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The role of periostin (POSTN) in remodeling the microenvironment surrounding solid tumors and its effect on the tumor cells in non-small-cell lung carcinoma (NSCLC) have not yet been fully understood. The aim of this study was to determine the relationship between POSTN expression (in tumor cells [NSCLC cells] and the tumor stroma) and pro-angiogenic factors (CD31, CD34, CD105, and VEGF-A) and microvascular density (MVD) in NSCLC. In addition, these associations were analyzed in individual histological subtypes of NSCLC (SCC, AC, and LCC) and their correlations with clinicopathological factors and prognosis were examined. Immunohistochemistry using tissue microarrays (TMAs) was used to assess the expression of POSTN (in tumor cells and cancer-associated fibroblasts [CAFs]) and the pro-angiogenic factors. A significant positive correlation was found between the expression of POSTN (in cancer cells/CAFs) and the expression of the analyzed pro-angiogenic factors (CD31, CD34, CD105, and VEGF-A) and MVD in the entire population of patients with NSCLC and individual histological subtypes (AC, SCC). In addition, this study found that POSTN expression (in tumor cells/CAFs) increased with tumor size (pT), histopathological grade (G), and lymph-node involvement (pN). In addition, a high expression of POSTN (in tumor cells and CAFs) was associated with shorter survival among patients with NSCLC. In conclusion, a high expression of POSTN (in cancer cells and CAFs) may be crucial for angiogenesis and NSCLC progression and can constitute an independent prognostic factor for NSCLC.

Abstract We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant chemotherapy (doxorubicin and cyclophosphamide), which suggests that the absence of this glycoprotein is associated with resistance of tumor cells to chemotherapy. Therefore, in the present study, we analyzed the impact of PIP expression on resistance of BC cells to anti-cancer drugs and its biological role in BC progression. Expression of PIP and apoptotic genes in BC cell lines was analyzed using real-time PCR and Western blotting. PIP was detected in BC tissue specimens using immunohistochemistry. The tumorigenicity of cancer cells was analyzed by the in vivo tumor growth assay. Apoptotic cells were detected based on caspase-3 activation, Annexin V binding and TUNEL assay. The interaction of PIP with BC cells was analyzed using flow cytometry. Using two cellular models of BC (i.e. T47D cells with the knockdown of the PIP gene and MDA-MB-231 cells overexpressing PIP), we found that high expression of PIP resulted in (1) increased sensitivity of BC cells to apoptosis induced by doxorubicin (DOX), 4-hydroperoxycyclophosphamide (4-HC), and paclitaxel (PAX), and (2) improved efficacy of anti-cancer therapy with DOX in the xenograft mice model. Accordingly, a clinical study revealed that BC patients with higher PIP expression were characterized by longer 5-year overall survival and disease-free survival. Subsequent studies showed that PIP up-regulated the expression of the following pro-apoptotic genes: CRADD, DAPK1, FASLG, CD40 and BNIP2. This pro-apoptotic activity is mediated by secreted PIP and most probably involves the specific surface receptor. This study demonstrates that a high expression level of PIP sensitizes BC cells to anti-cancer drugs. Increased sensitivity to chemotherapy is the result of pro-apoptotic activity of PIP, which is evidenced by up-regulation of specific pro-apoptotic genes. As high expression of PIP significantly correlated with a better response of patients to anti-cancer drugs, this glycoprotein can be a marker for the prognostic evaluation of adjuvant chemotherapy.

The tumor microenvironment is considered one of the main players in cancer development and progression and may influence the behavior of cancer cells. Periostin (POSTN) is an extracellular matrix protein, and its main functions are induction of fibrillogenesis, fibroblastic cell proliferation and migration, enhancing regeneration in normal tissue, and promoting metastasis in case of neoplasia. POSTN has already been studied in humans in several normal tissues, inflammatory processes, and neoplasms, revealing an important role in tumor progression in various types of cancer, such as colon, lung, head and neck, breast, ovarian, and prostate. In these latter, high levels of POSTN are usually associated with a more aggressive tumor behavior, tumor advanced stages, and poor prognosis, while in human bladder urothelial carcinoma (BUC), unlike in most tumors, POSTN expression seems to be downregulated. The expression of this marker has been poorly investigated in veterinary medicine; thus, this study aimed to immunohistochemically investigate the presence and the intensity of POSTN expression in canine BUCs and to determine a possible relationship between POSTN expression and histopathological features such as mitotic count and muscular and vascular invasions. For the present retrospective study, archived samples from 45 canine BUCs and 6 non-neoplastic canine bladders were considered for histological evaluation and immunohistochemical examination for the expression of POSTN. POSTN expression was semi-quantitatively assessed considering both the percentage of the neoplastic stroma positive for POSTN and the intensity of the immunohistochemical labeling. Histologically, 38 out of 45 tumors were papillary and 7 out of 45 were non-papillary. All tumors were infiltrating, being that 21 were muscle-invasive, and a significant correlation between this feature and vascular invasion emerged (P = 0.0001). In normal bladder tissue, as reported in humans, a thick and strongly positive belt of POSTN was visible, and in canine BUCs, stating that the expression is comparable with human benign as well as malignant bladder tissue, a general decrease in POSTN expression was observed except for a strongly labeled ring of POSTN observed around some neoplastic nodules infiltrating the muscle layer. Moreover, POSTN expression and mitotic count were significatively inversely correlated (P = 0.0015). The fact that POSTN protein is less expressed in urothelial carcinomas than in the normal bladder supports what was reported in human BUCs and, together with the negative correlation between mitotic count and protein expression that emerged in the present retrospective study, encourages further prospective follow-up studies to verify the possible role of POSTN in canine BUCs as a prognostic marker, and also as a possible target for the development of future anticancer therapies.

