Prognostic potential of PRMT5 and DSG2 proteins in pre-malignant cervical lesions.

Precancerous cervical lesions are metaplastic alterations of epithelial cells of the cervix, eventually developing into cervical cancer. Despite primary and secondary prevention, the burden of cervical cancer remains high globally. Protein arginine methyltransferases (PRMT) represent post-translational modifications that interact with multiple signalling pathways, playing a role in epithelial-mesenchymal transition. In complex with desmoglein-2 (DSG2), a cell adhesion protein, both participate in the progression of dysplastic changes with potential malignant development. The presented study was performed on archival paraffin-embedded blocks from adult women. The studied samples were categorised into low-grade and high-grade intraepithelial lesions. Immunohistochemical analysis was used to observe subcellular localisation, immunoreaction intensity, and percentage of PRMT5- and DSG2-expressing cells, followed by statistical analysis. Preliminary results identified statistically significant differences between the expression and subcellular localisation of proteins in question in low-grade and high-grade squamous intraepithelial lesions. The primary goal of the presented study is to perceive the involvement of PRMT5 and DSG2 in the initiation and progression of cervical lesions. Our observations indicate the potential of the assessed proteins as prognostic markers. However, further studies of PRMT5 and DSG2 are required to provide greater insight into cervical carcinogenesis.

Expression of fascin in association with p16 and Ki-67 in cervical lesions: immunohistochemical study.

Annual gynecological examination with cervical cancer screening and HPV vaccination ensures the appropriate prevention of the onset and progression of cervical cancer. Currently, efforts are being made to find new diagnostic and prognostic biomarkers. Fascin, an actin-bundling protein, promotes cellular migration. Its overexpression has been observed in many types of squamous carcinomas and was usually correlated with a worse prognosis and metastasis. However, the data on fascin expression in cervical lesions are limited. This study focuses on the quantitative evaluation of fascin expression, the immunoreaction intensity and subcellular localization of fascin expression in low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesions (HSIL) and squamous cell carcinomas (SCC). Fascin expression was also correlated with the routinely used diagnostic markers p16 and Ki-67. Biopsy specimens (n = 67) of LSIL, HSIL and SCC were taken from adult women in the age range 20-86 years. Fascin expression was detected by immunohistochemical analysis and quantified using morphometric software. Analysis of variance confirmed statistically significant differences in the percentage of fascin-positive cells between the LSIL, HSIL and SCC groups. Finally, the results showed a significant positive correlation between fascin expression and p16 and Ki-67 expression.

5-Fluorouracil Treatment of CT26 Colon Cancer Is Compromised by Combined Therapy with IMMODIN.

Due to the physiological complexity of the tumour, a single drug therapeutic strategy may not be sufficient for effective treatment. Emerging evidence suggests that combination strategies may be important to achieve more efficient tumour responses. Different immunomodulators are frequently tested to reverse the situation for the purpose of improving immune response and minimizing chemotherapy side effects. Immodin (IM) represents an attractive alternative to complement chemotherapy, which can be used to enhance the immune system after disturbances resulting from the side effects of chemotherapy. In the presented study, a model of CT26 tumor-bearing mice was used to investigate the effect of single IM or its combination with 5-fluorouracil (5-FU) on colon cancer cells. Our results highlight that the beneficial role of IM claimed in previous studies cannot be generalised to all chemotherapeutic drugs, as 5-FU toxicity was not increased. On the contrary, the chemotherapeutic anti-cancer efficacy of 5-FU was greatly compromised when combined with IM. Indeed, the combined treatment was significantly less effective regarding the tumour growth and animal survival, most probably due to the increased number of tumour-associated macrophages, and increased 5-FU cytotoxic effect related to kidneys and the liver.

Effects of Inhalation of STIM-Orai Antagonist SKF 96365 on Ovalbumin-Induced Airway Remodeling in Guinea Pigs.

