SKA3 Expression as a Prognostic Factor for Patients with Pancreatic Adenocarcinoma.

The spindle and kinetochore-associated complex subunit 3 (SKA3) is a protein essential for proper chromosome segregation during mitosis and thus responsible for maintaining genome stability. Although its involvement in the pathogenesis of various cancer types has been reported, the potential clinicopathological significance of SKA3 in pancreatic ductal adenocarcinoma (PDAC) has not been fully elucidated. Therefore, this study aimed to assess clinicopathological associations and prognostic value of SKA3 in PDAC. For this purpose, in-house immunohistochemical analysis on tissue macroarrays (TMAs), as well as a bioinformatic examination using publicly available RNA-Seq dataset, were performed. It was demonstrated that SKA3 expression at both mRNA and protein levels was significantly elevated in PDAC compared to control tissues. Upregulated mRNA expression constituted an independent unfavorable prognostic factor for the overall survival of PDAC patients, whereas altered SKA3 protein levels were associated with significantly better clinical outcomes. The last observation was particularly clear in the early-stage tumors. These findings render SKA3 a promising prognostic biomarker for patients with pancreatic ductal adenocarcinoma. However, further studies are needed to confirm this conclusion.

Overexpression of cyclin F/CCNF as an independent prognostic factor for poor survival in clear cell renal cell carcinoma.

Cyclin F (encoded by CCNF gene) has been reported to be implicated in the pathobiology of several human cancers. However, its potential clinical significance in clear cell renal cell carcinoma (ccRCC) remains unknown. The present study aimed to evaluate the potential significance of cyclin F, assessed by immunohistochemical (IHC) staining and molecular (bioinformatics) techniques, as a prognostic marker in ccRCC in relation to clinicopathological features and outcomes. IHC staining was performed using two independent ccRCC tissue array cohorts, herein called tissue macroarray (TMA)_1 and tissue microarray (TMA)_2, composed of 108 ccRCCs and 37 histologically normal tissues adjacent to the tumor (NAT) and 192 ccRCCs and 16 normal kidney samples, respectively. The mRNA expression data were obtained from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) public datasets, followed by bioinformatics analysis of biological mechanisms underlying prognosis. The relationship between immune cell infiltration level and CCNF expression in ccRCC was investigated using the Tumor Immune Estimation Resource 2.0 (TIMER2) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). Cyclin F expression was significantly elevated in ccRCC lesions compared to both NAT and normal renal tissues. Likewise, CCNF mRNA was markedly increased in ccRCCs relative to non-cancerous tissues. In all analyzed cohorts, tumors with features of more aggressive behavior were more likely to display cyclin F/CCNF-high expression than low. Furthermore, patients with high cyclin F/CCNF expression had shorter overall survival (OS) times than those with low expression. In addition, multivariable analysis revealed that cyclin F/CCNF-high expression was an independent prognostic factor for poor OS in ccRCC. Enrichment analysis for mechanistically relevant processes showed that CCNF and its highly correlated genes initiate the signaling pathways that eventually result in uncontrolled cell proliferation. CCNF expression was also correlated with immune cell infiltration and caused poor outcomes depending on the abundance of tumor-infiltrating immune cells in ccRCC. Our findings suggest that cyclin F/CCNF expression is likely to have an essential role in ccRCC pathobiology through regulating multiple oncogenic signaling pathways and affecting the tumor immune microenvironment and may serve as prognostic biomarker and promising therapeutic target in ccRCC.

Ubiquitin B, Ubiquitin C, and ß-Catenin as Promising Diagnostic and Prognostic Tools in Prostate Cancer.

Prostate cancer (PC) is a major global public health concern, imposing a significant burden on men and ranking as the second most prevalent malignancy. This study delves into the intricate world of ubiquitination processes and expression regulation, with a specific focus on understanding the roles of ubiquitin B (UBB), ubiquitin C (UBC), and ß-Catenin in PC development. We thoroughly analyze the expression profiles of UBB, UBC, and ß-Catenin, investigating their interactions and associations with clinical and histopathological data. These findings offer valuable insights into their potential as robust prognostic markers and their significance for patient survival. Our research uncovers the upregulation of UBB and UBC expression in PC tissues, and an even more pronounced expression in lymph node metastases, highlighting their pivotal roles in PC progression. Moreover, we identify a compelling correlation between high UBB and UBC levels and diminished overall survival in PC patients, emphasizing their clinical relevance. Additionally, we observe a significant reduction in membranous ß-Catenin expression in PC tissues. Importantly, abnormal ß-Catenin expression is strongly associated with shorter survival in PC patients and serves as a significant, independent prognostic factor for patient outcomes. Kaplan-Meier survival analysis indicates that patients with tumors characterized by simultaneous UBB and aberrant ß-Catenin expression exhibit the poorest overall survival. These collective insights underline the clinical importance of evaluating UBB, UBC, and ß-Catenin as combined prognostic markers in PC.

