinoma model. Statins co-administered with other suitable drug (e.g. melatonin) should be further evaluated for tumour-preventive properties.
Topics: Alkylating Agents; Animals; Antineoplastic Agents; Antioxidants; Apoptosis; Carcinoma in Situ; Carcinoma, Ductal, Breast; Carcinoma, Papillary; Disease Models, Animal; Drug Synergism; Female; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mammary Neoplasms, Experimental; Melatonin; Methylnitrosourea; Pravastatin; Rats, Sprague-Dawley
PubMed: 25270735
DOI: 10.1111/iep.12094 -
Molecular Carcinogenesis Dec 20159-cis-UAB30 (UAB30) and Targretin are well-known retinoid X receptor (RXR) agonists. They were highly effective in decreasing the incidence of methylnitrosourea...
9-cis-UAB30 (UAB30) and Targretin are well-known retinoid X receptor (RXR) agonists. They were highly effective in decreasing the incidence of methylnitrosourea (MNU)-induced mammary cancers. However, whether the anti-mammary cancer effects of UAB30 or Targretin originate from the activation of RXR is unclear. In the present study, we hypothesized that UAB30 and Targretin not only affect RXR, but likely influence one or more off-target proteins. Virtual screening results suggest that Src is a potential target for UAB30 and Targretin that regulates extracellular matrix (ECM) molecules and cell motility and invasiveness. In vitro kinase assay data revealed that UAB30 or Targretin interacted with Src and attenuated its kinase activity. We found that UAB30 or Targretin substantially inhibited invasiveness and migration of MCF-7 and SK-BR-3 human breast cancer cells. We examined the effects of UAB30 and Targretin on the expression of matrix metalloproteinases (MMP)-9, which are known to play an essential role in tumor invasion. We show that activity and expression of MMP-9 were decreased by UAB30 or Targretin. Western blot data showed that UAB30 or Targretin decreased AKT and its substrate molecule p70(s6k), which are downstream of Src in MCF-7 and SK-BR-3 cells. Moreover, knocking down the expression of Src effectively reduced the sensitivity of SK-BR-3 cells to the inhibitory effects of UAB30 and Targretin on invasiveness. Taken together, our results demonstrate that UAB30 and Targretin each inhibit invasion and migration by targeting Src in human breast cancer cells.
Topics: Bexarotene; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Fatty Acids, Unsaturated; Female; Humans; MCF-7 Cells; Matrix Metalloproteinase 9; Naphthalenes; Neoplasm Invasiveness; Oncogene Protein pp60(v-src); Proto-Oncogene Proteins c-akt; Retinoid X Receptors; Ribosomal Protein S6 Kinases, 70-kDa; Tetrahydronaphthalenes
PubMed: 25328014
DOI: 10.1002/mc.22232 -
PloS One 2016In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous...
In contrast to the mammalian retina, the zebrafish retina exhibits the potential for lifelong retinal neurogenesis and regeneration even after severe damage. Previous studies have shown that the transforming growth factor beta (TGFβ) signaling pathway is activated during the regeneration of different tissues in the zebrafish and is needed for regeneration in the heart and the fin. In this study, we have investigated the role of the TGFβ pathway in the N-methyl-N-nitrosourea (MNU)-induced chemical model of rod photoreceptor de- and regeneration in adult zebrafish. Immunohistochemical staining for phosphorylated Smad3 was elevated during retinal regeneration, and phosphorylated Smad3 co-localized with proliferating cell nuclear antigen and glutamine synthetase, indicating TGFβ pathway activation in proliferating Müller glia. Inhibiting the TGFβ signaling pathway using a small molecule inhibitor (SB431542) resulted in accelerated recovery from retinal degeneration. Accordingly, we observed increased cell proliferation in the outer nuclear layer at days 3 to 8 after MNU treatment. In contrast to the observations in the heart and the fin, the inhibition of the TGFβ signaling pathway resulted in increased proliferation after the induction of retinal degeneration. A better understanding of the underlying pathways with the possibility to boost retinal regeneration in adult zebrafish may potentially help to stimulate such proliferation also in other species.
Topics: Animals; Benzamides; Cell Proliferation; Dioxoles; Ependymoglial Cells; Methylnitrosourea; Regeneration; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Zebrafish; Zebrafish Proteins
PubMed: 27880821
DOI: 10.1371/journal.pone.0167073