Testin is a protein expressed in normal human tissues, being responsible, with other cytoskeleton proteins, for the proper functioning of cell–cell junction areas and focal adhesion plaques. It takes part in the regulation of actin filament changes during cell spreading and motility. Loss of heterozygosity in the testin-encoding gene results in altered protein expression in many malignancies, as partly described for cervical cancer. The aim of our study was the assessment of the immunohistochemical (IHC) expression of testin in cervical cancer and its analysis in regard to clinical data as well the expression of the Ki-67 antigen and p16 protein. Moreover, testin expression was assessed by Western blot (WB) in commercially available cell lines. The IHC analysis disclosed that the expression of testin inversely correlated with p16 (r = −0.2104, p < 0.0465) and Ki-67 expression (r = −0.2359, p < 0.0278). Moreover, weaker testin expression was observed in cancer cases vs. control ones (p < 0.0113). The WB analysis of testin expression in the cervical cancer cell lines corresponded to the IHC results and showed a weaker expression compared to that in the control cell line. When we compared the expression of testin in cervical cancer cell lines, we found a weaker expression in HPV-negative cell lines. In summary, we found that the intensity of testin expression and the number of positive cells inversely correlated with the expression of Ki-67 (a marker of proliferation) and p16 (a marker of cell cycle dysregulation). This study shows that the combined assessment of testin, Ki-67 and p16 expression may improve cervical cancer diagnostics.

Background: The potential involvement of zyxin (ZYX) in carcinogenesis has been investigated in many cancer types. However, there are a limited number of studies on the role of ZYX in the progression of non-small cell lung cancer (NSCLC). Since lung cancer is one of the most frequently diagnosed carcinomas, the aim of our study was to determine the localization and expression levels of ZYX in NSCLC and to correlate the results with the clinicopathological data. Materials and Methods: The expression of ZYX was assessed in NSCLC cases and in cell lines representing this tumor type. Levels of ZYX were determined in the clinical material using immunohistochemistry (IHC) and Western Blot. Real-time PCR was used to assess ZYX mRNA levels. The expression of ZYX was also checked in NSCLC cell lines using real-time PCR, Western Blot, and immunofluorescence/immunocytochemistry. Results: The results showed lower levels of ZYX in NSCLC cells compared with control tissues. This trend was observed at the protein and mRNA levels. The assays on the NSCLC model also demonstrated lower levels of ZYX in cancer cells compared with control cells. Conclusions: The decreased expression of ZYX in NSCLC may indicate a suppressor role of this protein in NSCLC.

The main aim of this study was to examine if a female mouse body in preimplantation pregnancy can distinguish between embryos of normal and impaired biological quality in the local and peripheral compartments. Normal (control group) and TNFα (tumor necrosis factor-α)-treated embryos (experimental group) at the morula stage were non-surgically transferred into the uteri of CD-1 strain [Crl:CD1(Icr)] female murine recipients. Twenty-four hours after the embryo transfer, females were euthanised, and uteri and spleens were dissected. In uterine tissues (local compartment), we assessed the expression of 84 genes comprising nine signal transduction pathways, using a modified RT2 Profiler PCR Array. In the spleen (peripheral compartment), we determined the proteome of splenic CD4+ lymphocytes using 2D protein electrophoresis with subsequent protein identification by mass spectrometry. Sample clustering and differential gene expression analyses within individual signal transduction pathways revealed differential expression of genes in the uteri of females after transplantation of normal vs. TNFα-treated embryos. The most affected signal transduction cascade was the NFKB (Nuclear factor NF-kappa-B) pathway, where 87.5% of the examined genes were significantly differentially expressed. Proteomic analysis of splenic CD4+ T lymphocytes revealed significant differential expression of 8 out of 132 protein spots. Identified proteins were classified as proteins influenced by cell stress, proteins engaged in the regulation of cytoskeleton stabilization and cell motility, and proteins having immunomodulatory function. These results support the hypothesis that even before embryo implantation, the body of pregnant female mice can sense the biological quality of an embryo both at the local and peripheral level.