Low Expression of MATR3 Is Associated with Poor Survival in Clear Cell Renal Cell Carcinoma.

Matrin 3 (MATR3) is one of the most abundant inner nuclear matrix proteins involved in multiple nuclear processes. However, to date, the biological role and prognostic relevance of MATR3 in human cancers still need to be explored. Therefore, the present study aimed to examine the expression levels and prognostic significance of MATR3 in clear cell renal cell carcinoma (ccRCC) patients. We assessed MATR3 immunohistochemical staining and RNA-seq data from publicly available data sets, and the results were analyzed with reference to clinicopathological characteristics and the overall survival of patients. Furthermore, the protein-protein interaction (PPI) network for MATR3 and its neighbors was constructed, functionally annotated, and screened for survival-related genes. MATR3 protein and mRNA levels were lower in tumor tissues compared to control tissues. Lower MATR3 protein (HR 2.36, 95%CI 1.41-3.97; p = 0.001) and mRNA (HR 2.01, 95%CI 1.46-2.75; p < 0.0001) expression levels were found to be a significant independent adverse prognostic factor for the patient's overall survival (OS). Moreover, of the candidate genes, the MRPL23 gene was identified as being the most predictive of OS, and combined MRPL23/MATR3 expression status predicted patient survival better than looking at each marker individually (HR 3.15, 95%CI 2.05-4.83; p < 0.0001). In conclusion, the results from the present investigation warrant further research into the biological and prognostic value of MATR3 and MRPL23 in ccRCC patients.

High expression of RUVBL1 and HNRNPU is associated with poor overall survival in stage I and II non-small cell lung cancer patients.

The present study aimed to investigate expression levels and prognostic significance of RUVBL1 and HNRNPU in stage I and II non-small-cell lung cancer (NSCLC) patients. Therefore, we evaluated immunohistochemical staining of RUVBL1 and HNRNPU, as well as RNA-seq data from public sources, and the results were evaluated concerning overall survival (OS) and clinicopathological features. We found that RUVBL1 and HNRNPU proteins and mRNA levels were higher in tumor tissues as compared to adjacent/normal tissues. RUVBL1 (p = 0.013) and HNRNPU (p = 0.021) high protein levels were independent prognostic factors for poor OS. Also, the multivariate analysis in the TCGA dataset revealed that high RUVBL1 (p = 0.064) and HNRNPU (p = 0.181) mRNA levels were not significantly associated with prognosis. However, the co-expression status of these markers (R + H +) was independently associated with poor OS both in the TCGA dataset (p = 0.027) and in our cohort (p = 0.001). In conclusion, combined and individual expression of RUVBL1 and HNRNPU proteins, as well as R + H + mRNA status, may serve as potential prognostic biomarkers for NSCLC. This study adds to the previous observations that RUVBL1 and HNRNPU might be novel and promising therapeutic targets and markers for prognostic evaluation.

Prognostic significance of MATR3 in stage I and II non-small cell lung cancer patients.

PURPOSE: Matrin 3 (MATR3) is a nuclear matrix protein involved in mRNA stabilization, nuclear retention of hyper-edited RNAs, and RNA splicing. The role of MATR3 in cancer is still unclear. The present study aimed to investigate expression levels and prognostic significance of MATR3 in stage I and II non-small cell lung cancer (NSCLC) patients. METHODS: We examined MATR3 protein immunohistochemically in tumoral and non-tumoral tissue sections from n = 67 NSCLC patients treated at hospital, and MATR3 mRNA from The Cancer Genome Atlas (TCGA) cohort with respect to valid prognostic and predictive features, as well as treatment outcome. RESULTS: Significantly higher immunohistochemical levels of MATR3 protein were found in tumor-adjacent tissue compared to cancer (p = 0.049). A decrease in MATR3 protein expression was found to be a significant independent adverse prognostic factor for patients overall survival (p = 0.007). By contrast, we observed higher MATR3 mRNA levels in tumoral tissue compared to control lung tissues (p < 0.001). Based on the TCGA dataset, we reported that high MATR3 mRNA level was significantly associated with worse OS of NSCLC patients (p < 0.001); however, it was not an independent prognostic marker (p = 0.156). The discrepancies in prognostic significance of MATR3 gene mRNA and protein levels imply a need for further investigation. CONCLUSION: In conclusion, the present study warrants further investigation into the biological and prognostic value of MATR3 as a potential prognostic marker in early-stage NSCLC patients.

