The Role of Irisin/FNDC5 Expression and Its Serum Level in Breast Cancer.

Irisin (Ir) is an adipomyokine formed from fibronectin type III domain-containing protein 5 (FNDC5), which can be found in various cancer tissues. Additionally, FNDC5/Ir is suspected of inhibiting the epithelial-mesenchymal transition (EMT) process. This relationship has been poorly studied for breast cancer (BC). The ultrastructural cellular localizations of FNDC5/Ir were examined in BC tissues and BC cell lines. Furthermore, we compared serum levels of Ir with FNDC5/Ir expression in BC tissues. The aim of this study was to examine the levels of EMT markers, such as E-cadherin, N-cadherin, SNAIL, SLUG, and TWIST, and to compare their expression levels with FNDC5/Ir in BC tissues. Tissue microarrays with 541 BC samples were used to perform immunohistochemical reactions. Serum levels of Ir were assessed in 77 BC patients. We investigated FNDC5/Ir expression and ultrastructural localization in MCF-7, MDA-MB-231, and MDA-MB-468 BC cell lines and in the normal breast cell line (Me16c), which was used as the control. FNDC5/Ir was present in BC cell cytoplasm and tumor fibroblasts. FNDC5/Ir expression levels in BC cell lines were higher compared to those in the normal breast cell line. Serum Ir levels did not correlate with FNDC5/Ir expression in BC tissues but were associated with lymph node metastasis (N) and histological grade (G). We found that FNDC5/Ir correlated moderately with E-cadherin and SNAIL. Higher Ir serum level is associated with lymph node metastasis and increased grade of malignancy. FNDC5/Ir expression is associated with E-cadherin expression level.

Association of Irisin/FNDC5 with ERRα and PGC-1α Expression in NSCLC.

The rapid growth and division of cancer cells are associated with mitochondrial biogenesis or switching to glycolysis. ERRα, PGC-1α and irisin/FNDC5 are some of the proteins that can influence these processes. The aim of this study was to determine the correlation of these proteins in non-small cell lung cancer (NSCLC) and to investigate their association with clinicopathological parameters. Immunohistochemistry reactions were performed on tissue microarrays (860 NSCLC, 140 non-malignant lung tissue). The normal fibroblast cell line (IMR-90) and lung cancer cell lines (NCI-H1703 and NCI-H522) were used as co-cultures. The mRNA levels of FNDC5 and ESRRA (encoding ERRα) were assessed in IMR-90 cells after co-culture with lung cancer cells. We observed a decreased level of ERRα with an increase in tumor size (T), stages of the disease, and lymph node metastases (N). In the adenocarcinoma (AC) subtype, patients with a higher ERRα expression had significantly longer overall survival. A moderate positive correlation was observed between FNDC5 mRNA and ESRRA mRNA in NSCLCs. The expression of FNDC5 mRNA in IMR-90 cells increased after 24 h, and ESRRA gene expression increased after 48 h of co-culture. The ERRα receptor with PGC-1α participates in the control of FNDC5/irisin expression. Normal fibroblasts revealed an upregulation of the FNDC5 and ESRRA genes under the influence of lung cancer cells.

Irisin Association with Ki-67, MCM3 and MT-I/II in Squamous Cell Carcinomas of the Larynx.

BACKGROUND: Current studies indicate irisin role in carcinogenesis. The aim of the study was to investigate the expression of irisin in LSCCs and to determine its association with clinicopathological factors, as well as recognized markers of proliferation, i.e., Ki-67 and MCM3,5,7 and MT-I/II proteins. MATERIAL AND METHODS: The research material consisted of 140 cases of LSCCs, 57 cases of laryngeal papillomas (BLs) and 14 controls (benign hypertrophic changes). Tissue microarrays were used to perform IHC. Western blot and immunofluorescence were performed in laryngeal cancer cell lines and normal keratinocytes. RESULTS: Irisin expression levels were significantly increased in LSCC compared to BLs (p < 0.0001) and controls (p = 0.001). We noted a positive moderate and weak correlation between irisin and Ki-67, MCM3 and MT-I/II. We observed an elevated level of irisin expression with increasing tumor size (T1-2 vs. T3-4; p = 0.0348). The levels of irisin were higher in N0 than in N1 and N2-3 (p = 0.0031 and p = 0.0457, respectively). Our in vitro study revealed a higher level of irisin in Larynx Epidermoid Carcinoma 2 (HEp-2) cells compared to the control Normal Human Keratinocyte (HaCat) cell line. CONCLUSIONS: Increased irisin expression levels in LSCC and its correlation with clinicopathological and proliferation factors may indicate the potential role of irisin as a biomarker in the diagnostic process of LSCC.

