N-methyl-N-nitrosourea Research Articles

Anti-Tumor Action, Clinical Biochemistry Profile and Phytochemical Constituents of a Pharmacologically Active Fraction of S. crispus in NMU-Induced Rat Mammary Tumour Model.

Cancer patients seek alternative remedies such as traditional medicinal plants for safe and effective treatment and help overcome the side effects of conventional therapy. Current knowledge indicates that extracts of Strobilanthes crispus of the Acanthaceae family exhibit potent anticancer properties in vitro and are non-toxic in vivo. S. crispus was also reported to be protective against chemical hepatocarcinogenesis. We previously showed that a bioactive fraction of S. crispus leaves also synergized with tamoxifen to cause apoptosis of human breast cancer cell lines without damaging non-malignant epithelial cells. The present study aimed to evaluate the antitumor effect of S. crispus dichloromethane fraction (F3) using N-methyl-N-Nitrosourea (NMU)-induced rat mammary tumor model. Tumor regression was observed in 75% of the rats following 8-week oral administration of F3 with no secondary tumour formation and no signs of anemia or infection. However, no improvement in the liver and renal function profiles was observed. Major constituents of F3 were identified as lutein, 131-hydroxy-132-oxo-pheophytin a, campesterol, stigmasterol, β-sitosterol, pheophytin a and 132-hydroxy-pheophytin a. These compounds however, may not significantly contribute to the antitumor effect of F3.

Abstract P5-11-01: Correlation of the effects of potential and known chemopreventive agents on proliferation rates in normal mammary glands and mammary cancers with their chemopreventive efficacy

Abstract Core needle biopsy, fine needle aspiration, or imaging (e.g., mammography) are currently used to examine chemopreventive agent efficacy in Phase II breast cancer trials. However, biomarker endpoints, such as proliferation rates using Ki67 in normal or "at risk" breast tissue, have not been formally validated relative to cancer outcome. The aim of this study was to validate Ki-67 in "normal" mammary tissue from the methylnitrosourea (MNU) rat mammary tumor model with mammary cancer multiplicity within the same animal as surrogate biomarker for agent efficacy. Multiple studies were performed in female Sprague-Dawley rats to correlate this proliferation biomarker in mammary tissue after two weeks of chemoprevention agent treatment with mammary cancer incidence and multiplicity at the end of the study. In brief, MNU was given at 50 days of age, one week later administration of the agent was started, and after two weeks a biopsy of the mammary gland was taken. Treatment with the agents continued for approximately 150 days, at which time the study was terminated and mammary tumor multiplicity was measured. Changes in proliferation and mammary tumor multiplicity were compared to a control group of untreated group of animals within the same experiment using a two-sided Student t-test. [table1]AgentNormal Mammary Gland Proliferation IndexFinal Mammary Cancer MultiplicityVorozole (1.25 mg/kg BW/day)88%↓90%↓Lipitor (125 mg/kg diet)38%↓16%↓Targretin (150 mg/kg diet)90%↓92%↓Naproxen (200 mg/kg diet)6%↑45%↑Iressa (10 mg/kg BW/day)52%↓93%↓Tamoxifen (3.3 mg/kg diet)77%↓100%↓Metformin (150 mg/kg BW/day)25%↓71%↑ In general, highly effective agents to preventing cancers (e.g., tamoxifen) also prevented normal mammary gland proliferation after only two weeks of treatment, while inactive agents (e.g., naproxen) had minimal effects on normal gland proliferation. The effects of the agents on established mammary cancers (in which the agents were given for 7 days to rats bearing small MNU-induced mammary cancers) showed similar correlations. Additional biomarkers, as well as the proliferation change and efficacy of these agents in rats fed a high-fat (Western) diet, will also be presented. In conclusion, determining the effect of a potential chemopreventive agent on cell proliferation following short-term treatment appears to be an effective method for predicting its efficacy in preventing mammary cancers. These data further confirm that Ki67 measurements are useful in Phase II prevention trials as a biomarker of agent efficacy. Citation Format: Brandy M Heckman-Stoddard, Clinton J Grubbs, Fariba Moeinpour, Vernon E Steele, Ronald A Lubet. Correlation of the effects of potential and known chemopreventive agents on proliferation rates in normal mammary glands and mammary cancers with their chemopreventive efficacy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-11-01.

