This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.
Uvod: Na experimentalnich modelech poskozeni periferniho nervu byly prokazany projevy zanětu ve strukturach periferni i centralni nervove soustavy. Předpokladame, že produkty Wallerovy degenerace mohou ovlivnit struktury nervove soustavy cestou hematolikvorove bariery anatomicky tvořene plexus choroideus (CP) mozkových komor. Buňky CP mohou na poskozeni periferniho nervu reagovat produkci zanětových mediatorů sloužicich k chemotaxi imunitnich buněk. Cilem nasi prace je popsat reakci buněk CP na chronicke poskozeni periferniho nervu. Pro vyjadřeni dynamiky invaze imunitnich buněk v zavislosti na delce poskozeni nervu jsme použili fluorescencně znacený dextran FluoroEmerald (FE) s různou dobou cirkulace. Metody: Experimenty byly provedeny na 20 laboratornich potkanech rodu Wistar (samci, 250 – 300 g). Anestezie byla provedena intraperitonealni aplikaci ketaminu a xylazinu. Na zviřatech byla provedena trojita unilateralni ligatura nervus ischiadicus, se kterou zviřata přeživala 3 dny (1. skupina; n=8) a 21 dnů (2. skupina; n=8). Třeti skupina zviřat (n=4) byla kontrolni bez poskozeni nervu. Každa z těchto tři skupin byla rozdělena do dvou podskupin podle doby cirkulace FE, ktera byla 5 nebo 18 hodin. Po uvedene době trvani poskozeni nervu byl zviřatům intravenozně aplikovan FE do vena jugularis externa sinistra. Po 5 nebo 18 hodinove cirkulaci FE byla zviřata usmrcena inhalaci CO2, perfundovana přes aortu fyziologickým roztokem s heparinem a nasledně Zamboniho fixacnim roztokem. Pote byl odebran mozek, který byl ponechan tři dny ve fixacnim roztoku. Po oplachovani v 10% sacharoze ve fosfatovem pufru byly zhotoveny kryostatove koronalni řezy o tlousťce 20 µm v urovni třeti a lateralni komory. Na kryostatových řezech byla sledovana distribuce FE v buňkach CP. Buňky vykazujici FE pozitivitu byly kvantifikovany poctem pozitivnich buněk na jednotku plochy CP. Na vybraných řezech byla provedena imunohistochemicka detekce rezidentnich makrofagů (ED2), aktivovaných makrofagů (ED1), dendritických buněk (OX-42), T-lymfocytů (OX-52) a antigen prezentujicich buněk (MHC II). Řezy byly vyhodnoceny ve fluorescencnim mikroskopu. Výsledky: Partikule FE byly pozorovany v kuboidalnich buňkach CP, imunitnich buňkach stromatu a epiplexových Kolmerových buňkach. Ve srovnani s naivnimi zviřaty byl statisticky významný vzestup poctu FE+ buněk zjistěn v CP operovaných zviřat po 18 hodinove, ale ne po 5 hodinove cirkulaci FE. Nejvice FE+ buněk bylo nalezeno v CP zviřat 2. skupiny po 18 hodinach cirkulace. Partikule FE byly zjistěny v cytoplazmě stromatalnich buněk CP, ktere vykazovaly imunofenotyp aktivovaných makrofagů (ED1+) a antigen prezentujicich buněk (MHCII+). Buňky, ktere vykazovaly imunofluorescenci s protilatkou OX42 (dendriticke buňky) nebyly soucasně pozitivni na FE. Pocet dendritických buněk v CP se zvysoval s delkou trvani komprese nervu. Kolmerovy buňky s partikulemi FE v cytoplasmě vykazovaly rovněž pozitivni reakci s protilatkami ED2 a OX-52. Naproti tomu Kolmerovy buňky, ktere neobsahovaly FE partikule, vykazovaly pozitivni imunohistochemickou reakci na ED1, MHCII a OX-42. Zavěr: CP na chronicke poskozeni periferniho nervu odpovida změnami, ktere jsme sledovali přitomnosti a imunofenotypizaci FE+ buněk ve stromatu CP. Na zakladě těchto změn lze předpokladat, že CP je možnou cestou, kterou se mohou siřit do centralni nervove soustavy prozanětlive molekuly vznikajici při poskozeni periferniho nervu. K těmto změnam dochazi na rozdil od ganglii spinalnich nervů až po delsi době poskozeni periferniho nervu.
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.
Background. Investigation of p53 immunoreactivity in formalin-fixed paraffin-embedded tissues of normal renal tissue and renal cell carcinoma with respect to histopathologic subtype and nuclear grade of RCC. Methods. 42 tissue sections of RCC and 5 samples of normal renal tissue were stained for p53 expression using immunohistochemical assay. The results were analyzed in relation to nuclear grade and histopathologic subtype. Results. In total, p53 expression was found to be 4 to 5 times higher (30.8%) in other types of RCC than in the clearcell type of RCC (6.9%). Further, there was no statistically significant difference in p53 overexpression among the histopathologic subtypes (P>0.05, P=0.063). No association was found between the expression of p53 and nuclear grade (P>0.05, P=0.17). Interestingly, our study also showed weak cytoplasmic positivity in renal tubular epithelium. Conclusion. Our findings suggest that p53 might play an important role in tumour development or progression and it might be used as a new predictor and therapeutic target for RCC.
The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro‑stimulating and anti‑apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non‑specific anti‑EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far‑western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix‑assisted laser desorption/ionization‑time‑of‑flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines.
Metabolic profile is an important biological marker of neoplastic processes not only in the tumor itself but also in the host organism. The neurohormone melatonin has been implicated in experiments as an oncostatic agent. Female Wistar:Han SPF rats (Velaz, Prague, Czech Republic) were irradiated continuously for 15 days using a daily gamma rays dose of 96 mGy. At the end of exposure one group of rats was administered 5 mg/kg b.w. of dimethylbenz/a/anthracene (DMBA) intragastrically. During the period of exposure to ionizing radiation a part of the animals was supplied with melatonin (M) at a concentration of 20 microliters/ml in drinking water. Selected parameters of lipid and carbohydrate metabolisms and levels of selected hormones were determined 2, 30 and 100 days post-irradiation. The irradiation itself caused only small changes in tissue lipids. The application of a single low dose (subthreshold from the point of view of induction of mammary tumors) of DMBA caused more pronounced changes in nonirradiated animals; of the changes observed an increase in lipids in the liver, triacylglycerols (TG) in the thymus and decrease in myocardial glycogen predominated. The intake (by drinking) of exogenous M prevented the biochemical pattern of fatty liver in animals administered DMBA in both groups, irradiated and nonirradiated. A prolonged effect of exogenous M, demonstrated by prevention of increase in TG in the thymus and of irradiated animals caused by administration of DMBA, was observed. The mechanism of metabolic effect of M is not known. Additional experiments are needed to explain the relationship between the beneficial effect of M on metabolic changes and its presumable oncostatic effect in rats.