Airway remodeling (AR) consists of wall thickening and hyperreactivity. STIM (stromal interaction molecule) and Orai protein pathways mediate extracellular Ca2+ signals involved in AR. This study aims to define the effects on AR of the STIM-Orai antagonist SKF 96365 given by inhalation in three increasing doses in ovalbumin-induced AR. In the control group, the antiasthmatic budesonide and salbutamol were given in the same model. The airway structure was evaluated by histological and immunohistochemistry and reactivity by specific airway resistance, contraction strength of isolated airway smooth muscles, and mucociliary clearance expressed by ciliary beating frequency. The immuno-biochemical markers of chronic inflammation were evaluated by BioPlex and ELISA assays. The AR was mediated by inflammatory cytokines and growth factors. The findings show significant anti-remodeling effects of SKF 96365, which were associated with a decrease in airway hyperreactivity. The anti-remodeling effect of SKF 96365 was mediated via the suppression of IL-4, IL-5, and IL-13 synthesis, and IL-12-INF-γ-TGF-ß pathway. The budesonide-related AR suppression had to do with a decrease in proinflammatory cytokines and an increase in the anti-inflammatory IL-10, with negligible influence on growth factors synthesis and mucous glands activity.

Immunohistochemical expression and significance of SATB2 protein in colorectal cancer.

In this study we evaluated the expression of SATB2 protein in colorectal cancer (CRC) and its association with microsatellite instability (MSI) status, inflammation and hypoxia. Immunohistochemical SATB2 expression was observed in 111 CRC samples. We assessed the correlation between SATB2 expression and clinico-morphological parameters, MSI, COX-2 and HIF-1α expression. SATB2 was noticed in 92.8% CRC. We observed nuclear staining with predominantly strong immunoreaction intensity (67.6%) and percentage of SATB-2 positive cells in more than 50% of cells (87.4%). The statistically significant associations were recorded between high SATB2 expression and low grade, negative lymph nodes and negative vascular invasion. Statistical analysis confirmed a significant correlation between SATB2 expression and microsatellite stability, tendency to correlate with COX-2 and no significant correlation with HIF-1α. SATB2 is overexpressed in CRC and its high expression is a marker of good prognosis. Moreover, SATB2 expression is significantly associated with microsatellite stability, there is tendency to correlate with pro-inflammatory COX-2 and there is no association with hypoxia.

Methionine Diet Evoked Hyperhomocysteinemia Causes Hippocampal Alterations, Metabolomics Plasma Changes and Behavioral Pattern in Wild Type Rats.

L-methionine, an essential amino acid, plays a critical role in cell physiology. High intake and/or dysregulation in methionine (Met) metabolism results in accumulation of its intermediate(s) or breakdown products in plasma, including homocysteine (Hcy). High level of Hcy in plasma, hyperhomocysteinemia (hHcy), is considered to be an independent risk factor for cerebrovascular diseases, stroke and dementias. To evoke a mild hHcy in adult male Wistar rats we used an enriched Met diet at a dose of 2 g/kg of animal weight/day in duration of 4 weeks. The study contributes to the exploration of the impact of Met enriched diet inducing mild hHcy on nervous tissue by detecting the histo-morphological, metabolomic and behavioural alterations. We found an altered plasma metabolomic profile, modified spatial and learning memory acquisition as well as remarkable histo-morphological changes such as a decrease in neurons' vitality, alterations in the morphology of neurons in the selective vulnerable hippocampal CA 1 area of animals treated with Met enriched diet. Results of these approaches suggest that the mild hHcy alters plasma metabolome and behavioural and histo-morphological patterns in rats, likely due to the potential Met induced changes in "methylation index" of hippocampal brain area, which eventually aggravates the noxious effect of high methionine intake.

Protective Effects of Flavonoids Against Mitochondriopathies and Associated Pathologies: Focus on the Predictive Approach and Personalized Prevention.

Multi-factorial mitochondrial damage exhibits a "vicious circle" that leads to a progression of mitochondrial dysfunction and multi-organ adverse effects. Mitochondrial impairments (mitochondriopathies) are associated with severe pathologies including but not restricted to cancers, cardiovascular diseases, and neurodegeneration. However, the type and level of cascading pathologies are highly individual. Consequently, patient stratification, risk assessment, and mitigating measures are instrumental for cost-effective individualized protection. Therefore, the paradigm shift from reactive to predictive, preventive, and personalized medicine (3PM) is unavoidable in advanced healthcare. Flavonoids demonstrate evident antioxidant and scavenging activity are of great therapeutic utility against mitochondrial damage and cascading pathologies. In the context of 3PM, this review focuses on preclinical and clinical research data evaluating the efficacy of flavonoids as a potent protector against mitochondriopathies and associated pathologies.

