Marzena Podhorska-Okolow

Melatonin stimulates the activity of protective antioxidative enzymes in myocardial cells of rats in the course of doxorubicin intoxication.

Abstract: The study aimed at determining the effect of melatonin on the activity of protective antioxidative enzymes in the heart and of lipid peroxidation products in the course of intoxication with doxorubicin (DOX). The rats were categorized into four groups, receiving: 0.9% NaCl i.p. (NaCl control); melatonin [20 mg/kg body weight (b.w.)] s.c. (control Mel); DOX (2.5 mg/kg b.w.) i.p.; melatonin plus DOX in doses as above. All the substances were administered once in a week for four consecutive weeks. Homogenates of heart tissue were examined for activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), levels of reduced glutathione (GSH) and of lipid peroxidation indices (MDA + 4‐HDA). Administration of melatonin alone did not induce alterations in levels of MDA + 4‐HDA, GSH, or in activity of GPx, SOD or CAT, as compared to the group receiving 0.9% NaCl. GSH levels decreased following DOX but remained at normal levels following DOX and melatonin. The level of MDA + 4‐HDA increased following DOX, as compared with the control, a change prevented by the combination of DOX+melatonin. Activities of GPx, SOD and CAT were higher in groups receiving DOX and/or DOX plus melatonin than in control groups. Activity of CAT and the level of GSH in the group receiving DOX plus melatonin were significantly higher than in the group intoxicated with DOX alone. The obtained results demonstrate that, when given in parallel with DOX, melatonin protects cardiomyocytes from damaging effects of the cytostatic drug (reflected by the levels of MDA + 4‐HDA). The protective effect resulted, in part from the augmented levels of GSH and from stimulation of CAT activity by melatonin in cardiomyocytes subjected to the action of DOX.

Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska‐Okolow M 
(2011) Histopathology 59, 1249–1260 
Podoplanin expression by cancer‐associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma

Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

Background:It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level.Methods:Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting.Results:Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann–Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann–Whitney U, P<0.01) as well as G3 and G1 (Mann–Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann–Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the ‘luminal epithelial-like’ phenotype did not express or weakly expressed UGT8, in contrast to malignant, ‘mesenchymal-like,’ cells forming metastases in nude mice.Conclusion:Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

Long term potentiation affects intracellular metalloproteinases activity in the mossy fiber — CA3 pathway

Matrix Metalloproteinases (MMPs) are a family of endopeptidases known to process extracellular proteins. In the last decade, studies carried out mainly on the Schaffer collateral-CA1 hippocampal projection have provided solid evidence that MMPs regulate synaptic plasticity and learning. Recently, our group has shown that MMP blockade disrupts LTP maintenance also in the mossy fiber-CA3 (mf-CA3) projection (Wojtowicz and Mozrzymas, 2010), where LTP mechanisms are profoundly different (NMDAR-independent and presynaptic expression site). However, how plasticity of this pathway correlates with activity and expression of MMPs remains unknown. Interestingly, several potential MMP substrates (especially of gelatinases) are localized intracellularly but little is known about MMP activity in this compartment. In the present study we have asked whether LTP is associated with the expression and activity of gelatinases in apparent intra- and extracellular compartments along mf-CA3 projection. In situ zymography showed that LTP induction was associated with increased gelatinases activity in the cytoplasm of the hilar and CA3 neurons. Using gelatin zymography, immunohistochemistry and immunofluorescent staining we found that this effect was due to de novo synthesis and activation of MMP-9 which, 2-3h after LTP induction was particularly evident in the cytoplasm. In contrast, MMP-2 was localized preferentially in the nuclei and was not affected by LTP induction. In conclusion, we demonstrate that LTP induction in the mf-CA3 pathway correlates with increased expression and activity of MMP-9 and provide the first evidence that this increase is particularly evident in the neuronal cytoplasm and nucleus.

