Agnieszka Gomulkiewicz

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.

Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyte culture intoxicated with α-amanitin

α-Amanitin (α-AMA) is the main toxin of Amanita phalloides and its subspecies (A. virosa and A. verna). The primary mechanism of α-AMA toxicity is associated with protein synthesis blocking in hepatocytes. Additionally, α-AMA exhibits prooxidant properties that may contribute to its severe hepatotoxicity. The aim of the present study was to assess the effect of α-AMA on lipid peroxidation and the activities of superoxide dismutase (SOD) and catalase (CAT) in human hepatocyte culture. The effects of benzylpenicillin (BPCN), N-acetyl-L-cysteine (ACC), and silibinin (SIL) on SOD and CAT activities and on lipid peroxidation in human hepatocyte culture intoxicated with α-AMA were also examined. In human hepatocyte culture, 48-hour exposure to α-AMA at a 2-μM concentration caused an increase in SOD activity, a reduction of CAT activity, and a significant increase in lipid peroxidation. Changes in SOD and CAT activity caused by α-AMA could probably enhance lipid peroxidation by increased generation of hydrogen peroxide combined with reduced detoxification of that oxygen radical. The addition of antidotes (ACC or SIL) to the culture medium provided more effective protection against lipid peroxidation in human hepatocytes intoxicated with α-AMA than the addition of BPCN, possessing no antioxidant properties.

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.

Influence of ezetimibe on ADMA-DDAH-NO pathway in rat liver subjected to partial ischemia followed by global reperfusion

BACKGROUND We evaluated effect of ezetimibe on selected parameters determining NO level in rat liver subjected to ischemia reperfusion (IR). METHODS Rats received ezetimibe (5 mg/kg) (groups E0 and EIR) or saline solution (groups C0 and CIR) intragastrically for 21 days. Then, the livers of CIR and EIR underwent ischemia (60 min) and reperfusion (4 h). Blood samples were obtained before surgery to estimate activities of aminotransferases, and just before ischemia and during reperfusion to estimate asymmetric and symmetric dimethylarginine (ADMA, SDMA) and arginine (Arg) levels. After IR, dimethylarginine dimethylaminohydrolase (DDAH) activity and endothelial nitric oxide synthase (eNOS) protein concentration were measured in liver homogenates. DDAH and protein arginine methyltransferase (PRMT) mRNA were quantified by real-time PCR in liver tissue samples. RESULTS In CIR, the ADMA level was significantly higher compared to all other groups in 30 min and to E0 group in 120 min of reperfusion. In EIR, ADMA was low, compared to non-ischemic groups. At 30 and 120 min of reperfusion, in non-ischemic groups the level of Arg and Arg/ADMA ratio were significantly higher than in ischemic groups and E0 was the group with the highest levels of those parameters of all. In CIR, eNOS protein concentration was significantly lower than in ezetimibe-treated groups. Activity of DDAH was significantly higher in E0 than in non-treated groups. In ischemic groups, DDAH mRNA expression was significantly higher than in non-ischemic ones and PRMT mRNA expression was significantly higher in C0 than in all other groups. CONCLUSIONS Influence of ezetimibe on ADMA/DDAH/NO pathway demonstrated in this work may suggest protective properties of this drug on rat livers injured by IR and, to a lower extent, on livers non-subjected to IR.

The Impact of Melatonin on Colon Cancer Cells’ Resistance to Doxorubicin in an in Vitro Study

Multi-drug resistance (MDR) is the main cause of low effectiveness of cancer chemotherapy. P-glycoprotein (P-gp) is one of the main factors determining MDR. Some studies indicate the potential role of melatonin (MLT) in MDR. In this study, we examined the effect of MLT on colon cancer cell’s resistance to doxorubicin (DOX). Using the sulforhodamine B (SRB), method the effect of tested substances on the survival of LoVo (colon cancer cells sensitive to DOX) and LoVoDX (colon cancer cells resistant to DOX) was rated. Using immunocytochemistry (ICC), the expression of P-gp in the LoVo and LoVoDX was determined. With the real-time PCR (RT-PCR) technique, the ABCB1 expression in LoVoDX was evaluated. Based on the results, it was found that MLT in some concentrations intensified the cytotoxicity effect of DOX in the LoVoDX cells. In the ICC studies, it was demonstrated that certain concentrations of MLT and DOX cause an increase in the percentage of cells expressing P-gp, which correlates positively with ABCB1 expression (RT-PCR). The mechanism of overcoming resistance by MLT is probably not only associated with the expression of P-gp. It seems appropriate to carry out further research on the use of MLT as the substance supporting cancer chemotherapy.