Effects of PDE3 Inhibitor Olprinone on the Respiratory Parameters, Inflammation, and Apoptosis in an Experimental Model of Acute Respiratory Distress Syndrome.

This study aimed to investigate whether a selective phosphodiesterase-3 (PDE3) inhibitor olprinone can positively influence the inflammation, apoptosis, and respiratory parameters in animals with acute respiratory distress syndrome (ARDS) model induced by repetitive saline lung lavage. Adult rabbits were divided into 3 groups: ARDS without therapy (ARDS), ARDS treated with olprinone i.v. (1 mg/kg; ARDS/PDE3), and healthy ventilated controls (Control), and were oxygen-ventilated for the following 4 h. Dynamic lung-thorax compliance (Cdyn), mean airway pressure (MAP), arterial oxygen saturation (SaO2), alveolar-arterial gradient (AAG), ratio between partial pressure of oxygen in arterial blood to a fraction of inspired oxygen (PaO2/FiO2), oxygenation index (OI), and ventilation efficiency index (VEI) were evaluated every hour. Post mortem, inflammatory and oxidative markers (interleukin (IL)-6, IL-1ß, a receptor for advanced glycation end products (RAGE), IL-10, total antioxidant capacity (TAC), 3-nitrotyrosine (3NT), and malondialdehyde (MDA) and apoptosis (apoptotic index and caspase-3) were assessed in the lung tissue. Treatment with olprinone reduced the release of inflammatory mediators and markers of oxidative damage decreased apoptosis of epithelial cells and improved respiratory parameters. The results indicate a future potential of PDE3 inhibitors also in the therapy of ARDS.

Rhus coriaria L. (Sumac) Demonstrates Oncostatic Activity in the Therapeutic and Preventive Model of Breast Carcinoma.

Comprehensive scientific data provide evidence that isolated phytochemicals or whole plant foods may beneficially modify carcinogenesis. The aim of this study was to evaluate the oncostatic activities of Rhus coriaria L. (sumac) using animal models (rat and mouse), and cell lines of breast carcinoma. R. coriaria (as a powder) was administered through the diet at two concentrations (low dose: 0.1% (w/w) and high dose: 1 % (w/w)) for the duration of the experiment in a syngeneic 4T1 mouse and chemically-induced rat mammary carcinoma models. After autopsy, histopathological and molecular analyses of tumor samples in rodents were performed. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were conducted. The dominant metabolites present in tested R. coriaria methanolic extract were glycosides of gallic acid (possible gallotannins). In the mouse model, R. coriaria at a higher dose (1%) significantly decreased tumor volume by 27% when compared to controls. In addition, treated tumors showed significant dose-dependent decrease in mitotic activity index by 36.5% and 51% in comparison with the control group. In the chemoprevention study using rats, R. coriaria at a higher dose significantly reduced the tumor incidence by 20% and in lower dose non-significantly reduced tumor frequency by 29% when compared to controls. Evaluations of the mechanism of oncostatic action using valid clinical markers demonstrated several positive alterations in rat tumor cells after the treatment with R. coriaria. In this regard, histopathological analysis of treated tumor specimens showed robust dose-dependent decrease in the ratio of high-/low-grade carcinomas by 66% and 73% compared to controls. In treated rat carcinomas, we found significant caspase-3, Bax, and Bax/Bcl-2 expression increases; on the other side, a significant down-regulation of Bcl-2, Ki67, CD24, ALDH1, and EpCam expressions and MDA levels. When compared to control specimens, evaluation of epigenetic alterations in rat tumor cells in vivo showed significant dose-dependent decrease in lysine methylation status of H3K4m3 and H3K9m3 and dose-dependent increase in lysine acetylation in H4K16ac levels (H4K20m3 was not changed) in treated groups. However, only in lower dose of sumac were significant decreases in the expression of oncogenic miR210 and increase of tumor-suppressive miR145 (miR21, miR22, and miR155 were not changed) observed. Finally, only in lower sumac dose, significant decreases in methylation status of three out of five gene promoters-ATM, PTEN, and TIMP3 (PITX2 and RASSF1 promoters were not changed). In vitro evaluations using methanolic extract of R. coriaria showed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). In conclusion, sumac demonstrated significant oncostatic activities in rodent models of breast carcinoma that were validated by mechanistic studies in vivo and in vitro.

