Altered expression of 17­ß­hydroxysteroid dehydrogenase type 2 and its prognostic significance in non­small cell lung cancer.

Numerous studies have reported that oestrogens may contribute to the development of non­small cell lung cancer (NSCLC). Although different steroidogenic enzymes have been detected in the lung, the precise mechanism leading to an exaggerated accumulation of active oestrogens in NSCLC remains unexplained. 17­ß­Hydroxysteroid dehydrogenase type 2 (HSD17B2) is an enzyme involved in oestrogen and androgen inactivation by converting 17­ß­oestradiol into oestrone, and testosterone into 4­androstenedione. Therefore, the enzyme serves an important role in regulation of the intracellular availability of active sex steroids. This study aimed to determine the expression levels of HSD17B2 in lung cancer (LC) and adjacent histopathologically unchanged tissues obtained from 161 patients with NSCLC, and to analyse the association of HSD17B2 with clinicopathological features. For that purpose, reverse transcription­quantitative PCR, western blotting and immunohistochemistry were conducted. The results revealed that the mRNA and protein expression levels of HSD17B2 were significantly decreased in LC tissues compared with matched controls (P<10­6). Conversely, strong cytoplasmic staining of HSD17B2 was detected in the unchanged respiratory epithelium and in glandular cells. Notably, a strong association was detected between reduced HSD17B2 expression and advanced tumour stage, grade and size. Furthermore, it was revealed that HSD17B2 may have potential prognostic significance in NSCLC. A log­rank test revealed the benefit of high HSD17B2 protein expression for the overall survival (OS) of patients (P=0.0017), and multivariate analysis confirmed this finding (hazard ratio=0.21; 95% confidence interval=0.07­0.63; P=0.0043). Stratified analysis in the Kaplan­Meier Plotter database indicated that patients with higher HSD17B2 expression presented better OS and post­progression survival. This beneficial effect was particularly evident in patients with adenocarcinoma and during the early stages of NSCLC. Decreased expression of HSD17B2 appears to be a frequent feature in NSCLC. Retrospective analysis suggests that the HSD17B2 mRNA and protein status might be independent prognostic factors in NSCLC and should be further investigated.

Klotho expression and nodal involvement as predictive factors for large cell lung carcinoma.

INTRODUCTION: Klotho has been recently described as a carcinogenesis suppressor. Large cell neuroendocrine lung carcinoma (LCNEC) is a rare, highly malignant neoplasm. In the light of increasing incidence of neuroendocrine tumours, biomarkers predicting survival are needed. We consider that Klotho might be one. MATERIAL AND METHODS: We analysed records of all patients diagnosed with LCNEC, atypical carcinoid and typical carcinoid operated on in our institution between 2007 and 2015. Initially, we found 134 cases. Forty-six specimens were unattainable and thus excluded from research. All patients diagnosed with LCNEC according to the WHO classification were included in the study. Immunohistochemical staining for Klotho was performed. We retrospectively reviewed patient charts and analysed multiple variables. RESULTS: Positive staining for Klotho was present in 36 tissue specimens, while 12 patients were Klotho-negative. Survival length was significantly higher in Klotho-positive cases (p = 0.024), while advanced nodal status (N1 and N2) represented a marker of poor outcome (p = 0.011). In multivariate analysis, both Klotho presence (p = 0.015; HR = 0.37; 95% CI: 0.17-0.86) and nodal involvement (p = 0.007; HR = 3.04; 95% CI: 1.37-6.82) were independent prognostic factors. Tumour vessel invasion and visceral pleura infiltration were not associated with worse treatment results. Klotho presence predicted a favourable prognosis in these groups (p = 0.018; p = 0.007). CONCLUSIONS: Our results suggest that Klotho might be a positive factor for predicting survival in LCNEC and nodal involvement a negative one. Thus, these two markers may assist in the selection of subjects with unfavourable prognosis and to personalise therapy regimens.

Decreased expression of connective tissue growth factor in non-small cell lung cancer is associated with clinicopathological variables and can be restored by epigenetic modifiers.

PURPOSE: Recent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC. METHODS: We used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay. RESULTS: We found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation. CONCLUSIONS: Decreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.

Increased expression of 17-beta-hydroxysteroid dehydrogenase type 1 in non-small cell lung cancer.

OBJECTIVES: Recent studies indicated that estrogens may influence the development of non-small cell lung cancer (NSCLC). The 17-beta-hydroxysteroid dehydrogenase type 1 (HSD17B1) catalyzes the reduction of estrone (E1) to the highly potent E2. Although the significance of aromatase in an intratumoral E2 production in NSCLC is well established, the role of HSD17B1 remains largely unknown. Therefore, we investigated the expression of HSD17B1 in lung cancerous and corresponding histopathologically unchanged tissues from NSCLC patients and the association between HSD17B1 expression and clinicopathological features. Than, we examined the biological significance of HSD17B1 in NSCLC cells in vitro. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) on HSD17B1 expression and activity. MATERIALS AND METHODS: We used Real Time quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry to evaluate HSD17B1 expression in tissues obtained from 48 patients with NSCLC. The methylation status of the promoter region of HSD17B1 in A549 and Calu-1 cells was evaluated by bisulfite sequencing. We investigated the effect of 5-dAzaC on HSD17B1 transcript levels (by RT-qPCR) and on HSD17B1 enzyme activity by measuring the conversion of E1 to E2. The xCELLigence System was used for monitoring of cell proliferation. RESULTS: We found a substantial increase of HSD17B1 mRNA and protein amount in NSCLC tissues compared with histopathologically unchanged tissues in the group of male patients. An overexpression of HSD17B1 was associated with squamous cell carcinoma and with lung cancer stage 3A. We showed that 5-dAzaC induces DNA demethylation of HSD17B1 promoter, leading to increased HSD17B1 mRNA levels and protein activity in NSCLC cells. It resulted in enhanced E2 production in both cell lines and supported the proliferation of Calu-1 cells but not A549 cells. CONCLUSION: Increased expression of HSD17B1 in NSCLC may contribute to an elevated intratissue level of E2 and consequently may support the development and spread of cancer.