Transnitrosation of non-mutagenic N-nitrosoproline forms mutagenic N-nitroso-N-methylurea

N-Nitroso-N-methylurea (NMU) is a potent carcinogen and suspected as a cause of human cancer. In this study, mutagenic NMU was detected by HPLC after the transnitrosation of non-mutagenic N-nitrosoproline (NP) to N-methylurea in the presence of thiourea (TU) under acidic conditions. The structure of NMU was confirmed by comparing 1H NMR and IR spectra with that of authentic NMU after fractionation by column chromatography. Furthermore, a fraction containing NMU formed by transnitrosation was mutagenic in Salmonella typhimurium TA1535.NMU was formed in the reaction of NP and N-methylurea in the presence of 1,1,3,3-tetramethylthiourea (TTU) or 1,3-dimethylthiourea in place of TU as an accelerator. The reaction rate constants (k) for NMU formation were correlated with their nucleophilicity of sulfur atom in thioureas. The N-methylurea concentration did not affect the NMU formation, whereas the rate of NMU formation correlated linearly with concentrations of NP, TTU and oxonium ion. The observed kinetics suggests a mechanism by which the nitroso group was transferred directly from the protonated NP to the thiourea then to N-methylurea to form NMU. The rate-determining step was the formation of the complex with the protonated NP and thiourea.

Lack of effect of metformin on mammary carcinogenesis in nondiabetic rat and mouse models.

Epidemiologic studies have shown that diabetics receiving the biguanide metformin, as compared with sulfonylureas or insulin, have a lower incidence of breast cancer. Metformin increases levels of activated AMPK (AMP-activated protein kinase) and decreases circulating IGF-1; encouraging its potential use in both cancer prevention and therapeutic settings. In anticipation of clinical trials in nondiabetic women, the efficacy of metformin in nondiabetic rat and mouse mammary cancer models was evaluated. Metformin was administered by gavage or in the diet, at a human equivalent dose, in standard mammary cancer models: (i) methylnitrosourea (MNU)-induced estrogen receptor-positive (ER(+)) mammary cancers in rats, and (ii) MMTV-Neu/p53KO ER(-) (estrogen receptor-negative) mammary cancers in mice. In the MNU rat model, metformin dosing (150 or 50 mg/kg BW/d, by gavage) was ineffective in decreasing mammary cancer multiplicity, latency, or weight. Pharmacokinetic studies of metformin (150 mg/kg BW/d, by gavage) yielded plasma levels (Cmax and AUC) higher than humans taking 1.5 g/d. In rats bearing small palpable mammary cancers, short-term metformin (150 mg/kg BW/d) treatment increased levels of phospho-AMPK and phospho-p53 (Ser20), but failed to reduce Ki67 labeling or expression of proliferation-related genes. In the mouse model, dietary metformin (1,500 mg/kg diet) did not alter final cancer incidence, multiplicity, or weight. Metformin did not prevent mammary carcinogenesis in two mammary cancer models, raising questions about metformin efficacy in breast cancer in nondiabetic populations.

Immunohistochemical evaluation of chemopreventive effect of hydro-alcoholic extract of garlic (Allium sativum) in N-methyl-N-nitrosourea (NMU) induced rat mammary tumours

Consumption of diets rich in vegetables and herbs is often associated with a reduced risk of developing cancer,particularly breast cancer.Many different cultures throughout the world have recognized the potential use of garlic for prevention and treatment of different diseases.The aim of the present study was to evaluate the chemopreventive effect ofhydro-ethanolic extract of Allium sativum(AS)on the expression pattern of biomolecular markers such as, proliferating cell nuclear antigen (PCNA),vascular endothelial growth factor (VEGF), and caspase-3 during N-methyl-N-nitrosourea (NMU) induced mammary carcinogenesis in Wistar rats.The NMU was injected at 50, 80, and 110days of age in both treatment and preventive group. In untreated control animals, 18 numbers of mammary tumours were successfully induced in 6 (50%) rats with average latency period of 85.84±2.47 days. In preventive group, the average latency period was 141.66±4.09 and total four tumours developed in 2 (16.67%) rats. Immunohistochemical analysis revealed over expression of PCNAand VEGF, and decreased expression of caspase-3 in the mammary tissues of NMU treated rats. Oral administration of hydro-ethanolic extract of AS in NMU treated rats significantly down regulated the expression of PCNA, VEGF and up regulated the expression of caspase-3. In conclusion, the hydro-ethanolic extract of AS treatment reduced cellular proliferation, angiogenesis, and induced more apoptosis in NMU induced mammary carcinogenesis in Wistar rats.