Chemopreventive and Therapeutic Efficacy of Cinnamomum zeylanicum L. Bark in Experimental Breast Carcinoma: Mechanistic In Vivo and In Vitro Analyses.

Comprehensive oncology research suggests an important role of phytochemicals or whole plant foods in the modulation of signaling pathways associated with anticancer action. The goal of this study is to assess the anticancer activities of Cinnamomum zeylanicum L. using rat, mouse, and cell line breast carcinoma models. C. zeylanicum (as bark powder) was administered in the diet at two concentrations of 0.1% (w/w) and 1% (w/w) during the whole experiment in chemically induced rat mammary carcinomas and a syngeneic 4T1 mouse model. After autopsy, histopathological and molecular evaluations of mammary gland tumors in rodents were carried out. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were performed. The dominant metabolites present in the tested C. zeylanicum essential oil (with relative content over 1%) were cinnamaldehyde, cinnamaldehyde dimethyl acetal, cinnamyl acetate, eugenol, linalool, eucalyptol, limonene, o-cymol, and α-terpineol. The natural mixture of mentioned molecules demonstrated significant anticancer effects in our study. In the mouse model, C. zeylanicum at a higher dose (1%) significantly decreased tumor volume by 44% when compared to controls. In addition, treated tumors showed a significant dose-dependent decrease in mitotic activity index by 29% (0.1%) and 45.5% (1%) in comparison with the control group. In rats, C. zeylanicum in both doses significantly reduced the tumor incidence by 15.5% and non-significantly suppressed tumor frequency by more than 30% when compared to controls. An evaluation of the mechanism of anticancer action using valid oncological markers showed several positive changes after treatment with C. zeylanicum. Histopathological analysis of treated rat tumor specimens showed a significant decrease in the ratio of high-/low-grade carcinomas compared to controls. In treated rat carcinomas, we found caspase-3 and Bax expression increase. On the other hand, we observed a decrease in Bcl-2, Ki67, VEGF, and CD24 expressions and MDA levels. Assessment of epigenetic changes in rat tumor cells in vivo showed a significant decrease in lysine methylation status of H3K4m3 and H3K9m3 in the high-dose treated group, a dose-dependent increase in H4K16ac levels (H4K20m3 was not changed), down-regulations of miR21 and miR155 in low-dose cinnamon groups (miR22 and miR34a were not modulated), and significant reduction of the methylation status of two out of five gene promoters-ATM and TIMP3 (PITX2, RASSF1, PTEN promoters were not changed). In vitro study confirmed results of animal studies, in that the essential oil of C. zeylanicum displayed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using MTS, BrdU, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). As a conclusion, C. zeylanicum L. showed chemopreventive and therapeutic activities in animal breast carcinoma models that were also significantly confirmed by mechanistic evaluations in vitro and in vivo.

Effect of Methionine Diet on Metabolic and Histopathological Changes of Rat Hippocampus.

Hyperhomocysteinemia (hHcy) is regarded as an independent and strong risk factor for cerebrovascular diseases, stroke, and dementias. The hippocampus has a crucial role in spatial navigation and memory processes and is being constantly studied for neurodegenerative disorders. We used a moderate methionine (Met) diet at a dose of 2 g/kg of animal weight/day in duration of four weeks to induce mild hHcy in adult male Wistar rats. A novel approach has been used to explore the hippocampal metabolic changes using proton magnetic resonance spectroscopy (1H MRS), involving a 7T MR scanner in combination with histochemical and immunofluorescence analysis. We found alterations in the metabolic profile, as well as remarkable histo-morphological changes such as an increase of hippocampal volume, alterations in number and morphology of astrocytes, neurons, and their processes in the selective vulnerable brain area of animals treated with a Met-enriched diet. Results of both methodologies suggest that the mild hHcy induced by Met-enriched diet alters volume, histo-morphological pattern, and metabolic profile of hippocampal brain area, which might eventually endorse the neurodegenerative processes.

Anticancer Activities of Thymus vulgaris L. in Experimental Breast Carcinoma in Vivo and in Vitro.

