Comparative assessment of CacyBP/SIP, ß-catenin and cannabinoid receptors in the adrenals of hypertensive rats.

Taking into account homeostatic disorders resulting from arterial hypertension and the key importance of CacyBP/SIP, ß-catenin and endocannabinoids in the functioning of many organs, it was decided to assess the presence and distribution of CacyBP/SIP, ß-catenin, CB1 and CB2 in the adrenal glands of hypertensive rats of various aetiology. The study was conducted on the adrenal glands of rats with spontaneous and renovascular hypertension. The expression of CacyBP/SIP, ß-catenin, CB1 and CB2 was detected by immunohistochemistry and real-time PCR method. The results of the present study revealed both lower gene expression and immunoreactivity of CacyBP/SIP in the adrenal glands of all hypertensive groups compared to the normotensive rats. This study demonstrated a reduction in the immunoreactivity and expression of the ß-catenin, CB1 and CB2 genes in the adrenals of 2K1C rats. While in SHR, the reaction showing ß-catenin and CB1 was very weak or negative, and the expression of CB2 in the adrenal glands of these rats increased. The results of this study show, for the first time, marked differences in the expression of CacyBP/SIP, ß-catenin and CB1 and CB2 cannabinoid receptors in the adrenal glands of rats with primary (SHR) and secondary hypertension (2K1C).

Changes in adhesion molecules: ß-catenin, E-cadherin and Galectin-3 in cells of testicular seminoma.

Introduction: The most common testicular tumors are seminomas. They are characterized by rapid growth and a very high potential for metastasis to other organs. Mutual interactions of tumor cells play an important role in the invasiveness and metastatic capacity, in which complexes of adhesion proteins play a special role. There is a lack of studies on changes in these molecules and their behaviour in testicular cancer. The aim of the study was immunohistochemical identification and evalutaion of adhesive molecules ß-catenin, E-cadherin, galectin-3 in testicular cancer - seminoma. Methods: Tests were performed on sections of testicular cancer - seminoma in comparison with unchanged tissue samples as a control. Material was taken from 30 patients who underwent orchiectomy. Immunohistochemistry and PCR were used to identify ß-catenin, E-cadherin and galectin-3 and gene expression. Results: Immunoreactivity and expression of ß-catenin and E-cadherin in seminomas were markedly decreased compared to non-cancerous testicular tissue. Galectin-3 immunoreactivity was found in both control and cancerous tissue, but in different location. In non-cancerous tissue, it was localized in the cytoplasm of the cells of the seminiferous tubules, in seminomas it was localized mainly in the endothelium. The expression of the Lgals3 gene encoding galectin-3 in seminomas was slightl higher in relation to the tissue unchanged by the carcinogenetic process. Conclusions: The results of the study suggest a significant role of ß-catenin, E-cadherin and galectin-3 in the carcinogenesis of seminomas and may indicate new aspects of the patomechanism of seminomas formation, and thus time lead to better understand the biology of these tumors.

The Effect of CacyBP/SIP on the Phosphorylation of ERK1/2 and p38 Kinases in Clear Cell Renal Cell Carcinoma.

The prognosis for patients with RCC is very poor because this cancer is diagnosed mainly in the metastatic stage and is resistant to radio- and chemotherapy. According to recent research, CacyBP/SIP exhibits phosphatase activity against MAPK and may be involved in many cellular processes. This function has not been studied in RCC so far, so we decided to test whether CacyBP/SIP has phosphatase function against ERK1/2 and p38 in high-grade clear cell RCC. The research material consisted of fragments of clear cell RCC, whereas the comparative material consisted of the adjacent normal tissues. Immunohistochemistry and qRT-PCR were used to identify the expression of CacyBP/SIP, ERK1/2, and p38. The studies showed an increase in immunoreactivity and gene expression of the parameters examined in clear cell RCC compared with normal tissues. Only in the case of ERK1/2 was it shown that the expression of the MAPK3 gene was downregulated and the MAPK1 gene was higher in clear cell RCC. These studies demonstrated that CacyBP/SIP lacked phosphatase function against ERK1/2 and p38 in high-grade clear cell RCC. Further research is needed because a better understanding of the role of CacyBP/SIP and MAPK offers hope for the treatment of urological cancer.