Correlation of AgNOR count with biological behavior of N-Nitroso-N-Methylurea (NMU)-induced squamous cell carcinoma in Wistar rats

Gastric squamous cell carcinoma is one of the most common malignancies in human beings throughout the world. Assessment of proliferative aggressiveness of squamous cell carcinoma plays an important role in diagnostic, prognostic and therapeutic decisions on them. It is determined by both proliferative fraction and speed of neoplastic cells. AgNOR count is an important easily feasible histochemical technique to assess both proliferative fraction and speed of tumour growth. The present study was aimed at evaluating the ability of AgNOR count to assess proliferative speed of a tumour by correlating with pathomorphological parameters of tumour growths (grading, tumour volume, multinodularity, total number of neoplastic cells and survival period) induced in seven Wistar rats by intraperitoneal administration of N-Nitroso-N-Methylurea (NMU) at the dose rate of 50mg/kg b.wt. once per week for four weeks. Tumours were harvested on necropsy after the natural death of experimental rats. Out of total seven cases of gastric squamous cell carcinoma, four tumours were grade II and other three were of grade III. The t-test showed significantly higher mean values of mAgNOR count, mitotic figure count and different pathomorphological parameters of biological behavior i.e., tumour volume, multinodularity and total cell count in Grade-III compared to Grade-II gastric squamous cell carcinomas, indicating their biologically more aggressive growth. It was observed that mAgNOR count may be helpful in predicting real proliferative nature of a tumour and may help in taking diagnostic, prognostic and therapeutic decisions on gastric squamous cell carcinomas.

The mutational landscapes of genetic and chemical models of Kras-driven lung cancer.

SUMMARYNext-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding factors that induce driver mutations, and the processes that shape their selection during tumourigenesis. We performed whole-exome sequencing (WES) on adenomas from three mouse models of non-small cell lung cancer (NSCLC), induced by exposure to carcinogens (Methylnitrosourea (MNU) and Urethane), or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single nucleotide variants (SNVs), compared to only 6 in tumours from the KrasLA2 model. In contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations (CNAs) compared to the carcinogen-induced tumours, suggesting that carcinogen and genetically-engineered models adopt different routes to tumour development. The wild type (WT) allele of Kras has been shown to act as a tumour suppressor in mouse models of NSCLC. We demonstrate that urethane-induced tumours from WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous animals carry mostly (92%) Q61L mutations, indicating a major role of germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C>T mutations at CpG sites. These data provide a basis for understanding the conclusions from human tumour genome sequencing that identified two broad categories based on relative frequency of SNVs and CNAs1, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

Resveratrol enhances the chemopreventive effect of celecoxib in chemically induced breast cancer in rats.