Advances in Microscopic Studies of Tendinopathy: Literature Review and Current Trends, with Special Reference to Neovascularization Process.

Tendinopathy is a process of chaotic extracellular matrix remodeling followed by increased secretion of enzymes and mediators of inflammation. The histopathological assessment of tendinous tissue is crucial to formulate the diagnosis and establish the severity of tendon degeneration. Nevertheless, the microscopic analysis of tendinous tissue features is often challenging. In this review, we aimed to compare the most popular scales used in tendon pathology assessment and reevaluate the role of the neovascularization process. The following scores were evaluated: the Bonar score, the Movin score, the Astrom and Rausing Score, and the Soslowsky score. Moreover, the role of neovascularization in tendon degeneration was reassessed. The Bonar system is the most commonly used in tendon pathology. According to the literature, hematoxylin and eosin with additional Alcian Blue staining seems to provide satisfactory results. Furthermore, two observers experienced in musculoskeletal pathology are sufficient for tendinopathy microscopic evaluation. The control, due to similar and typical alterations in tendinous tissue, is not necessary. Neovascularization plays an ambiguous role in tendon disorders. The neovascularization process is crucial in the tendon healing process. On the other hand, it is also an important component of the degeneration of tendinous tissue when the regeneration is incomplete and insufficient. The microscopic analysis of tendinous tissue features is often challenging. The assessment of tendinous tissue using the Bonar system is the most universal. The neovascularization variable in tendinopathy scoring systems should be reconsidered due to discrepancies in studies.

SPDL1 Is an Independent Predictor of Patient Outcome in Colorectal Cancer.

Spindle Apparatus Coiled-Coil Protein 1 (SPDL1) is a relatively recently identified coiled-coil domain containing protein and an important determinant of DNA fidelity by ensuring faithful mitosis. Hence, SPDL1 is suspected to underlie genomic (in-)stability in human cancers, yet its exact roles in these diseases remain largely underexplored. Given that genomic instability (GIN) is a crucial feature in colorectal cancer (CRC), we primarily asked whether the expression of this protein may account for differences in clinicopathological features and survival rates of CRC patients. Protein expression was evaluated by immunohistochemistry in the institutional tissue microarray (TMA), and gene expression by the analysis of publicly available datasets. To place the prognostic relevance in a predicted biological context, gene co-expression set around SPDL1 identified by public data mining was annotated and assessed for enrichment in gene ontology (GO) categories, BRITE hierarchies, and Reactome pathways. The comparison with adjacent normal tissue revealed a high expression of SPDL1 protein in a subset of tumor cases (48.84%), and these had better prognosis than the SPDL1-low expression counterpart even after adjustment for multiple confounders. SPDL1-high expression within tumors was associated with a median 56-month survival advantage, but not with any clinicopathological characteristics of our cohort. In the TCGA cohort, SPDL1 was overexpressed in tumor tissue and positively associated with improved survival, chromosome instability phenotype, and various GIN markers. In addition to the genes critically involved in the cell cycle and mitosis, a gene set co-expressed with SPDL1 contained checkpoint members of both chromosome segregation and DNA replication, as well as those associated with defective DNA repair, and retrograde vesicle-mediated transport. In conclusion, SPDL1 is an independent predictor of CRC patient survival in a possible connection with chromosomal instability.

Prognostic Significance of TLR2, SMAD3 and Localization-dependent SATB1 in Stage I and II Non-Small-Cell Lung Cancer Patients.