Abstract Background We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Since GalCer is a precursor molecule in the synthesis of sulfatides, the present study was aimed to define the role of sulfatides in apoptosis and breast cancer progression. Methods Expression of GAL3ST1 in breast cancer cell lines and breast cancer tissue specimens was analysed using real-time PCR, western blotting and immunohistochemistry analysis. The amount of sulfatide, GalCer and ceramide was analysed by thin-layer chromatography binding assay and by the modified hydrophilic interaction liquid chromatography coupled with electrospray mass spectrometry methodology. The tumourigenicity of cancer cells was analysed by an in-vivo tumour growth assay. Apoptotic cells were detected based on caspase-3 activation and the TUNEL assay. The interaction of breast cancer cells with P-selectin or E-selectin was analysed using the flow adhesion assay. The ability of sulfatide-expressing cells to activate and aggregate platelets was studied using the flow-cytometry-based aggregation assay. Results Using two models of breast cancer, T47D cells with blocked synthesis of sulfatide and MDA-MB-231 cells with neosynthesis of this glycosphingolipid, we showed that high sulfatide levels resulted in increased sensitivity of cancer cells to apoptosis induced by hypoxia and doxorubicin in vitro, and decreased their tumourigenicity after transplantation into athymic nu/nu mice. Accordingly, a clinical study on GAL3ST1 expression in invasive ductal carcinoma revealed that its elevated level is associated with better prognosis. Using MDA-MB-231 cells with neosynthesis of sulfatide we also showed that sulfatide is responsible for adhesion of breast cancer cells to P-selectin-expressing cells, including platelets. Sulfatide also acted as an activating molecule, increasing the expression of P-selectin. Conclusions This study demonstrates that increased synthesis of sulfatide sensitises cancer cells to microenvironmental stress factors such as hypoxia and anticancer drugs such as doxorubicin. However, sulfatide is probably not directly involved in apoptotic cascades, because its increased synthesis by GAL3ST1 decreased the amounts of its precursor, GalCer, a known anti-apoptotic molecule. On the other hand, our data support the view that sulfatides are malignancy-related adhesive molecules involved in activating and binding P-selectin-expressing platelets to breast cancer cells.

Cervical cancer is one of the most common malignant cancers in women worldwide. The 5-year survival rate is 65%; nevertheless, it depends on race, age, and clinical stage. In the oncogenesis of cervical cancer, persistent HPV infection plays a pivotal role. It disrupts the expression of key proteins as Ki-67, p16, involved in regulating the cell cycle. This study aimed to identify the potential role of testin in the diagnosis of cervical precancerous lesions (CIN). The study was performed on selected archival paraffin-embedded specimens of CIN1 (31), CIN2 (75), and CIN3 (123). Moderate positive correlation was observed between testin and Ki-67 as well as testin and p16 expression in all dysplastic lesions (r = 0.4209, r = 0.5681; p < 0.0001 for both). Statistical analysis showed stronger expression of the testin in dysplastic lesions vs. control group (p < 0.0001); moreover, expression was significantly higher in HSIL than LSIL group (p < 0.0024). In addition, a significantly stronger expression of testin was observed in CIN3 vs. CIN1 and CIN3 vs. CIN2. In our study, expression of Ki-67, p16, and testin increased gradually as the lesion progressed from LSIL to HSIL. The three markers complemented each other effectively, which may improve test sensitivity and specificity when used jointly.

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

The global epidemic of cardiovascular diseases leads to increased morbidity and mortality caused mainly by myocardial infarction and stroke. Atherosclerosis is the major pathological process behind this epidemic. We designed a novel model of atherosclerosis in swine. Briefly, the first group (11 pigs) received normal pig feed (balanced diet group-BDG) for 12 months, the second group (9 pigs) was fed a Western high-calorie diet (unbalanced diet group-UDG) for 12 months, the third group (8 pigs) received a Western type high-calorie diet for 9 months later replaced by a normal diet for 3 months (regression group-RG). Clinical measurements included zoometric data, arterial blood pressure, heart rate and ultrasonographic evaluation of femoral arteries. Then, the animals were sacrificed and the blood serum, urine and skeletal muscle tissue were collected and 1H NMR based metabolomics studies with the application of fingerprinting PLS-DA and univariate analysis were done. Our results have shown that the molecular disturbances might overlap with other diseases such as onset of diabetes, sleep apnea and other obesity accompanied diseases. Moreover, we revealed that once initiated, molecular changes did not return to homeostatic equilibrium, at least for the duration of this experiment.