Comparison of TMA Technique and Routine Whole Slide Analysis in Evaluation of Proliferative Markers Expression in Laryngeal Squamous Cell Cancer.

BACKGROUND/AIM: Comparison of the expression of Ki-67, MCM3, 5, 7 and MTI/II proteins using immunohistochemistry (IHC) on whole section (WS) and tissue microarray (TMA) of laryngeal squamous cell carcinoma (LSCC) samples. MATERIALS AND METHODS: A total of 51 archival paraffin blocks of LSCC were used. TMAs were prepared from 1.5 mm core punches. IHC reactions were performed using antibodies against Ki-67, minichromosome maintenance proteins (MCM3, 5, 7) and metallothionein (MTI/II). RESULTS: Spearman rank correlation test revealed moderate positive correlation in the case of Ki-67: WS vs. TMA (r=0.38, p=0.07) and strong positive correlation in regard to the rest of tested markers: MCM3, WS vs. TMA (r=0.49, p=0.0004); MCM5, WS vs. TMA (r=0.61, p<0.0001); MCM7, WS vs. TMA (r=0.59, p<0.0001); MTI/II, WS vs. TMA (r=0.66, p<0.0001). Mann Whitney U-test showed no significant differences in the case of Ki-67 and MCM5. Moreover, Bland-Altman test showed a low level of bias in regard to Ki-67, WS vs. TMA and MCM5, WS vs. TMA. CONCLUSION: TMA may be an effective and reliable method of assessment of Ki-67 and MCM5 expression in LSCC.

Expression of Irisin/FNDC5 in Cancer Cells and Stromal Fibroblasts of Non-small Cell Lung Cancer.

Background: Recent in vitro studies have indicated that irisin inhibits proliferation, migration and epithelial-mesenchymal transition. Irisin expression has not been studied in tumour tissues of non-small cell lung cancer (NSCLC) patients yet. The aim of the study was to determine the irisin expression in NSCLCs in comparison to the clinicopathological factors and expression of TTF-1, p63 and Ki-67. Material and methods: Tissue microarrays with 729 NSCLC and 140 non-malignant lung tissue (NMLT) were used to perform immunohistochemical reactions. Laser Capture Microdissection (LCM) was used to collect cancer and stromal cells from NSCLCs. FNDC5 expression was tested for LCM samples, 75 NSCLCs and 25 NMLTs with the RT-PCR technique. Western-blot, immunofluorescence reaction and RT-PCR assays were performed on lung cancer cell lines. Results: Irisin expression was observed in NSCLC cancer cells and stromal fibroblasts. In cancer cells, irisin expression was decreased in higher grades (G) of malignancy, tumour size (T) and according to lymph node metastasis. In stromal cells, irisin expression was increased in higher G and advanced T. A shorter overall survival was observed in patients with higher irisin expression in NSCLC stromal cells. Conclusions: Irisin expression in stromal fibroblasts may influence cancer cell proliferation and may be a prognostic factor for survival in NSCLC.

MCM5 Expression Is Associated With the Grade of Malignancy and Ki-67 Antigen in LSCC.

BACKGROUND/AIM: The minichromosome maintenance proteins (MCMs) may be potential biomarkers of cancer cell proliferation. They are essential to initiate DNA replication. The aim of the study was to investigate the level of MCM5 expression in benign lesions (BLs) and laryngeal squamous cell cancer (LSCC). MATERIALS AND METHODS: Immunohistochemical (IHC) analysis was carried out on 83 LSCCs and 10 BLs. Western-blot, immunofluorescence analysis (IF) and real-time PCR (RT-PCR) were performed using HEp-2 cancer cells and HaCaT keratinocytes. RESULTS: The expression of MCM5 was higher in LSCC than in the BLs (p<0.0001) and was higher in subsequent malignancies of LSCC. Positive correlations were demonstrated between the expression levels of MCM5 and the Ki-67 antigen. In vitro studies have confirmed that the expression of MCM5 is elevated in cancer cells. CONCLUSION: MCM5 protein may be used as a potential marker of cancer cell proliferation in LSCC.

Role of PD-L1 Expression in Non-Small Cell Lung Cancer and Their Prognostic Significance according to Clinicopathological Factors and Diagnostic Markers.