Naturally-occurring mixtures of phytochemicals present in plant foods are proposed to possess tumor-suppressive activities. In this work, we aimed to evaluate the antitumor effects of Thymus vulgaris L. in in vivo and in vitro mammary carcinoma models. Dried T. vulgaris (as haulm) was continuously administered at two concentrations of 0.1% and 1% in the diet in a chemically-induced rat mammary carcinomas model and a syngeneic 4T1 mouse model. After autopsy, histopathological and molecular analyses of rodent mammary carcinomas were performed. In addition, in vitro evaluations using MCF-7 and MDA-MB-231 cells were carried out. In mice, T. vulgaris at both doses reduced the volume of 4T1 tumors by 85% (0.1%) and 84% (1%) compared to the control, respectively. Moreover, treated tumors showed a substantial decrease in necrosis/tumor area ratio and mitotic activity index. In the rat model, T. vulgaris (1%) decreased the tumor frequency by 53% compared to the control. Analysis of the mechanisms of anticancer action included well-described and validated diagnostic and prognostic markers that are used in both clinical approach and preclinical research. In this regard, the analyses of treated rat carcinoma cells showed a CD44 and ALDH1A1 expression decrease and Bax expression increase. Malondialdehyde (MDA) levels and VEGFR-2 expression were decreased in rat carcinomas in both the T. vulgaris treated groups. Regarding the evaluations of epigenetic changes in rat tumors, we found a decrease in the lysine methylation status of H3K4me3 in both treated groups (H3K9m3, H4K20m3, and H4K16ac were not changed); up-regulations of miR22, miR34a, and miR210 expressions (only at higher doses); and significant reductions in the methylation status of four gene promoters-ATM serin/threonine kinase, also known as the NPAT gene (ATM); Ras-association domain family 1, isoform A (RASSF1); phosphatase and tensin homolog (PTEN); and tissue inhibitor of metalloproteinase-3 (TIMP3) (the paired-like homeodomain transcription factor (PITX2) promoter was not changed). In vitro study revealed the antiproliferative and proapoptotic effects of essential oils of T. vulgaris in MCF-7 and MDA-MB-231 cells (analyses of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS); 5-bromo-20-deoxyuridine (BrdU); cell cycle; annexin V/PI; caspase-3/7; Bcl-2; PARP; and mitochondrial membrane potential). T. vulgaris L. demonstrated significant chemopreventive and therapeutic activities against experimental breast carcinoma.

Association of Induced Hyperhomocysteinemia with Alzheimer's Disease-Like Neurodegeneration in Rat Cortical Neurons After Global Ischemia-Reperfusion Injury.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder that results in massive hippocampal and neocortical neuronal loss leading to dementia and eventual death. The exact cause of Alzheimer's disease is not fully explored, although a number of risk factors have been recognized, including high plasma concentration of homocysteine (Hcy). Hyperhomocysteinemia (hHcy) is considered a strong, independent risk factor for stroke and dementia. However, the molecular background underlying these mechanisms linked with hHcy and ischemic stroke is not fully understood. Paper describes rat model of global forebrain ischemia combined with the experimentally induced hHcy. Global ischemia-reperfusion injury (IRI) was developed by 4-vessels occlusion lasting for 15 min followed by reperfusion period of 72 h. hHcy was induced by subcutaneous injection of 0.45 µmol/g of Hcy in duration of 14 days. The results showed remarkable neural cell death induced by hHcy in the brain cortex and neurodegeneration is further aggravated by global IRI. We demonstrated degeneration of cortical neurons, alterations in number and morphology of tissue astrocytes and dysregulation of oxidative balance with increased membrane protein oxidation. Complementary to, an immunohistochemical analysis of tau protein and ß-amyloid peptide showed that combination of hHcy with the IRI might lead to the progression of AD-like pathological features. Conclusively, these findings suggest that combination of risk factor hHcy with IRI aggravates neurodegeneration processes and leads to development of AD-like pathology in cerebral cortex.

Metformin and melatonin inhibit DMBA-induced mammary tumorigenesis in rats fed a high-fat diet.