Basal cell carcinoma (BCC) is the most frequent skin cancer, with many different histological subtypes.Recent studies have investigated the expression of proliferative markers, but little is known about theexpression of metallothioneins (MT) in different histological subtypes of this cancer and their impact on proliferationintensity in BCC. In this study, we examined MT-I/II expression by immunohistochemistry in 58 differenthistological subtypes of BCC (38 nodular, six adenoid, eight infiltrative, and six metatypic cases) and correlatedits expression with tumor size and Ki-67 proliferation rate. Statistical analysis revealed no significant differencesin the expression of studied markers in regard to the histological subtype. A positive correlation betweenMT and Ki-67 expression was observed for all the studied cases (r = 0.26; p = 0.049), but was evenstronger in the metatypic subtype of BCC (r = 0.85; p = 0.033). MT and Ki-67 expression did not correlate withtumor size. In conclusion, it seems that metallothioneins may have an impact on the proliferation rate of BCC,but further studies are required to determine whether MT may be a risk factor of recurrences. Basal cell carcinoma (BCC) is the most frequent skin cancer, with many different histological subtypes.Recent studies have investigated the expression of proliferative markers, but little is known about theexpression of metallothioneins (MT) in different histological subtypes of this cancer and their impact on proliferationintensity in BCC. In this study, we examined MT-I/II expression by immunohistochemistry in 58 differenthistological subtypes of BCC (38 nodular, six adenoid, eight infiltrative, and six metatypic cases) and correlatedits expression with tumor size and Ki-67 proliferation rate. Statistical analysis revealed no significant differencesin the expression of studied markers in regard to the histological subtype. A positive correlation betweenMT and Ki-67 expression was observed for all the studied cases (r = 0.26; p = 0.049), but was evenstronger in the metatypic subtype of BCC (r = 0.85; p = 0.033). MT and Ki-67 expression did not correlate withtumor size. In conclusion, it seems that metallothioneins may have an impact on the proliferation rate of BCC,but further studies are required to determine whether MT may be a risk factor of recurrences.

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

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.

Polyphenols are present in several edible plants and for many years induce high interest mainly due to their antioxidative and anti-inflammatory influence. At present, numerous studies are conducted on antineoplastic effects of the compounds. One of most effective biopolyphenols involves the flavonol quercetin. Our studies aimed at evaluation of antiproliferative and pro-apoptotic effects of quercetin alone and in combinations with daunorubicin on cells of human pancreatic carcinoma lines. The experiments were conducted on two cell lines, sensitive to daunorubicin EPP85-181P line, and its resistant variant EPP85-181RDB. Effect of studied substances on cell proliferation was detected using sulphorhodamine B (SRB) protein staining method. Apoptotic damage was estimated using comet and TUNEL techniques. Our data demonstrated that quercetin exerted cytotoxic action on cells of the both neoplastic cell lines in concentration-dependent manner. In the case of EPP85-181RDB cell line, quercetin seemed to sensitize resistant cells to daunorubicin. In parallel, the effect of both substances on the sensitive cell line was synergistic. Results of the studies confirmed that quercetin may probably break resistance of neoplastic cells to chemotherapy. On the other side, studied flavonol augmented action of cytostatic drug in case of sensitive tumour cells what suggest, that it might allow to decrease dosage of cytostatic drugs and reduce negative side effects of the treatment.

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.

Lung cancer represents one of the most frequent causes of death due to neoplastic disease in Poland and around the world. The high mortality which accompany neoplastic diseases used to be ascribed mainly to dissemination of cancerous cells. Studies on animal models suggest that tumour lymphangiogenesis represents the principal factor in the process of metastases formation. Lymphangiogenesis involves a process of formation of new lymphatic vessels from already existing lymphatic capillaries. Lymphangiogenesis is stimulated by vascular endothelial growth factors (VEGF) and other, recently reported factors, such as, e.g., cyclooxygenase 2, fibroblast growth factor 2, angiopoetin-1 and the insulin-resembling growth factor. In lymphangiogenesis a key role is played by neutropilin 2 or podoplanin and this promoted development of studies on lymphangiogenesis. Activation of VEGF-C/VEGF-D/VEGFR-3 axis increases motility and invasiveness of neoplastic cells, promotes development of metastases in several types of tumours such as, e.g., lung cancer, mammary carcinoma, cancers of the neck, prostate and large intestine. In recent years lymphangiogenesis provided topic of many studies. A positive correlation was detected between expressions of VEGF-C/D and VEGFR-3 in non-small cell lung cancer. In patients with lung cancer with high expression of VEGF-C a markedly abbreviated survival was noted. Positive correlation was detected between expression of VEGF-C and VEGF-D on one hand and expression of LYVE-1 on the other in sentinel lymph nodes with metastases of neoplastic cells in patients with non-small cell lung cancer. Also, high density of lymphatic vessels and high density of intraneoplastic microvessels proved to be independent poor prognostic indices in patients with non-small cell lung cancer. Extensive hope is linked to studies on inhibitors of lymphangiogenesis, which may improve results of treatment also in tumour patients.