This study aimed to explore the prognostic value of SATB1, SMAD3, and TLR2 expression in non-small-cell lung carcinoma patients with clinical stages I-II. To investigate, we evaluated immunohistochemical staining to each of these markers using tissue sections from 69 patients from our cohort and gene expression data for The Cancer Genome Atlas (TCGA) cohort. We found that, in our cohort, high expression levels of nuclear SATB1n and SMAD3 were independent prognostic markers for better overall survival (OS) in NSCLC patients. Interestingly, expression of cytoplasmic SATB1c exhibited a significant but inverse association with survival rate, and it was an independent predictor of unfavorable prognosis. Likewise, TLR2 was a negative outcome biomarker for NSCLC even when adjusting for covariates. Importantly, stratification of NSCLCs with respect to combined expression of the three biomarkers allowed us to identify subgroups of patients with the greatest difference in duration of survival. Specifically, expression profile of SATB1n-high/SMAD3high/TLR2low was associated with the best OS, and it was superior to each single protein alone in predicting patient prognosis. Furthermore, based on the TCGA dataset, we found that overexpression of SATB1 mRNA was significantly associated with better OS, whereas high mRNA levels of SMAD3 and TLR2 with poor OS. In conclusion, the present study identified a set of proteins that may play a significant role in predicting prognosis of NSCLC patients with clinical stages I-II.

CRISPR-Based Activation of Endogenous Expression of TPM1 Inhibits Inflammatory Response of Primary Human Coronary Artery Endothelial and Smooth Muscle Cells Induced by Recombinant Human Tumor Necrosis Factor α.

Tumor necrosis factor α (TNFα) is one of the most important proinflammatory cytokines, which affects many processes associated with the growth and characteristics of endothelial, smooth muscle, and immune system cells. However, there is no correlation between most in vivo and in vitro studies on its role in endothelial cell proliferation and migration. In this study, we examined the effect of recombinant human (rh) TNFα produced in HEK293 cells on primary human coronary artery endothelial cells (pHCAECs) in the context of F-actin organization and such processes as migration and adhesion. Furthermore, we evaluated the possibility of the inhibition of the endothelial inflammatory response by the CRISPR-based regulation of TPM1 gene expression. We showed that TNFα-induced activation of pHCAECs was related to the reorganization of the actin cytoskeleton into parallel-arranged stress fibers running along the longer axis of pHCAECs. It allowed for the directed and parallel motion of the cells during coordinated migration. This change in F-actin organization promoted strong but discontinuous cell-cell contacts involved in signalization between migrating cells. Moreover, this form of intercellular connections together with locally increased adhesion was related to the formation of migrasomes and further migracytosis. Stabilization of the actin cytoskeleton through the CRISPR-based activation of endogenous expression of TPM1 resulted in the inhibition of the inflammatory response of pHCAECs following treatment with rh TNFα and stabilization of cell-cell junctions through reduced cleavage of vascular endothelial cadherin (VE-cadherin) and maintenance of the stable levels of α- and ß-catenins. We also showed that CRISPR-based activation of TPM1 reduced inflammatory activation, proliferation, and migration of primary human coronary artery smooth muscle cells. Therefore, products of the TPM1 gene may be a potential therapeutic target for the treatment of proinflammatory vascular disorders.

Prognostic Impact and Functional Annotations of KIF11 and KIF14 Expression in Patients with Colorectal Cancer.

Genomic instability (GIN) has an important contribution to the pathology of colorectal cancer (CRC). Therefore, we selected mitosis and cytokinesis kinesins, KIF11 and KIF14, as factors of potential clinical and functional value in CRC, as their aberrant expression has been suspected to underlie GIN. We examined the expression and the prognostic and biological significance of KIF11 and KIF14 in CRC via in-house immunohistochemistry on tissue microarrays, public mRNA expression datasets, as well as bioinformatics tools. We found that KIF11 and KIF14 expression, at both the protein and mRNA level, was markedly altered in cancer tissues compared to respective controls, which was reflected in the clinical outcome of CRC patients. Specifically, we provide the first evidence that KIF11 protein and mRNA, KIF14 mRNA, as well as both proteins together, can significantly discriminate between CRC patients with better and worse overall survival independently of other relevant clinical risk factors. The negative prognostic factors for OS were high KIF11 protein, high KIF11 protein + low KIF14 protein, low KIF11 mRNA and low KIF14 mRNA. Functional enrichment analysis revealed that the gene sets related to the cell cycle, DNA replication, DNA repair and recombination, among others, were positively associated with KIF11 or KIF14 expression in CRC tissues. In TCGA cohort, the positive correlations between several measures related to GIN and the expression of KIFs were also demonstrated. In conclusion, our results suggest that CRC patients can be stratified into distinct risk categories by biological and molecular determinants, such as KIF11 and KIF14 expression and, mechanistically, this is likely attributable to their role in maintaining genome integrity.