BACKGROUND: The latest immunotherapy, used in the treatment of non-small cell lung cancer (NSCLC), uses monoclonal antibodies directed against programmed death ligand 1 (PD-L1) to inhibit its interaction with the PD-1 receptor. Elevated levels of PD-L1 expression were observed on NSCLC cells. The association between PD-L1 expression and clinicopathological features is still unclear. Therefore, we examined this relationship and also compare PD-L1 expression levels with Ki-67, p63 and TTF-1. METHODS: 866 samples of NSCLCs were used to prepare tissue microarrays (TMAs) on which immunohistochemical (IHC) reactions were performed. Changes in the level of CD274 (PD-L1) gene expression in 62 NSCLC tumors were tested in relation to 14 normal lung tissues by real-time PCR reactions (RT-PCR). RESULTS: PD-L1 expression was observed in 32.6% of NSCLCs. PD-L1 expression was increased in higher malignancy grades (G) (p < 0.0001) and in higher lymph node status (pN) (p = 0.0428). The patients with low PD-L1 expression had longer overall survival compared to the group with high expression (p = 0.0332) in adenocarcinoma (AC) only. CONCLUSIONS: PD-L1 expression seems to be associated with increased tumor proliferation and aggressiveness as well as shorter patient survival in NSCLC, predominantly in the AC group.

The role of human papillomavirus in oncogenic transformation and its contribution to the etiology of precancerous lesions and cancer of the larynx: A review.

Human papillomavirus (HPV) belongs to the Papillomaviridae family and infects squamous cells and mucous membranes of humans. Various studies conducted over the last years have shown a correlation between HPV infection and carcinogenesis process. The DNA of the virus is detected in approximately 20% of cancers of the upper respiratory tract. The presence of HPV in cancerous lesion of the larynx varies depending on the procedure applied for sample collection and the viral DNA detection method. The high variance in the frequency of HPV detection is observed even among results obtained with the use of PCR reaction. It varies between 3 and 85%. HPV is also the etiological factor of laryngeal papillomas in both children and adults. However, a considerable amount of research demonstrates that 1-7% of the larynx papillomas in adults undergo transformation into squamous cell carcinoma. The aim of the study was to summarize the current state of knowledge regarding the presence of the HPV virus in the larynx as well as its participation in malignant transformation.

Expression of Cell Cycle-related Proteins p16, p27 and Ki-67 Proliferating Marker in Laryngeal Squamous Cell Carcinomas and in Laryngeal Papillomas.

BACKGROUND: The deregulation of the cell cycle is crucial for the processes of carcinogenesis and tumor progression. The cell cycle is under the control of cyclin-dependent kinases (CDKs) and their inhibitors (CDKIs). Proteins p16 and p27 are CDKIs, and their altered expression has been noticed in many types of cancers. The aim of this study has been to compare the expression levels of these proteins in laryngeal papillomas and carcinomas in order to confirm their predictive value in cancer development. MATERIALS AND METHODS: Eighty-three cases of laryngeal lesions were analyzed: cancers (51) and papillomas (32). Immunohistochemical (IHC) reactions were performed in order to detect the expression levels of p16, p27 and Ki-67. Immunofluorescence (IF) and western blot were performed on HEp-2 cell line. RESULTS: The expression of p16 and p27 was decreased in cancer cells in comparison to papillomas. Additionally, we observed cytoplasmic expression of p16 in cancers and lack of such expression in papillomas. The same pattern of expression of p16 was also detected by IF and western blot analysis in HEp-2 cell line. The expression of Ki-67 protein revealed no significant differences in the case of papilloma compared with cancer, however a significant correlation between tumor grade and the nuclear expression of Ki-67 was observed. CONCLUSION: There is a necessity in finding biomarkers, which would predict the risk level of malignant transformation. Our study has confirmed that altered expressions of the p16 and p27 proteins might be useful biomarkers in the progression of laryngeal squamous cell carcinomas (LSCC).

Expression of Cell Cycle-Related Proteins p16, p27, p53 and Ki-67 in HPV-positive and -negative Samples of Papillomas of the Upper Respiratory Tract.

BACKGROUND: The role of human papillomavirus (HPV) infection as an etiological factor of respiratory tract papillomas has been described in numerous studies, however its role in malignant transformation has not been clearly defined. Depending on their oncogenic potential they have been classified as low- and high-risk HPVs. We analyzed the expression of four cell cycle-related proteins in order to understand the processes leading to malignant transformation. MATERIALS AND METHODS: Fifty-six cases of pharyngeal and laryngeal papillomas were analyzed. Nested multiplex polymerase chain reactions to detect the presence of the HPV types, as well as immunohistochemical reactions were performed for the detection of cell cycle-related proteins p16, p27, p53 and Ki-67. RESULTS: The presence of HPVs 6/11 and 16 was confirmed in 10/56 cases. The expression of all analyzed cell cycle-related proteins was increased in HPV-infected papillomas. CONCLUSION: HPV infection of the upper respiratory tract may influence the expression of cell cycle-related proteins, that could indicate its possible role in the process of malignant transformation.

The role of human papillomavirus in the malignant transformation of cervix epithelial cells and the importance of vaccination against this virus.