P-glycoprotein (P-gp) is one of the ABC transporters responsible for the resistance of several tumours to successful chemotherapy. Numerous agents are capable of interfering with the P-gp-mediated export of drugs but unfortunately most of them produce serious side effects. Some plant polyphenols, including the flavonol quercetin (Q), manifest anti-neoplastic activity mainly due to their influence on cell cycle control and apoptosis. Reports are also available which show that Q may intensify action of cytostatic drugs and suppress the multidrug resistance (MDR) phenomenon. The study aimed at determination if Q sensitizes cells resistant to daunorubicin (DB) through its effect on P-gp expression and action. The experiments were conducted on two cell lines of human pancreatic carcinoma, resistant to DB EPP85-181RDB and sensitive EPP85-181P as a comparison. Cells of both lines were exposed to selected concentrations of Q and DB, and then membranous expression of P-gp and its transport function were examined. The influence on expression of gene for P-gp (ABCB1) was also investigated. Results of the studies confirmed that Q affects expression and function of P-gp in a concentration-dependent manner. Moreover it decreased expression of ABCB1. Thus, Q may be considered as a potential modulator of P-gp.

Contents of estrogen receptors (ER) and progesterone receptors (PgR) in cells of breast cancers represent strong predictive factors. The higher is the contents of ER and PgR in breast cancer, the higher is a probability of obtaining a response to hormonal therapy and prognosis for the patient is better. In a routine manner, all tumours of mammary gland are subjected to evaluation of ER and PgR expression using immunohistochemistry. Forty ductal breast cancers (pT2N0) were subjected to an immunohistochemical evaluation (IHC) aimed at detection of ER and PgR expression. From every tumour three samples were taken for immunohistochemical studies: the lateral one from the side of axilla (ER-1; PgR-1); the median one (ER-2; PgR-2) and the medial one from the side of sternum (ER-3; PgR-3). The levels of both ER and PgR expression proved to be highly differentiated between the medial zone of the tumour and its periphery. The distinct expression of ER and PgR in ductal breast cancers, dependent on evaluated zone of the tumour, confirms its heterogeneous character and exerts an effect on the type of applied treatment.

OBJECTIVESPresence of lymphatics in adventitia of major arteries remains controversial. Presence of lymphatics in adventitia of internal carotid artery was not documented and its relation to atherosclerosis was not studied. The aim of our study was to evaluate presence of lymphatic vessels in adventitia of internal carotid artery in healthy and atherosclerotic arteries.METHODSFragments of arterial wall of internal carotid artery were obtained during the surgical eversion endarterectomy in 15 patients with internal carotid artery stenosis and 2 healthy organ donors. 21 arteries were studied. Patients age ranged from 56 to 77 years. Fragments of arterial wall were embeded in paraffin. Lymphatics of arterial adventitia were visualized with immunohistochemistry using LYVE-1 and anty-podoplanin antibodies.RESULTSThe lymphatic vessels were visualized in adventitia of 20 carotid arteries. The serial sections have revealed that both LYVE-1 and podoplanin have identical specificity for lymphatic endothelium Number of lymphatics in adventitia significantly correlated with thickness of intima (pCONCLUSIONSLymphatics are present in adventitia of internal carotid artery. Number of adventitial lymphatics increases with severity of atherosclerosis measured as intimal thickness.

In progression of tumours, resulting from, i.e., release of cells from the parental tumour and development of metastases, expression of cell adhesion molecules (CAM) plays a significant role. CAM, including E-cadherin and the linked to it beta-catenin, determine the extent of adhesion between normal and neoplastically altered cells. Moreover, the unbound form of beta-catenin in a cell nucleus may affect the rate of cell proliferation This study aimed at demonstrating intensity and localisation of E-cadherin and beta-catenin expression as related to expression of the proliferation-associated antigen, Ki-67 in mammary adenocarcinomas of bitches. The study was performed on 35 cases of the above mentioned tumours. On paraffin sections immunohistochemical reactions were performed using monoclonal antibodies directed against E-cadherin, beta-catenin and Ki-67 antigen. In the studies a membranous expression of E-cadherin, a cytoplasmic-nuclear expression of beta-catenin and nuclear expression of Ki-67 antigen were demonstrated. Statistical calculations using Spearman's test demonstrated a pronounced positive correlation between expression of beta-catenin and Ki-67 antigen and absence of correlation between expression of E-cadherin and Ki-67 antigen. No correlation could be detected between expression intensities of E-cadherin and beta-catenin.