Correlation between metallothionein (MT) expression and selected prognostic factors in ductal breast cancers

Our study aimed at examining significance of metallothionein (MT) expression in ductal breast cancers by determination of a relationship between expression of MT protein (MT-1/2) and selected prognostic factors, including grade of histological differentiation (G), expression of Ki-67 proliferative antigen, expression of estrogen receptors (ER) and progesterone receptors (PgR) and expression of HER-2 receptor. Material for the studies involved 54 samples of invasive ductal breast cancer, manifesting malignancy grades of G1-G3. In paraffin sections of examined tumours immunohistochemical reactions were performed using specific antibodies directed to MT, Ki-67, ER, PgR or HER-2. Intensity of MT-specific immunohistochemical reactions was measured using the semiquantitative IRS scale of Remmele. Intensity of colour reactions targeted at Ki-67, ER, PgR was evaluated scoring proportions of positive cells, while HER-2-specific reactions were evaluated in the scale of 0-3 points. The lowest level of MT expression was detected in breast cancer cases of G1 malignancy grade (G1 vs G3 p=0.020). A positive correlation between MT and Ki-67 antigen expression (r=0.32, p=0.019) was disclosed. Moreover, MT expression exhibited negative correlations with expression of ER (r=-0.35, p=0.008) and PgR (r=-0.27, p=0.046). No relationships could be detected between expression of MT and expression of HER-2 (r=0.12, p=0.37). The obtained results suggest that MT expression might be helpful in prognostic evaluation of ductal breast cancers.

alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

Benzylpenicillin, acetylcysteine and silibinin as antidotes in human hepatocytes intoxicated with α-amanitin

Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to alpha-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to alpha-AMA at concentration 2microM. Exposure of the cells to alpha-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to alpha-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in alpha-AMA toxicity.

Early morphological and functional alterations in canine hepatocytes due to α-amanitin, a major toxin of Amanita phalloides.

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of α-amanitin (α-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of α-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

Correlation of Ki-67 and MCM-2 proliferative marker expression with grade of histological malignancy (G) in ductal breast cancers.

The study aimed at examining a relationship between expression of Ki-67 antigen and minichromosome maintenance 2 protein (MCM-2) and a grade of histological malignancy G in ductal breast cancers. The function of widely used marker of proliferation Ki-67 is still not clear. In contrast, the MCM-2 protein is well known to play an important role in controlling the cell cycle. Both proteins represent small protein molecules, which manifest nuclear expression only during cell division of normal and neoplastic cells. Their expression is noted in several malignant tumours. These studies were conducted on 56 archival paraffin blocks of ductal breast cancers. Immunohistochemical reactions were performed using monoclonal Ki-67- and MCM-2-specific antibodies. Statistical analysis demonstrated a positive correlation between expressions of two proteins (r=0.6; p<0.05). The most intense expression of these two markers was demonstrated in G3 grade cancers. Statistical analysis showed more pronounced expression of Ki-67 antigen in G3 grade cancers as compared to cancers of G1 and G2 grades (p<0.001) and, in the case of MCM-2 protein, a more pronounced expression in G3 grade cancers, as compared to those of G1 (p<0.05) or G2 grade (p<0.01). The results obtained in our study suggest that MCM-2 could be used as a marker of proliferation in breast carcinomas.

Angiotensin II type 1 receptor (AT-1R) expression correlates with VEGF-A and VEGF-D expression in invasive ductal breast cancer.

Recent studies point to the involvement of angiotensin II (Ang II) receptor type 1 (AT-1R) on processes of metastasing, stimulation of invasiveness and angiogenesis in tumours. In this study, the correlation between intensity of AT-1R expression and expression of lymph- and angiogenesis markers in invasive ductal breast cancers (IDC) was examined. Immunohistochemical studies (IHC) were performed on archival material of 102 IDC cases. Only 28 (27.5%) cases manifested low AT-1R expression while 74 (72.5%) cases demonstrated a moderate or pronounced AT-1R expression. Expression intensity of AT-1R was found to correlate with expressions of VEGF-A (r = 0.26; p = 0.008) and VEGF-D (r = 0.24; p = 0.015). Out of the examined markers of angiogenesis and lymphangiogenesis only the pronounced expression of VEGF-C was found to correlate with patient poor clinical outcome (p = 0.009). The positive correlation between AT-1R and VEGF-A and VEGF-D could point to stimulatory action of Ang II on their expression what might result in augmented lymph- and angiogenesis in IDC.