Evaluation of the Expression and Localization of the Multifunctional Protein CacyBP/SIP and Elements of the MAPK Signaling Pathway in the Adrenal Glands of Rats with Primary and Secondary Hypertension.

Hypertension is a global civilization disease and one of the most common causes of death in the world. Organ dysfunction is a serious health consequence of hypertension, which involves damage to the heart, kidneys and adrenals. The interaction of recently discovered multifunctional protein-CacyBP/SIP with ERK1/2 and p38 kinases by regulating the activity and intracellular localization of these kinases may play an important role in the signaling pathways involved in the pathogenesis of hypertension. Due to the lack of data on this subject, we decided to investigate the localization, expression and possible relationship between the studied parameters in the adrenals under arterial hypertension. The study was conducted on the adrenals of rats with spontaneous and DOCA-salt hypertension. The expression of CacyBP/SIP, p-ERK1/2 and p-p38 was detected by immunohistochemistry and qRT-PCR. The results show a statistically significant decrease in CacyBP/SIP expression in the adrenal glands of hypertensive rats. With ERK1/2, there was a decrease in cortical immunoreactivity and an increase in the adrenal medulla of primary hypertensive rats. In contrast, in the adrenals of DOCA-salt rats, ERK1/2 immunoreactivity increased in the cortex and decreased in the medulla. In turn, p38 expression was higher in the adrenal glands of rats with primary and secondary hypertension. The obtained results may suggest the involvement of CacyBP/SIP in the regulation of signaling pathways in which MAP kinases play an important role and provide new insight into molecular events in hypertension. Moreover, they show the participation of CacyBP/SIP in response to oxidative stress.

Wnt/ß-catenin signaling in the adrenal glands of rats in various types of experimental hypertension.

Wnt/ß-catenin signaling plays a key role in maintaining homeostasis, which is disturbed in hypertension. Taking into account the lack of literature describing changes in the Wnt/ß-catenin pathway in the adrenal glands under conditions of elevated arterial pressure, here we compare the expression of WNT4, WNT10A, ß-catenin, and GSK-3ß in the adrenal glands of hypertensive rats of various etiologies. The studies were carried out on the adrenal glands of rats with spontaneous hypertension (SHR), renalvascular (2K1C), and deoxycorticosterone acetate (DOCA)-salt. Immunohistochemical and PCR methods were used to identify the molecular components of the canonical signaling pathway and to evaluate gene expression. Immunoreactivity and expression of WNT4, WNT10A, ß-catenin, and GSK-3ß in adrenals of SHR was decreased, compared to control rats. In adrenals of 2K1C rats, intensity of immunohistochemical reaction and expression of WNT4 and ß-catenin was lower, while immunoreactivity and expression of WNT10A and GSK-3ß were higher, compared to normotensive animals. Significantly stronger immunoreaction and expression of WNT4, ß-catenin and GSK-3ß but weaker immunoreactivity and expression of WNT10A were noted in adrenals in DOCA-salt rats, compared to control rats. In conclusion, our data provide new molecular information indicating that the canonical WNT pathway is disrupted in the adrenal glands of hypertensive rats. They show that the dysregulation of the WNT pathway depends on the etiology of hypertension.

Influence of Inhibition of COX-2-Dependent Lipid Metabolism on Regulation of UVB-Induced Keratinocytes Apoptosis by Cannabinoids.