Using immunohistochemistry, Fas/Apo-1 protein expression was investigated in thyroid cancers of 67 patients. Thyroid biopsies from twenty eight patients with benign thyroid diseases were also examined. The patients with thyroid cancer manifested a variable histology of the cancer, including 14 patients with follicular carcinoma, 48 with papillary carcinoma, 5 patients with medullary carcinoma. The benign thyroid disease involved nodular goitre in 11 patients and follicular adenoma in other 17 patients. The study aimed at examining immunohistochemical expression of Fas protein in order to determine whether the level of its expression correlated with histological diagnosis. In individual patients Fas expression was more prevalent in thyroid carcinomas as compared to benign tumors (p=0.001). A marked increase in Fas expression was found in papillary carcinoma, as compared to follicular and medullary carcinomas (p=0.02). In conclusion, Fas was significantly more frequently overexpressed in thyroid cancer, indicating its role in thyroid tumorigenesis.

Langerhans cell histiocytoses (LCH) represent rare diseases of unclear etiology and pathogenesis. Most of the cases include children, 1 to 15 years of age, and various organs are involved (bones, skin, liver, lymph nodes, bone marrow and other). The diagnosis of LCH used to be established by biopsy of the inflamed tissue and demonstration of expression of markers specific for Langerhans cells: CD1a and langerin. The diagnosis can be ultimately confirmed by demonstration of Birbeck's granules in the electron microscopy. The present study was aimed at immunocytochemical demonstration, in the examined LCH material (skin, bones, lymph nodes), of the specific antigen expression and at comparing it with the presence of Birbeck's granules. In the examined 11 cases co-expression of CD1a with langerin and with the presence of Birbeck's granules was noted. Also in all examined biopsies the expression of S-100 protein on inflammatory cells was found. The results corroborate the usefulness of immunocytochemical studies on CD 1 a and langerin expression in diagnosis of LCH.

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

The induction of exercise-induced apoptosis in not actively involved in exercise organs, such as kidney could be a result of oxidative stress. Metallothionein (MT) exerts a protective effect in the cell against oxidative stress and apoptosis. We have previously demonstrated an increased incidence of apoptosis in distal tubular cells and collecting ducts in rat kidney after acute exercise. The present study was designed to test the hypothesis that MT may play a protective role in rat renal tubules against exercise-induced apoptosis after the acute exercise and regular training. Male Wistar rats were divided into control, acute exercised and 8-wk regularly trained groups. The kidneys were removed after a rest period of 6 h and 96 h. The ultrastructure of renal tubular cells was examined by electron microscopy. Apoptosis was detected in paraffin sections by the TUNEL technique. Expression of MT was examined by immunohistochemistry. The level of lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was assayed in renal tissue homogenates. After acute exercise, the occurrence of apoptosis was restricted to distal tubules and collecting ducts of rat kidney, whereas the proximal tubules remained unaffected. The 8-wk training did not result in increased apoptosis in tubular cell. MT expression was confined exclusively to proximal tubules in all groups. However, it was significantly increased in acutely exercised animals, as compared to control and trained rats. After the 8-wk training, MT expression remained unaltered as compared to the control group. TBARS levels were significantly increased after acute exercise, while after regular training they remained unchanged. A significant correlation between TBARS level and MT expression was demonstrated. The findings could suggest a protective role of MT against oxidative stress and apoptosis in proximal tubular cells.

Lymphatic vasculature in solid tumors may serve as the pathway for metastatic spread of the cancer to the regional lymph nodes and to distant organs. Controversy still exists whether tumors metastasize through existing lymphatics or through newly formed vessels (lymphangiogenesis). The role of lymphangiogenesis in lymphoma spread and proliferation is not clearly established. VEGF-C is the most potent inducer of lymphangiogenesis. LYVE-1 was shown to be a specific marker for lymphatic vessels in normal and tumor tissue. The aim of the present study was the evaluation of lymph node LYVE-1-positive lymphatic sinus density (LSD) and VEGF-C expression in patients with non-Hodgkin's lymphoma (nHL) and in reactive lymph nodes. Sixty paraffin-embedded lymph nodes from newly diagnosed patients with B-cell nHL were evaluated. Twelve lymph node biopsy specimens from adult patients with reactive lymphonodulitis were used as controls. Sections of lymph nodes were stained immunohistochemically for LYVE-1 and VEGF-C. VEGF-C expression in lymph nodes of nHL patients was low and not significantly different from that in the control (p = 0.6). Moreover, VEGF-C expression did not differ significantly between aggressive and indolent lymphomas (p = 0.53). Similarly we did not find differences in LSD in aggressive nHL and in indolent nHL (p=0.49). The mean LSD in reactive lymph nodes was higher than in nHL (p = 0.03). Only in 2 out of 12 reactive lymph nodes LYVE-1-positive vessels were absent. In all groups we demonstrated a strong positive correlation between VEGF-C and LYVE-1-expression (p = 0.0001). Higher LSD in reactive lymph nodes as compared to those of nHL patients suggests that lymphoma proliferation leads to the destruction of the existing lymphatics rather than to lymphangiogenesis within lymph nodes. NHL are not associated with increased expression of VEGF-C nor increased LYVE-1-positive lymphatic sinuses density within lymph nodes.