Resveratrol and celecoxib were used as chemopreventive agents in animal models of carcinogenesis, and exert antiproliferative and proapoptotic effects on cancer cells. Therefore, the aim of this study was to evaluate whether combining resveratrol with celecoxib may exert more potent anticarcinogenic effects than the single agents. Mammary carcinogenesis was initiated in 70 female Sprague-Dawley rats with N-methyl-N-nitrosourea (NMU). The chemoprevention with resveratrol, celecoxib, and their combination started 2 weeks before the first carcinogen dose and lasted until the end of the experiment. Tumor incidence and frequency, latency period, tumor volume, the expression of cyclooxygenase 2 (COX2) and growth differentiation factor 15 (GDF15), and also the formation of reactive oxygen species were analyzed using different methods. In addition, the levels of resveratrol and its metabolites in blood and selected tumor tissues were determined by high-performance liquid chromatography. Finally, the anticancer effects of the reagents were studied in the human breast cancer cell line MCF-7. Celecoxib as a single agent significantly decreased tumor frequency, prolonged tumor latency, and decreased the total number of malignant tumors compared with the NMU conditions. Tumor volume was nonsignificantly reduced (0.68±0.25 vs. 0.93±0.28 cm3). Importantly, the addition of resveratrol to celecoxib reduced tumor volume by 60% compared with celecoxib alone (from 0.68±0.25 to 0.27±0.07 cm3, P<0.05). Furthermore, the combination of resveratrol and celecoxib reduced tumor frequency by 29% compared with celecoxib alone (P=0.53). Tumor latency was not influenced by this combination compared with celecoxib alone (126.56±3.45 vs. 120.71±4.08 days). In addition, COX2 mRNA and immunoreactive protein stained on tumor sections were reduced and GDF15 protein increased significantly by the combination studied compared with the NMU conditions. In agreement with these data, a significant reduction in reactive oxygen species in blood lymphocytes of the combination was detected, which may have contributed toward the cancer-preventive effects of this application. This study showed that in NMU-induced mammary cancer in rats, the combination of resveratrol and celecoxib led to a significant reduction in all tumor parameters. In addition, in terms of tumor volume, the combination was more efficient than celecoxib as a single agent.

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research. A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes. In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Src is a novel potential off‐target of RXR agonists, 9‐cis‐UAB30 and Targretin, in human breast cancer cells

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.

Abstract 2419: Protective role of thioredoxin-1 in base excision repair under redox modulation

Abstract Thioredoxin-1 (Trx1) is an antioxidant enzyme with a protective role in the removal of reactive oxygen species (ROS). We investigated the mechanism by which the redox modulator Trx1 affects base excision repair (BER) activity to understand the protective role of Trx1. We constructed a Trx1 knockdown system to demonstrate the specific mechanism of Trx1. DNA damage in terms of relative intensity of the DNA tail and γ-H2AX foci was markedly higher in the Trx1 shRNA cells compared with that in the wild type cells, leading to increased cellular susceptibility to a sublethal dose of BER-inducible toxicant, nitrosomethylurea (NMU). In addition, we observed a modulatory role of Trx1 in the BER pathway via the p53 downstream gene, growth arrest, and DNA-damage-inducible protein 45 α (Gadd45a). The protein level and function of p53, a Trx1 downstream gene, coincidently decreased in the Trx1 shRNA cells. Furthermore, Trx1 shRNA cells showed decreased Gadd45a expression and interaction of Gadd45a with apurynic/apyrimidinic endonuclease 1 (APE1) as well as APE1 activity. In conclusion, Trx1 might cooperate in the control of APE1 function by modulating the p53-mediated BER via the protein-protein interaction between Gadd45a and APE1, providing insight into the novel role of redox factor Trx1 in modulation of BER. Note: This abstract was not presented at the meeting. Citation Format: Hye Lim Kim, Preeyaporn Koedrith, Sang Min Lee, Yeo Jin Kim, Young Rok Seo. Protective role of thioredoxin-1 in base excision repair under redox modulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2419. doi:10.1158/1538-7445.AM2014-2419

Correlation of tumour response with starting tumour size and dose of tamoxifen in an N-methyl-N-nitrosourea (NMU)-induced rat mammary cancer model.

The aim of this preliminary study was to address variations of responses observed with different starting tumor sizes of 10 and 15 mm, and the effects of different doses of tamoxifen (TAM) on experimental rat mammary tumors. Thirty-five inbred female Sprague Dawley rats aged 43 days were administered with three weekly doses of N-methyl-N-nitrosourea (NMU) intraperitoneally (ip) at 50 mg/kg body weight. Animals were randomized (beginning from 10 mm tumor size) into four TAM-treated (50, 100, 200 and 500 μg/day) groups of six animals each, and another group (n=6) treated with TAM 100 μg/day at starting tumour size of 15 mm. The animals were treated by oral gavage daily for 8 weeks before sacrifice. Serum urea and creatinine, and overall physical tumor burden were significantly modulated in animals treated with variable doses of TAM compared to the untreated controls (n=5). Final body weight and tumor number were significantly different in the 10 mm-treated animals compared to those treated at 15 mm. There were no significant differences in histopathological features among all the groups. Our findings suggest the importance of standardizing tumour size and drug doses before initiation of treatment, particularly in the direct comparison of basic end-tumour physical parameters.