Human papillomavirus (HPV) belongs to the diverse group of sexually transmitted viruses that manifest affinity to the squamous epithelia of the skin and mucous membranes. Over 100 types of HPV have been described and identified in human tissues, and it has been proved that persistent infection with high-risk types of the virus (types 16 and 18 in particular) could lead to cervical cancer. High-risk HPV types have been found in approximately 70% of all cases of cervical cancer worldwide. The aim of this study was to describe the role of HPV in the process of neoplastic transformation in epithelial cells and to emphasize the prophylactic significance of vaccinations against the virus.

Prognostic significance of metallothionein, p53 protein and Ki-67 antigen expression in laryngeal cancer.

BACKGROUND: This study aimed at the evaluation of the expression of metallothionein (MT) in laryngeal carcinoma and its correlation with the expression of Ki-67 antigen and p53 protein and selected clinical and pathological variables in view of their potential prognostic significance. MATERIALS AND METHODS: Sixty-five laryngeal cancer patients were retrospectively analysed. Expression of MT, Ki-67 and p53 in tumour tissue samples were assessed by immunohistochemistry. RESULTS: In laryngeal cancer a significantly augmented expression of MT, Ki-67 and p53 was noted, as compared to the control group (p < 0.001) and a significantly increased expression of MT in low malignancy tumours (G1) as compared to the control group (p < 0.001). The expression of Ki-67 antigen was positively correlated with the expression of p53 protein (r = 0.477; p < 0.05) and expression of either marker was positively correlated with malignancy grade (r = 0.47, p < 0.05; r = 0.31, p < 0.05; for Ki-67 and p53, respectively). Shortened survival was noted in patients with high expression of Ki-67 antigen and p53 protein. CONCLUSION: The intensity of MT expression was not related to prognosis in laryngeal cancer. Nevertheless, it may provide a significant index indicating the malignant transformation of benign lesions of laryngeal epithelium.

Exercise-induced apoptosis in renal tubular cells of the rat.

A number of studies have shown that acute physical exercise is associated with the induction of apoptosis not only in skeletal muscle but also in many distant organs. One of the pathogenic agents responsible for exercise-induced damage in many tissues is the generation of oxygen free radicals. The aim of the present study was to examine the influence of exercise-induced oxidative stress on the rat kidney. The analysis was performed on the kidneys of rats subjected to treadmill running until exhaustion. Our results demonstrated that acute exercise led to apoptotic damage of the renal distal tubular cells, although this was not a result of oxidative stress.

The detection of Helicobacter pylori in paraffin sections using the PCR technique and various primers as compared to histological techniques.

Helicobacter pylori is thought to represent a significant etiopathogenic factor in diseases of the upper gastrointestinal tract. It seems, therefore, important to elaborate effective techniques for its detection. The aim of the present study was to evaluate the effectiveness of Helicobacter pylori detection using the PCR technique on paraffin sections with various pairs of primers and to compare the results with those of a histological appraisal. Material for the studies involved 50 paraffin blocks with gastric mucosa biopsies fixed in 4% buffered formalin. In this material 4 tests were performed with the aim of diagnosing Helicobacter pylori infection: 1) H+E staining, 2) staining by the Giemsa technique, 3) an immunocytochemical technique with antibodies against H. pylori and 4) the PCR technique with various primers. In the present study the most reliable results for H. pylori detection as well as the most pronounced correlation were obtained by using the PCR technique with primers for the ureC gene, immunohistochemistry and staining according to Giemsa. Less compatible results were obtained employing the two PCR techniques which utilise various primers. The experiments confirmed the usefulness of the PCR technique in the detection of Helicobacter pylori in paraffin sections by using a suitable pair of primers, and also indicated that Giemsa staining and immunohistochemistry should be taken into account.

Influence of exogenous melatonin on doxorubicin-evoked effects in myocardium and in transplantable Morris hepatoma in rats.

BACKGROUND: Melatonin is one of the most effective oxygen free radical scavengers. Its cardio- and nephro-protective effects were demonstrated in the course of anthracycline administration. The present study aimed at examining the cytostatic efficiency of doxorubicin (DOX) applied together with melatonin to rats with transplantable Morris hepatoma. MATERIALS AND METHODS: Animals were divided into four groups receiving 0.9% NaCl, melatonin, DOX and melatonin together with DOX, respectively. Three weeks after transplantation of the tumour cells, the heart and tumour were isolated. The extent of tumour necrosis and the number of apoptotic cells were evaluated in paraffin-embedded sections. RESULTS: Administration of melatonin together with DOX decreased the extent of tumour necrosis and the apoptotic tumour cells but, on the other hand, decreased the number of apoptotic cardiomyocytes. CONCLUSION: Melatonin weakens the cytotoxic activity of DOX by the decreased proportions of necrotic and apoptotic cells of transplantable Morris hepatoma. Melatonin protects also cardiomyocytes by decreasing DOX-induced apoptosis in the cells.