Angiogenesis represents an important process manifested in tumour growth and in development of metastases. Using immunohistochemistry, the authors evaluated number of vessels in various nodular lesions of the thyroid (54 cases). Expression of CD34 antigen and microvessel density were evaluated in sections of archival paraffin blocks originating from the Department of Pathological Anatomy, University Medical School and the Lower Silesia Centre of Oncology in WrocĹaw, Poland. Microvessel density was assessed in ten different fields per section in "hot spots". Expression of CD34 was quantified using computerised image analysis and, then, mean micrvessel count (MVC) and microvessel area (MVA) were calculated. In thyroid tissue with benign lesions, the MVC (31.7) was higher than in neoplastic lesions (22.3), although no differences in MVA were observed. This observation points to differences in the size of newly formed vessels in individual nodular lesions of the thyroid.

Angiogenesis is important in development, maintenance and progression of haematological malignancies. Some clinical observations have indicated that in non-Hodgkin's lymphoma (nHL) tumour microvessel density (MVD) may correlate with tumour staging and outcome. The aim of the study was to examine relationship between MVD as a parameter of tumour angiogenesis measured by expression of CD34 and the grade of nHL histological malignancy as determined by REAL classification. 40 lymph node samples of patients with newly diagnosed nHL (17 women, 23 men; aged 48-70 yrs, median age 64 yrs; stage III and IV) and treated at the Department of Haematology, WrocĹaw Medical University in 1999-2002 were fixed in 10% buffered formalin and embedded in paraffin. In all the studied cases, sections were incubated with antibodies against CD34. The slides were stained with hematoxylin and eosin and evaluated histopathologically. Patients were divided into two groups according to histological malignancy: indolent nHL (19 patients) and aggressive nHL (21 patients). Mean MVD measured by expression of CD34 in aggressive and indolent nHL groups amounted to 19.45 +/- 11.24 vessels/0.375 mm2 and 21.7 +/- 12.4 vessels/0.375 mm2, respectively. Statistical analysis of microvessel staining demonstrated no correlation between tumour MVD and grade of histological malignancy in lymph nodes of nHL patients. Nevertheless, angiogenesis observed in nHL provides rationale for use of angiogenesis inhibitors in lymphoma therapy.

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ.

Abstract Chitinase-like proteins have multiple biological functions that promote tumor growth, angiogenesis and metastasis. Expression of CHI3L2, which is similar in structure to CHI3L1, is detected in glioma cells and tumor-associated macrophages (TAMs) in glioma and breast cancer. However, its exact role remains unclear. We analyzed the expression of CHI3L2 in 74 invasive ductal breast carcinoma (IDC) tumors, breast cancer and macrophages cell cultures using immunohistochemistry, immunofluorescence, Western blot and PCR methods. Clinicopathologic data were included in the analysis. The results obtained show that CHI3L2 expression decreases with increasing degree of tumor grade and negative status of estrogen (ER) and progesterone receptors (PR). Furthermore, CHI3L2 is significantly and positively correlated with phosphorylation of STAT-3 and ERK1/2 signaling pathways, but negatively correlated with macrophage infiltration. CHI3L2 is expressed both in the cytoplasm of cancer cells and in macrophages and may regulate STAT-3 and ERK1/2 phosphorylation in breast cancer cell lines. Analysis of the clinicopathologic data revealed that CHI3L2 levels had no effect on patient survival. CHI3L2 expression may be specific for cancer cells in IDC and involved in cross-talk with the tumor microenvironment. Our study has shown that IDC cancer cells express the CHI3L2 protein, possibly indicating a novel function of this protein.