Intake of anthocyanidins pelargonidin and cyanidin reduces genotoxic stress in mice induced by diepoxybutane, urethane and endogenous nitrosation

Pelargonidin (PEL) and cyanidin (CYN) are among the six most abundant anthocyanidins which provide red, blue and purple colors to fruits and vegetables. Health benefits associated with intake of anthocyanins have been attributed mainly to antioxidant activity of these color pigments. The aim of our present study was to assess in mice the impact of PEL and CYN intake on genotoxic stress induced by DNA damaging environmental toxicants. These anthocyanidins were administered by gavage to mice before exposure to genotoxic carcinogens diepoxybutane (DEB) and urethane (URE). In addition, the inhibitory effect of PEL and CYN on endogenous nitrosation was evaluated by using a model nitrosation reaction mixture consisting of methyl urea (MU)+sodium nitrite (SN) which reacts in the stomach to form the carcinogenic methyl nitrosourea (MNU). All the test doses of PEL (2.5–20mg/kg) and CYN (1–4mg/kg) significantly reduced the genotoxicity of DEB. A dose-related increase was observed for antigenotoxicity of PEL against URE. The lowest test-dose of CYN showed maximum protection against URE. Co-administration of PEL/CYN with the nitrosation reaction mixture led to reduction in genotoxicity. CYN was more effective as an inhibitor of endogenous nitrosation. Combination of PEL with ascorbic acid (AA) enhanced the antinitrosating effect when compared to that with each phytochemical alone. The results of our present study indicate that common anthocyanidins PEL and CYN can play a major role in reducing genotoxic stress induced by environmental toxicants.

In vitro chemical mutagenesis in exclusively vegetatively-propagated rose-scented geranium (Pelargonium spp.)

SummaryRose-scented geranium (Pelargonium spp.) is an important, vegetatively-propagated, aromatic plant. In such plants, genetic variation may be generated through somaclonal variation and/or induced mutagenesis. Internode and petiole explants of two clones, ‘Bourbon’ and ‘Narmada’, were treated with 0, 0.25, or 0.60 mM N-nitroso-N-methylurea (NMU) to generate variations in oil yield, oil components, and to study the effectiveness of selection for herbage yield and oil concentration. Somaclones were regenerated and evaluated for six oil yield components in the SC2 generation (i.e., the second vegetative generation after the in vitro propagation phase) and in the M1V2 generation (i.e., the second vegetative generation after in vitro mutagen treatment). Significant variations were observed in the majority of the oil yield components studied following all three NMU treatments. Of the 36 somaclones selected for higher herbage yields or oil concentrations in the SC2 and M1V2 generations, 14 and 15 somaclones exhibited significantly higher herbage yields and oil concentrations, respectively, and gave 38 – 137% higher oil yields than their respective parental clones in the next vegetative generation. As only one somaclone with a higher oil yield than the parental clone was obtained from the 0 NMU treatment, in vitro mutagenesis with NMU could be more useful than somaclonal variation for genetic improvement in rose-scented geranium.

Investigations on potential co-mutagenic effects of formaldehyde

The genotoxicity and mutagenicity of formaldehyde (FA) has been well-characterized during the last years. Besides its known direct DNA-damaging and mutagenic activity in sufficiently exposed cells, FA at low concentrations might also enhance the mutagenic and carcinogenic effects of other environmental mutagens by interfering with the repair of DNA lesions induced by these mutagens. To further assess potential co-mutagenic effects of FA, we exposed A549 human lung cells to FA in combination with various mutagens and measured the induction and removal of DNA damage by the comet assay and the production of chromosomal mutations by the cytokinesis-block micronucleus assay (CBMN assay). The mutagens tested were ionizing radiation (IR), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), N-nitroso-N-methylurea (methyl nitrosourea; MNU) and methyl methanesulfonate (MMS). FA (10–75μM) did not enhance the genotoxic and mutagenic activity of these mutagens under the test conditions applied. FA alone and in combination with MNU or MMS did not affect the expression (mRNA level) of the gene of the O6-methylguanine-DNA methyltransferase (MGMT) in A549 cells. The results of these experiments do not support the assumption that low FA concentrations might interfere with the repair of DNA damage induced by other mutagens.