Prognostic Significance of KIF11 and KIF14 Expression in Pancreatic Adenocarcinoma.

Available biomarkers for pancreatic adenocarcinoma (PAC) are inadequate to guide individual patient prognosis or therapy. Therefore, herein we aimed to verify the hypothesis that differences in the expression of KIF11 and KIF14, i.e., molecular motor proteins being primarily implicated in cell division events could account for the differences in the clinical outcome of PAC patients. In-house immunohistochemistry was used to evaluate the protein expressions of KIF11 and KIF14 in PAC, whereas RNA-seq datasets providing transcript expression data were obtained from public sources. IHC and mRNA results were correlated with clinicopathological features and overall survival (OS). Furthermore, the genes co-expressed with KIF11 or KIF14 were predicted and functionally annotated. In our series, malignant ducts displayed more intense but less abundant KIF11 staining than normal-appearing ducts. The former was also true for KIF14, whereas the prevalence of positive staining was similar in tumor and normal adjacent tissues. Based on categorical immunoreactive scores, we found KIF11 and KIF14 to be frequently downregulated or upregulated in PAC cases, respectively, and those with elevated levels of either protein, or both together, were associated with better prognosis. Specifically, we provide the first evidence that KIF11 or KIF14 proteins can robustly discriminate between patients with better and worse OS, independently of other relevant clinical risk factors. In turn, mRNA levels of KIF11 and KIF14 were markedly elevated in tumor tissues compared to normal tissues, and this coincided with adverse prognosis, even after adjusting for multiple confounders. Tumors with low predicted KIF11 or KIF14 expression were seen to have enrichment for circadian clock, whereas those with high levels were enriched for the genomic instability-related gene set. KIF11 and KIF14 were strongly correlated with one another, and CEP55, ASPM, and GAMT were identified as the main hub genes. Importantly, the combined expression of these five genes emerged as the most powerful independent prognostic indicator associated with poor survival outcome compared to classical clinicopathological factors and any marker alone. In conclusion, our study identifies novel prognostic biomarkers for PAC, which await validation.

Expression of the Body-Weight Signaling Players: GDF15, GFRAL and RET and their clinical relevance in Gastric Cancer.

The existence, the functional role and clinical relevance of GDF15 and its signaling through a GFRAL/RET-dependent complex in gastric cancer (GC) and other human tumors remain to be elucidated, despite the widespread recognition of obesity as an important cancer-predisposing factor. Therefore, we aimed to analyze the expression levels of GDF15, GFRAL and RET in GC tissues in relation to each other and clinicopathological features, including patient survival, in order to establish a potential implication of the body-weight signaling pathway in the pathology and clinical outcome of GC. Protein expression was examined by immunohistochemistry on tissue microarrays containing 104 and 30 consecutive GC and normal gastric mucosa samples, whereas gene expression data for The Cancer Genome Atlas cohort of 413 GC patients were obtained from public sources. We found that the protein expression of GDF15, GFRAL and RET was significantly elevated and positively correlated in our set of GC tissues, which was reflected in their tendency to be overexpressed in low-grade and intermediate-grade tumors rather than high-grade ones. No other relationships between the expression status of the examined proteins and clinicopathological characteristics of GC patients were found. Through in silico data analysis, we showed that high GDF15 expression was associated with better overall survival (OS) of GC patients, whereas the opposite was true for high levels of GFRAL or RET. Specifically, GFRAL and RET emerged as independent prognostic factors associated with poor OS. Furthermore, high combined expression of the three markers: GDF15+GFRAL+RET was significantly associated with reduced OS, and it was an independent prognostic factor of borderline significance in terms of OS, when adjusted for covariates. If validated in large-scale studies, the individual and combined expression of GDF15, GFRAL and RET may provide significant clinical implications for the prognosis prediction of GC patients.

Expression of Genomic Instability-Related Molecules: Cyclin F, RRM2 and SPDL1 and Their Prognostic Significance in Pancreatic Adenocarcinoma.