The data from in-vitro and in-vivo studies show that both peroral antidiabetic metformin (MF) and pineal hormone melatonin (MT) inhibit the growth of many cancers, including breast cancer. However, most in-vivo studies used standard-type diet with low fat content. Therefore, in this study, we evaluated the chemopreventive effect of MF and MT in an in-vivo model of breast cancer in rats on a high-fat diet (10% of total fat). Mammary carcinogenesis was induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Chemoprevention with MF (administered in a diet, 0.2%) and MT (administered in tap water, 20 mg/l) was induced 20 days before the carcinogen administration through the termination of the experiment (14 weeks after carcinogen administration). Tumor growth parameters were analyzed together with histopathological examination and immunohistochemical detection of KI67 (proliferation marker), caspase-3, BAX, BCL-2 (apoptosis markers), and CD24 and CD44 (cancer stem cell markers) in mammary tumor samples. The combination of chemopreventive agents decreased tumor incidence by 29%. Cumulative tumor volume was lower in all groups treated with chemoprevention. Histopathology did not show significant changes in high-grade/low-grade tumor ratio. Immunohistochemistry showed increased expression of BAX in the combination group, and caspase-3 expression increased in both MT and combination groups. MT, and particularly the MF and MT combination, inhibited DMBA-induced mammary tumor growth in rats by apoptosis stimulation in cancer cells. Our results indicate that MT supplements in patients treated with MF may have a considerable effect on the incidence of breast cancer.

Antineoplastic effects of clove buds (Syzygium aromaticum L.) in the model of breast carcinoma.

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF-7 cells was carried out. Dietary administered CLO caused the dose-dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl-2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase-3 and ALDH1 expression increase after high-dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro-apoptotic effects of CLO extract in MCF-7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase-7, Bcl-2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.

Oregano demonstrates distinct tumour-suppressive effects in the breast carcinoma model.

PURPOSE: There has been a considerable interest in the identification of natural plant foods for developing effective agents against cancer. Thus, the anti-tumour effects of oregano in the in vivo and in vitro breast cancer model were evaluated. METHODS: Lyophilized oregano (ORE) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration. At autopsy, mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation in MCF-7 cells was carried out. RESULTS: Low-dose ORE suppressed tumour frequency by 55.5 %, tumour incidence by 44 %, and tumour volume by 44.5 % compared to control animals. Analysis of rat tumour cells showed Ki67, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase after low-dose ORE treatment. High-dose ORE lengthened tumour latency by 12.5 days; moreover, Bcl-2, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase in carcinoma cells were observed. Histopathological analysis revealed a decrease in the ratio of high-/low-grade carcinomas in both treated groups. In vitro studies showed that ORE decreased survival and proliferation of MCF-7 cells. In ORE-treated MCF-7 cells, an increase in cells expressing sub-G 0/G 1 DNA content and an increase in the percentage of annexin V/PI positive MCF-7 cells were observed. In vitro, both caspase-dependent and possible non-caspase-dependent apoptotic pathways were found. The deactivation of anti-apoptotic activity of Bcl-2, a decrease in mitochondrial membrane potential, and the activation of mitochondrial apoptosis pathway were observed in the ORE-treated MCF-7 cells. CONCLUSIONS: Our results demonstrate, for the first time, a distinct tumour-suppressive effect of oregano in the breast cancer model.

Survivin in breast lesions: immunohistochemical analysis of 196 cases.

We examined the survivin expression pattern by immunohistochemistry in 43 fibroadenomas and 153 ductal carcinomas of the breast. The subcellular localization of survivin and the intensity of immunoreaction were assessed. We analyzed the differences of survivin expression between fibroadenomas and carcinomas. We also correlated the survivin expression pattern in carcinomas with other clinicomorphological parameters such as the age of patients, the grade and size of primary tumor as well as the lymph node metastasis. Overall, survivin was detected in 107/153 carcinomas (69.9%) and in 26/43 fibroadenomas (60.5%). Statistical analysis confirmed significant correlations between the assessed parameters in fibroadenomas and carcinomas. Grade of carcinomas was significantly related to survivin expression in both subcellular localization and the intensity of immunoreaction. Tumor grade 3 was associated with nuclear positivity and combined nuclear and cytoplasmic localization. Carcinomas larger than 20 mm showed nuclear and combined localization in 81% of cases and higher intensity of survivin immunoreaction was also notably related to larger carcinomas. Statistically significant differences were also observed between subcellular survivin localization and intensity of immunoreaction. Our result suggest that nuclear accumulation of survivin is associated with proliferative fenotype and survivin was shown to be a worse prognostic marker in breast ductal carcinoma.