Abstract P5-05-09: Extract ERr731® does not mimic estrogen stimulated growth of MNU-induced breast cancer in ovariectomized Sprague-Dawley rats

Abstract Background: ERr731® (trade names Phyto-Strol® or femi-loges®), an extract isolated from the roots of the Siberian rhubarb (Rheum rhaponticum), is used as an alternative treatment option for menopausal complaints. In addition, it is regularly used for the treatment of oligomenorrhoea or amenorrhea. The efficacy of the extract on treatment of menopausal complaints was shown in clinical trials and follow-up studies. Although clinical trials did not show changes of mammographic density, we addressed the question whether ERr731 is capable to stimulate experimental breast cancer in an estrogen dependent manner. Objective: The aim of the study was to assess whether or not the special extract ERr731® in a dosage relevant to human applications is capable to stimulate the growth of methyl-nitroso-urea (MNU) induced experimental breast cancer in ovariectomized Sprague-Dawley (SD) rats. Experimentals: 32 female SD rats were obtained at the age of postnatal day (PND) 40. On PND 49 and 56 all animals received an intra peritoneal MNU injection of 50 mg/kg body weight (BW). 28 days after the second injection 24 animals were ovariectomized (OvEx) and randomly allocated to 3 experimental groups, 8 animals were SHAM operated and served as positive control. SHAM operated animals (first positive control) and one group of OvEX animals (negative control) were fed a (phyto)estrogen-free diet. A second positive control group of OvEx animals received the phytoestrogen-free diet supplemented with 10 mg/kg diet of estradiol-benzoate. To mimic human exposure, ERr731®, in a dose of 0.067 mg/kg BW/d was chosen which is equivalent to 2.5 mg of the extract/kg chow. The experiment was terminated 25 weeks after MNU injection. Tumor-specific parameters, as well as uterine wet weights (UWW) were assessed. Results: Ovariectomy reduced the UWW &amp;gt; 6fold. Estradiol-benzoate restored UWW almost completely, whereas ERr731® did not lead to an increase in UWW. Tumor incidence in estrogenized animals was 62.5% (5/8 animals) in the SHAM group and 50% (4/8 animals) in the estradiol-benzoate treated group. No tumors were detected in OvEx negative controls and OvEx animals supplemented with the human relevant dose of ERr731®. The total numbers of tumors were 17 in the group of SHAM operated animals and 5 in the estradiol-benzoate treatment group. Conclusions: The human dose of ERr731® did neither exhibit estrogenic properties in the uterotrophic assay nor did it stimulate growth of MNU-induced breast cancers in OvEx SD rats, whereas estradiol supplementation at a low dose as well as estradiol levels in animals with a regular estrus cycle significantly promoted tumor growth. In this experimental model the used human dosage of ERr731® therefore appears safe regarding estrogen dependent growth stimulation of experimental MNU breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-09.

Quantitative assessment of the dose–response of alkylating agents in DNA repair proficient and deficient ames tester strains

Mutagenic and clastogenic effects of some DNA damaging agents such as methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) have been demonstrated to exhibit a nonlinear or even "thresholded" dose-response in vitro and in vivo. DNA repair seems to be mainly responsible for these thresholds. To this end, we assessed several mutagenic alkylators in the Ames test with four different strains of Salmonella typhimurium: the alkyl transferases proficient strain TA1535 (Ogt+/Ada+), as well as the alkyl transferases deficient strains YG7100 (Ogt+/Ada-), YG7104 (Ogt-/Ada+) and YG7108 (Ogt-/Ada-). The known genotoxins EMS, MMS, temozolomide (TMZ), ethylnitrosourea (ENU) and methylnitrosourea (MNU) were tested in as many as 22 concentration levels. Dose-response curves were statistically fitted by the PROAST benchmark dose model and the Lutz-Lutz "hockeystick" model. These dose-response curves suggest efficient DNA-repair for lesions inflicted by all agents in strain TA1535. In the absence of Ogt, Ada is predominantly repairing methylations but not ethylations. It is concluded that the capacity of alkyl-transferases to successfully repair DNA lesions up to certain dose levels contributes to genotoxicity thresholds.