Inflammation and apoptosis are regulated by similar factors, including ultraviolet B (UVB) radiation and cannabinoids, which are metabolized by cyclooxygenase-2 (COX-2) into pro-apoptotic prostaglandin derivatives. Thus, the aim of this study was to evaluate the impact of cyclooxygenase-2 inhibition by celecoxib on the apoptosis of keratinocytes modulated by UVB, anandamide (AEA) and cannabidiol (CBD). For this purpose, keratinocytes were non-treated/treated with celecoxib and/or with UVB and CBD and AEA. Apoptosis was evaluated using microscopy, gene expressions using quantitate reverse-transcriptase polymerase chain reaction; prostaglandins using liquid chromatography tandem mass spectrometry and cyclooxygenase activity using spectrophotometry. UVB enhances the percentage of apoptotic keratinocytes, which can be caused by the increased prostaglandin generation by cyclooxygenase-2, or/and induced cannabinoid receptor 1/2 (CB1/2) expression. AEA used alone intensifies apoptosis by affecting caspase expression, and in UVB-irradiated keratinocytes, cyclooxygenase-2 activity is increased, while CBD acts as a cytoprotective when used with or without UVB. After COX-2 inhibition, UVB-induced changes are partially ameliorated, when anandamide becomes an anti-apoptotic agent. It can be caused by observed reduced generation of anandamide pro-apoptotic derivative prostaglandin-ethanolamide by COX. Therefore, products of cyclooxygenase-dependent lipid metabolism seem to play an important role in the modulation of UVB-induced apoptosis by cannabinoids, which is particularly significant in case of AEA as inhibition of cyclooxygenase reduces the generation of pro-apoptotic lipid mediators and thus prevents apoptosis.

Warthy-Basaloid Squamous Cell Carcinoma of Penile - Case Report.

OBJECTIVE: The aim of the study was to present a case of penile squamous cell carcinoma and immunohistochemical identification and evaluation of E-cadherin and ß-catenin expression. METHODS: We are presenting a 70-year old man with a variant of penile squamous cell carcinoma with mixed warty and basaloid features. After diagnosis, the patient underwent partial penectomy. Samples taken from the material after surgery were subjected to basic histological staining and immunohistochemical identification of E-cadherin and ß-catenin. A Real-time PCR study was conducted to investigate the expression of E-cadherin and ß-catenin. RESULTS: Routine histopathological examinations revealed the characteristic features of warty-basaloid squamous cell carcinoma. In the case studied, a positive immunohistochemical reaction was observed for E-cadherin and ß-catenin. QRT-PCR analysis showed a statistically significant decrease in E-cadherin expression in tumor samples compared to healthy tissue. In contrast, expression of the gene encoding ß-catenin was slightly higher in tumor samples compared to normal tissue. CONCLUSIONS: The reduced level of the complex of adhesive elements, E-cadherin-ß-catenin, disturbs cell differentiation, promotes a more invasive phenotype-stromal infiltration and the formation of distant metastases. In the described case of the penile tumor, a decrease in E-cadherin expression was noted, which could be related to the occurrence of neoplastic infiltration of the spongy body space. In summary, E-cadherin and ß-catenin expression and the immunoreactivity of these proteins are expressed at different levels in tumor cells and in penile interstitial cells. Regulation of expression during various physiological and pathophysiological processes indicates a potentially important role of E-cadherin and ß-catenin in cell proliferation and adhesion.

Canonical Wnt signaling in the kidney in different hypertension models.

There is a close relationship between the kidney and blood pressure. On the one hand, kidney dysfunction causes an increase in blood pressure; on the other hand, high blood pressure causes kidney dysfunction. Wnt/ß-catenin signaling is a key pathway that regulates various cellular processes and tissue homeostasis and is also involved in damage and repair processes. In healthy organs, Wnt/ß-catenin signaling is muted, but it is activated in pathological states. The purpose of the present study was to immunohistochemically evaluate and compare the expression of WNT4, WNT10A, Fzd8, ß-catenin, and GSK-3ß (glycogen synthase kinase 3ß) in the kidneys of rats with essential arterial hypertension (SHR), renal-renal hypertension (2K1C), and DOCA-salt-induced hypertension. The study was performed on five male WKY rats, seven SHRs, and twenty-four (n = 24) young male Wistar rats. The main results showed that during hypertension, there are changes in Wnt/ß-catenin signaling in the kidneys of rats, and the severity of these changes depends on the type of hypertension. This study is the first to assess the levels of some elements of the canonical Wnt/ß-catenin signal transduction pathway in various types of arterial hypertension by immunohistochemistry and may form the basis for further molecular and functional studies of this pathway in hypertension.