In the present study, we aimed to assess the selected components of cell cycle machinery, checkpoint, DNA repair, and synthesis, namely RRM2, cyclin F, and SPDL1 in pancreatic adenocarcinomas (PAC) by in-house immunohistochemistry (IHC) and bioinformatic analysis of public datasets, in terms of expression, correlation with clinicopathological parameters, and patient survival. Sixty eight patients with pancreatic ductal adenocarcinoma (PDAC) were included in our cohort study, and IHC was performed on tissue macroarrays. RNA-Seq-based transcriptome data for 177 PACs were retrieved from the Cancer Genome Atlas (TCGA). We found cyclin F, RRM2, and SPDL1 to be overexpressed at both protein and mRNA levels in tumor tissues compared to respective controls. Based on TCGA dataset, we have demonstrated that CCNF, RRM2, and SPDL1 are potent independent prognostic markers for poor overall survival, both by themselves and even more in combination with each other. Furthermore, high CCNF mRNA expression was associated with features of cancer progression. By contrast, overexpression of cyclin F or SPDL1 proteins denoted a good prognosis in PDAC patients; however, in the case of the former protein, the results did not reach statistical significance. Specifically, high levels of SPDL1 protein emerged as the most powerful independent prognostic factor associated with a better outcome. If validated, the CCNF/RRM2/SPDL1 three-gene panel developed in this study, as well as SPDL1 protein, may provide significant clinical implications for the prognosis prediction of PAC patients.

Detection of BRAF V600E Mutation in Ganglioglioma and Pilocytic Astrocytoma by Immunohistochemistry and Real-Time PCR-Based Idylla Test.

The BRAF V600E mutation is an important oncological target in certain central nervous system (CNS) tumors, for which a possible application of BRAF-targeted therapy grows continuously. In the present study, we aim to determine the prevalence of BRAF V600E mutations in a series of ganglioglioma (GG) and pilocytic astrocytoma (PA) cases. Simultaneously, we decided to verify whether the combination of fully automated tests-BRAF-VE1 immunohistochemistry (IHC) and Idylla BRAF mutation assay-may be useful to accurately predict it in the case of specified CNS tumors. The study included 49 formalin-fixed, paraffin-embedded tissues, of which 15 were GG and 34 PA. Immunohistochemistry with anti-BRAF V600E (VE1) antibody was performed on tissue sections using the VentanaBenchMark ULTRA platform. All positive or equivocal cases on IHC and selected negative ones were further assessed using the Idylla BRAF mutation assay coupled with the Idylla platform. The BRAF-VE1 IHC was positive in 6 (6/49; 12.3%) and negative in 39 samples (39/49; 79.6%). The interpretation of immunostaining results was complicated in 4 cases, of which 1 tested positive for the Idylla BRAF mutation assay. Therefore, the overall positivity rate was 14.3%. This included 2 cases of GG and 5 cases of PA. Our study found that BRAF V600E mutations are moderately frequent in PA and GG and that for these tumor entities, IHC VE1 is suitable for screening purposes, but all negative, equivocal, and weak positive cases should be further tested with molecular biology techniques, of which the Idylla system seems to be a promising tool.

HER2, NF-κB, and SATB1 Expression Patterns in Gastric Cancer and Their Correlation with Clinical and Pathological Parameters.

Gastric cancer (GC) is currently recognized as one of the most common and fatal tumor worldwide. The identification of novel biomarkers in relation to clinical information as well as extending the knowledge on a multiple crosstalk between various oncogenic pathways implicated in GC carcinogenesis seems pivotal to limit the disease-associated mortality. Therefore, we assessed the expression of HER2, NF-κB, and SATB1 in a total of 104 gastric adenocarcinomas and 30 normal gastric samples and correlated the expression patterns with each other and with some clinicopathological variables. Protein expression was examined by immunohistochemistry (IHC) on tissue microarrays (TMAs), and fluorescence in situ hybridization (FISH) was employed to detect HER2 amplification. In the studied group, HER2 and SATB1 were found to be overexpressed in gastric cancer tissue in comparison to normal gastric mucosa. The expression status of the former protein was seen to differ according to some clinicopathological features, but without statistical significance, whereas the expression of the latter was not importantly associated with any of them. In turn, the NF-κB protein level was significantly related to the presence of lymph node metastasis. HER2 expression was not significantly correlated with that of other proteins, but a positive correlation was found between the expression of SATB1 and NF-κB. Further studies with a larger group of patients combined with in vitro mechanistic experiments are required to fully elucidate the role and relationship of HER2, NF-κB, and SATB1 expression in gastric cancer progression. However, to the best of our knowledge, this study is the first look at a simultaneous evaluation of these three markers in the samples of gastric cancer patients.