Young Barley Indicates Antitumor Effects in Experimental Breast Cancer In Vivo and In Vitro.

The effect of dietary administered young barley containing a mixture of phytochemicals to female rats for the prevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis was evaluated. After carcinogen administration (14 wk), mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation of possible mechanisms in MCF-7 breast cancer cell line was performed. Barley (0.3%) demonstrated mild antitumor effect in mammary carcinogenesis, yet 3% barley did not further improve this effect. Immunohistochemical analysis of rat tumor cells in treated groups showed significant increase in caspase-3 expression and significant reduction in Ki67 expression. In addition, 3% barley significantly decreased dityrosine levels versus control. Barley in higher dose significantly decreased serum low-density lipoprotein-cholesterol in rats. In vitro studies showed that barley significantly decreased survival of MCF-7 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and significantly decreased 5-bromo-20-deoxyuridine incorporation versus control. Barley prevented cell cycle progression and extended incubation with barley showed significant increase in the percentage of annexin V/propidium iodide-positive MCF-7 cells. Our results propose an antitumor effect for the mixture of phytochemicals present in young barley in a breast cancer model.

Fruit peel polyphenols demonstrate substantial anti-tumour effects in the model of breast cancer.

PURPOSE: Fruit and vegetable intake is inversely correlated with cancer; thus, it is proposed that an extract of phytochemicals as present in whole fruits, vegetables, or grains may have anti-carcinogenic properties. Thus, the anti-tumour effects of fruit peel polyphenols (Flavin7) in the chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. METHODS: Lyophilized substance of Flavin7 (F7) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration, and mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. In addition, using an in vitro cytotoxicity assay, apoptosis and proliferation after F7 treatment in human breast adenocarcinoma (MCF-7) cells were performed. RESULTS: High-dose F7 suppressed tumour frequency by 58 % (P < 0.001), tumour incidence by 24 % (P < 0.05), and lengthened latency by 8 days (P > 0.05) in comparison with the control rats, whereas lower dose of F7 was less effective. Histopathological analysis of tumours showed significant decrease in the ratio of high-/low-grade carcinomas after high-dose F7 treatment. Immunohistochemical analysis of rat carcinoma cells in vivo found a significant increase in caspase-3 expression and significant decrease in Bcl-2, Ki67, and VEGFR-2 expression in the high-dose group. Both doses demonstrated significant positive effects on plasma lipid metabolism in rats. F7 significantly decreased survival of MCF-7 cells in vitro in MTT assay by dose- and time-dependent manner compared to control. F7 prevented cell cycle progression by significant enrichment in G1 cell populations. Incubation with F7 showed significant increase in the percentage of annexin V-/PI-positive MCF-7 cells and DNA fragmentation. CONCLUSIONS: Our results reveal a substantial tumour-suppressive effect of F7 in the breast cancer model. We propose that the effects of phytochemicals present in this fruit extract are responsible for observed potent anti-cancer activities.

Differential expression of doublecortin and microglial markers in the rat brain following fractionated irradiation.

Ionizing radiation induces altered brain tissue homeostasis and can lead to morphological and functional deficits. In this study, adult male Wistar rats received whole-body exposure with fractionated doses of gamma rays (a total dose of 5 Gy) and were investigated 30 and 60 days later. Immunohistochemistry and confocal microscopy were used to determine proliferation rate of cells residing or derived from the forebrain anterior subventricular zone (SVZa) and microglia distributed along and/or adjacent to subventricular zone-olfactory bulb axis. Cell counting was performed in four anatomical parts along the well-defined pathway, known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm of the RMS. Different spatiotemporal distribution pattern of cell proliferation was seen up to 60 days after irradiation through the migratory pathway. A population of neuroblasts underwent less evident changes up to 60 days after treatment. Fractionated exposure led to decline or loss of resting as well as reactive forms of microglia until 60 days after irradiation. Results showed that altered expression of the SVZa derived cells and ultimative decrease of microglia may contribute to development of radiation-induced late effects.