Bone Marrow-Derived Cells May Not Be the Original Cells for Carcinogen-Induced Mouse Gastrointestinal Carcinomas

AimIt has been reported that bone marrow-derived cells (BMDC) can be original cells of mouse gastric cancers induced by Helicobacter felis (H. felis) infection. However, it is unknown whether BMDCs are also the original cells of mouse gastrointestinal cancers induced by gastric carcinogens N-nitroso-N-methylurea (NMU) and H. felis infection.MethodsC57BL/6 recipient mice were initially irradiated with 10Gy X-ray, reconstituted with bone marrow cells from the C57BL/6-Tg (CAG-EGFP) donor mice to label BMDCs with green fluorescence protein (GFP). After 4 weeks of recovery, the bone marrow-transplanted mice were given NMU in drinking water (240 ppm) and subsequently infected with H. felis by gavage. Eighty weeks later, all mice were euthanized for pathological examination. The BMDCs expressing GFP were detected in tissues using direct GFP fluorescence confocal microscopy analysis and immunohistochemistry staining (IHC) assays.ResultsNeoplastic lesions were induced by NMU treatment and/or H. felis infection at the antrum of the glandular stomach and small intestine. In the direct GFP fluorescence confocal assay, GFP(+) epithelial cell cluster or glands were not observed in these gastrointestinal tumors, however, most GFP(+) BMDCs sporadically located in the tumor stromal tissues. Some of these GFP(+) stromal BMDCs co-expressed the hematopoietic marker CD45 or myofibroblasts markers αSMA and SRF. In the indirect GFP IHC assay, similar results were observed among 11 gastric intraepithelial neoplasia lesions and 2 small intestine tumors.ConclusionThese results demonstrated that BMDCs might not be the source of gastrointestinal tumor cells induced by NMU and/or H. felis infection.

Concordant Effects of Aromatase Inhibitors on Gene Expression in ER+ Rat and Human Mammary Cancers and Modulation of the Proteins Coded by These Genes

Aromatase inhibitors are effective in therapy/prevention of estrogen receptor-positive (ER⁺) breast cancers. Rats bearing methylnitrosourea (MNU)-induced ER⁺ mammary cancers were treated with the aromatase inhibitor vorozole (1.25 mg/kg BW/day) for five days. RNA expression showed 162 downregulated and 180 upregulated (P < 0.05 and fold change >1.5) genes. Genes modulated by vorozole were compared with published data from four clinical neoadjuvant trials using aromatase inhibitors (anastrozole or letrozole). More than 30 genes and multiple pathways exhibited synchronous changes in animal and human datasets. Cell-cycle genes related to chromosome condensation in prometaphase [anaphase-prometaphase complex (APC) pathway, including Aurora-A kinase, BUBR1B, TOP2, cyclin A, cyclin B CDC2, and TPX-2)] were downregulated in animal and human studies reflecting the strong antiproliferative effects of aromatase inhibitors. Comparisons of rat arrays with a cell culture study where estrogen was removed from MCF-7 cells showed decreased expression of E2F1-modulated genes as a major altered pathway. Alterations of the cell cycle and E2F-related genes were confirmed in a large independent set of human samples (81 pairs baseline and two weeks anastrozole treatment). Decreases in proliferation-related genes were confirmed at the protein level for cyclin A2, BuRB1, cdc2, Pttg, and TPX-2. Interestingly, the proteins downregulated in tumors were similarly downregulated in vorozole-treated normal rat mammary epithelium. Finally, decreased expression of known estrogen-responsive genes (including TFF, 1,3, progesterone receptor, etc.) were decreased in the animal model. These studies demonstrate that gene expression changes (pathways and individual genes) are similar in humans and the rat model.