Mechanism of pro-apoptotic action of prosthetic restorations on oral mucosa cells.

PURPOSE: The aim of this study was to check the pro-apoptotic mechanism of prosthetic reconstruction on epithelial cells of the oral mucosa. PATIENTS AND METHODS: The research was carried out on the saliva of healthy patients using prostheses. The sample swabs were stained using the May-Grünwald-Giemsa method and processed by immunohistochemistry for nuclear factor kappa B (NF-κB; p65) and caspase-3. Western blots were used to detect caspase-3, NF-κB, p53 and COX-2 expression. RESULTS: We found an increased expression of caspase-3, NF-κB and p53 in the oral epithelial cells of patients using prosthetic restorations compared to the subjects from the control group. No differences in COX-2 expression were found between the groups. The strongest immunoreactivity and expression of caspase-3, NF-κB and p53 were observed in patients using full prosthesis for less than two years. CONCLUSIONS: The results of the conducted research indicate that prosthetic restorations may affect the process of apoptosis of oral mucosa epithelial cells. Lack of difference in expression of COX-2 in the saliva of the studied patients suggests that apoptosis is not caused by inflammatory factors.

Sex differences in distribution of cannabinoid receptors (CB1 and CB2), S100A6 and CacyBP/SIP in human ageing hearts.

BACKGROUND: Women live about 4 years longer due to lower prevalence of cardiovascular complication with ageing. However, the mechanisms involved in the preservation of heart functionality in women have not been fully elucidated. The endocannabinoid system fulfils a significant role in the regulation of cardiovascular system functioning. Cannabinoids, acting through specific receptors (CB1 and CB2), influence on blood pressure, heart rate and myocardial contractility. The function of cardiac muscle cells is strictly dependent on calcium ions. Calcium homeostasis in cardiomyocytes is subjected to complex regulation via calcium-binding proteins. Among them, increasing attention has been paid to the recently discovered S100A6 and CacyBP/SIP. In order to better understand sex differences in the regulation of cardiomyocyte function during ageing, we undertook the present research aimed at immunohistochemical identification and comparative evaluation of cannabinoid receptors, S100A6 and CacyBP/SIP, in the myocardium of ageing men and women. METHODS: The study was conducted on the hearts of 12 men and 10 women (organ donors) without a history of cardiovascular disease. The subjects were divided into two age groups: subjects older than 50 years and subjects under 50 years old. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors, S100A6 and CacyBP/SIP. In the heart samples from each study, participant's expression of genes coding for CB1, CB2, S100A6 and CacyBP/SIP using real-time PCR method was measured. RESULTS: CB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of subjects over 50 was weaker than in younger individuals. In the heart of younger men, CB1-immunoreactivity was weaker and CB2-immunoreaction was stronger compared to women. In the hearts of older men, the CB1-immunostaining was more intense and CB2-immunoreactivity was weaker than in women. Immunodetection of CB1 shoved the presence of receptor in the intercalated discs, but only in the hearts of individuals over the 50 years old. In the hearts of older individuals, stronger immunolabelling was observed for S100A6 and CacyBP/SIP. Male hearts had greater S100A6-immunoreactivity (both age groups) but less CacyBP/SIP immunostaining (individuals over 50 years) compared to the age-matched women. The expression of genes coding CB1, CB2, S100A6 and CacyBP/SIP in the human heart was sex and age-dependent. Observed changes between men and women as well as between subject under and over 50 years were consistent with immunohistochemically stated changes in peptide content. CONCLUSION: Together, the data presented here indicate a close interaction between ageing and sex on the distribution and levels of cannabinoid receptors (CB1, CB2), S100A6 and CacyBP/SIP in the human heart.