Detection and significance of cytotoxic cell subsets in biopsies of HCV-infected human livers.

Chronic viral hepatitis C still remains the clinical challenge. Attempts of the immune system to cope with this infection are unsatisfactory. There is a conviction that the main site of interaction between virus (Hepatitis C virus, HCV) and immune system is in situ, i.e., in liver. Natural killer (NK) cells appeared relevant in the acute hepatitis. Less is known about the immune response in the chronic HCV infection. The aim of this study was to evaluate the prevalence of various cytotoxic cell subsets in chronic HCV+ liver tissue and to seek links between them and laboratory data of patients. Sections from paraffin blocks of liver biopsy tissues of HCV+ untreated patients were subjected to the reaction with antibodies vs. cytotoxic cell subsets and immunohistochemistry. Positive cells were searched in cellular infiltrates in portal areas and in liver parenchyma. They were classified on the "Yes" or "No" basis. Majority of liver biopsies exhibited cellular infiltrates in portal spaces and as single cells in liver parenchyma. Infiltrates consisted of CD8+ T cells, CD56+ NK ones, including CD158i+ and CD158b+. The latter were rarely seen. There were also granzyme B+ cells. The most abundant were NKG2D+ cells, much more common than NK CD56+ ones. It implied that NKG2D was also expressed on T cells. Prevalence of NKG2D+ cells correlated with high activity of liver enzymes such as alanine aminotransferase, aspartate aminotransferase and a greater histological severity of liver injury. NKG2D+ cells form the bulk of cells infiltrating HCV-infected human liver. Correlation of NKG2D+ cells with some laboratory parameters of patients suggests their role in hepatitis C pathogenesis.

Selected markers of proliferation and apoptosis in the parathyroid lesions: a spatial visualization and quantification.

The aim of the paper was to apply a method for quantitative assessment of proliferation and apoptosis markers, based on their 3D visualization, in cases of parathyroid adenoma and hyperplasia. Material was obtained from 49 patients (32 females and 17 males) with primary hyperparahyroidism. Quantitative immunohistochemistry studies of Ki-67, proliferating cell nuclear antigen (PCNA) and bcl-2 were performed on digital microscopy images with the use of 3D visualization. The use of spatial visualization method allowed us to perform objective quantitative assessment of the studied immunohistochemical markers. The average cell nuclear fraction of Ki67+ was 1.8% in hyperplasia and 1.9% in adenoma cases while 3.5% in the controls. The highest expression of PCNA was found in parathyroid hyperplasia (22.9%) and significantly decreased in adenoma (12.5%) and in the control group (16.8%). The lower expression of bcl-2 in hyperplasia cases (mean area fraction of 0.172 per 1 mum(2), in contrast to 0.643 in adenomas and 0.648 in control) suggested that principal cells can be ready for apoptosis and may confirm the important role of bcl-2 protein in etiopathogenesis of hyperplasia of the parathyroid gland while PCNA might be a useful marker for differentiating adenoma from early hyperplasia in primary hyperparahyroidism cases.

Effect of salt and osmotic stress on changes in polyamine content and arginine decarboxylase activity in Lupinus luteus seedlings.

The effects of NaCl (260 mM) and sorbitol (360 mM) isoosmotic stresses on polyamine titers in lupin (Lupinus luteus L. var. Ventus) in relation to organ-specific responses were investigated. Analysis showed that during the first few hours (4 h) of salt and osmotic stress higher amounts of putrescine (Put) and spermidine (Spd) were accumulated in the roots and leaves of lupin seedlings. After exposing the plants to a longer duration (24 h) of exposure to NaCl, the level of free Put decreased in roots and cotyledons by about 48% and 54%, respectively, and increased in hypocotyls and leaves by about 27% and 73%, respectively. The Level of free Spd also decreased in roots by about 50%, in contrast to the increase of Spd observed in hypocotyls and leaves by about 50% and 70%, respectively. The effect of non-ionic stress on the level of Put and Spd in studied organs of lupin was similar to that of NaCl. Free spermine was at an undetectable level in examined organs. However, in the roots of lupin growing for 24 h in the presence of NaCl and/or sorbitol, the activity of arginine decarboxylase (ADC) (EC 4.1.1.19) increased by about 66% and 80%, respectively. ADC activity in leaves was similar to that observed in the control. Additionally, in the roots and leaves of lupin growing under the stress condition (NaCl or sorbitol), a higher level of polyamines (PAs) bound to microsomal membranes was observed. It is probable that PAs bound to microsomal membranes prevent stress-induced damage. We conclude that both stresses induce biosynthesis of Put and other PAs in the roots, as well as Put accumulation in the leaves, and this may indicate translocation of Put from the roots to the shoot. The possible role of PAs in adaptive mechanisms to stress is discussed.