BackgroundBasal Cell Carcinoma (BCC) is the most common form of skin cancer, characterized by its low metastatic potential yet considerable diversity in clinical and dermatoscopic presentation. Advances in dermatoscopy have significantly improved the early detection of BCC, revealing specific patterns that guide diagnosis and management. Parallelly, immunohistochemical markers have been explored for their potential to elucidate the underlying tumor biology and prognosis, with particular focus on angiogenesis, melanocytic activity, and lymphangiogenesis.ObjectiveThis study aims to investigate the correlations between dermatoscopic features and the immunohistochemical expressions of CD34, CD31, Melan-A, and D2-40 in BCC, through a comprehensive analysis of 100 cases We sought to determine whether visual dermatoscopic patterns correlate with the molecular characteristics defined by immunohistochemical staining, potentially enhancing diagnostic accuracy.MethodsA total of 100 cases of clinically and histopathologically confirmed BCC were prospectively analyzed, employing standard dermatoscopic techniques for lesion evaluation and immunohistochemical staining for CD34, CD31, Melan-A, and D2-40 to assess tumor angiogenic potential, melanocytic activity, and lymphangiogenesis. The study was conducted with adherence to ethical standards and informed consent from all participants.ResultsDermatoscopic examination revealed a variety of vascular patterns and pigmented features across different BCC anatomical locations. However, the comprehensive correlation analysis predominantly found a lack of significant associations between dermatoscopic appearances and expressions of the targeted immunohistochemical markers, with the notable exception of a correlation between observed hemorrhage and the Melan-A marker.ConclusionsThe lack of significant correlations between dermatoscopic features and immunohistochemical marker expressions in BCC suggests that the biological behavior and angiogenic, melanocytic, and lymphangiogenic activities within BCC lesions may be influenced by factors beyond those assessed in this study. Despite the exploratory nature of these findings, they underscore the complexity of BCC biology and highlight the need for further research incorporating additional markers and advanced imaging techniques.

Abstract Nanosecond pulsed electric field (nsPEF) has emerged as a promising approach for inducing cell death in melanoma, either as a standalone treatment or in combination with chemotherapeutics. However, to date, there has been a shortage of studies exploring the impact of nsPEF on the expression of cancer-specific molecules. In this investigation, we sought to assess the effects of nsPEF on melanoma-specific MAGE (Melanoma Antigen Gene Protein Family) expression. To achieve this, melanoma cells were exposed to nsPEF with parameters set at 8 kV/cm, 200 ns duration, 100 pulses, and a frequency of 10 kHz. We also aimed to comprehensively describe the consequences of this electric field on melanoma cells' invasion and proliferation potential. Our findings reveal that following exposure to nsPEF, melanoma cells release microvesicles containing MAGE antigens, leading to a simultaneous increase in the expression and mRNA content of membrane-associated antigens such as MAGE-A1. Notably, we observed an unexpected increase in the expression of PD-1 as well. While we did not observe significant differences in the cells' proliferation or invasion potential, a remarkable alteration in the cells' metabolomic and lipidomic profiles towards a less aggressive phenotype was evident. Furthermore, we validated these results using ex vivo tissue cultures and 3D melanoma culture models. Our study demonstrates that nsPEF can elevate the expression of membrane-associated proteins, including melanoma-specific antigens. The mechanism underlying the overexpression of MAGE antigens involves the initial release of microvesicles containing MAGE antigens, followed by a gradual increase in mRNA levels, ultimately resulting in elevated expression of MAGE antigens post-experiment. These findings shed light on a novel method for modulating cancer cells to overexpress cancer-specific molecules, thereby potentially enhancing their sensitivity to targeted anticancer therapy.

Abstract Heart failure (HF) is an end-stage of many cardiac diseases and one of the main causes of death worldwide. The current management of this disease remains suboptimal. The adult mammalian heart was considered a post-mitotic organ. However, several reports suggest that it may possess modest regenerative potential. Adult cardiac progenitor cells (CPCs), the main players in the cardiac regeneration, constitute, as it may seem, a heterogenous group of cells, which remain quiescent in physiological conditions and become activated after an injury, contributing to cardiomyocytes renewal. They can mediate their beneficial effects through direct differentiation into cardiac cells and activation of resident stem cells but majorly do so through paracrine release of factors. CPCs can secrete cytokines, chemokines, and growth factors as well as exosomes, rich in proteins, lipids and non-coding RNAs, such as miRNAs and YRNAs, which contribute to reparation of myocardium by promoting angiogenesis, cardioprotection, cardiomyogenesis, anti-fibrotic activity, and by immune modulation. Preclinical studies assessing cardiac progenitor cells and cardiac progenitor cells-derived exosomes on damaged myocardium show that administration of cardiac progenitor cells-derived exosomes can mimic effects of cell transplantation. Exosomes may become new promising therapeutic strategy for heart regeneration nevertheless there are still several limitations as to their use in the clinic. Key questions regarding their dosage, safety, specificity, pharmacokinetics, pharmacodynamics and route of administration remain outstanding. There are still gaps in the knowledge on basic biology of exosomes and filling them will bring as closer to translation into clinic.