Cellular and molecular alterations induced by low­dose fisetin in human chronic myeloid leukemia cells.

The present study aimed to evaluate the cellular and molecular effects of low concentrations of the flavonoid, fisetin, on K562 human chronic myeloid leukemia cells, in the context of both potential anti­proliferative and anti­metastatic effects. Thiazolyl blue tetrazolium bromide assay, Trypan blue exclusion assay, Annexin V/propidium iodide test, cell cycle analysis, Transwell migration and invasion assays, the fluorescence staining of ß­catenin and F­actin as well as reverse transcription­quantitative polymerase chain reaction were performed to achieve the research goal. Furthermore, the nature of the interaction between fisetin and arsenic trioxide in the K562 cells was analyzed according to the Chou­Talalay median­effect method. We found that low concentrations of fisetin had not only a negligible effect on the viability and apoptosis of the K562 cells, but also modulated the mRNA levels of selected metastatic­related markers, accompanied by an increase in the migratory and invasive properties of these cancer cells. Although some markers of cell death were significantly elevated in response to fisetin treatment, these were counterbalanced through anti­apoptotic and pro­survival signals. With decreasing concentrations of fisetin and arsenic trioxide, the antagonistic interactions between the 2 agents increased. On the whole, the findings of this study suggest that careful consideration should be taken when advising cancer patients to take fisetin as a dietary supplement and when considering fisetin as a potential candidate for the treatment of chronic myeloid leukemia. Further more detailed studies are required to confirm our findings.

Expression of cyclin B1, D1 and K in non­small cell lung cancer H1299 cells following treatment with sulforaphane.

Sulforaphane (SFN) was first isolated from broccoli sprout and it is present at high concentrations in plants belonging to the Cruciferae family. The chemotherapeutic and anti­cancerogenic capacities of SFN have been demonstrated by inhibition of cancer cell proliferation in several cancer cell lines. The aim of the present study was to evaluate the effect of SFN on apoptosis, cell cycle and expression of selected cell cycle­associated proteins: Cyclin B1, cyclin D1 and cyclin K in the H1299 cell line. The non­small cell lung cancer cell line H1299 was treated with increasing concentrations of SFN (5, 10 and 15 µM) for two days. After incubation, the percentage of cells in the individual cell cycle phases, as well as the percentage of necrotic and apoptotic cells, were estimated using flow cytometry. The expression of cyclins was examined by immunofluorescence staining, flow cytometry, western blot analysis and qRT­PCR. Cyclin K was characterized by nuclear localization and increased expression after treatment with SFN. The expression data were confirmed by qRT­PCR. SFN­induced cell cycle arrest was associated with a decrease in cyclin B1 expression. Cells treated with SFN were also characterized by higher cyclin D1 and cyclin K expression. These data suggest the involvement of cyclin K in response to SFN. Moreover, we investigated the prognostic value of cyclin K, CDK12 and CDK13 in adenocarcinoma patients using 'The Kaplan­Meier plotter' (KM plotter) database. It was shown that high expression of CDK12 and CDK13 but no cyclin K proteins is associated with worse overall survival among adenocarcinoma patients.

Lidocaine induces protective autophagy in rat C6 glioma cell line.

Malignant glioma is the most common type of brain cancer with poor prognosis. Surgical resection, chemotherapy and radiotherapy are the main therapeutic options; however, in addition to their insufficient efficacy, they are associated with the pain experienced by patients. To relieve pain, local anesthetics, such as lidocaine can be used. In the present study, the effects of lidocaine on the C6 rat glioma cell line were investigated. An MTT assay and Annexin V/propidium iodide analysis indicated the increase in the percentage of apoptotic and necrotic cells in response to lidocaine. Furthermore, light microscopy analysis on the ultrastructural level presented the occurrence of vacuole­like structures associated with autophagy, which was supported by the analysis of autophagy markers (microtubule­associated protein 1A/1B­light chain 3, acridine orange and Beclin­1). Additionally, reorganization of the cytoskeleton was observed following treatment with lidocaine, which serves an important role in the course of autophagy. To determine the nature of autophagy, an inhibitor, bafilomycin A1 was applied. This compound suppressed the fusion of autophagosomes with lysosomes and increased the percentage of apoptotic cells. These results demonstrated that lidocaine may induce cytoprotective autophagy and that manipulation of this process could be an alternative therapeutic strategy in the treatment of cancer.

Evaluation of Anti-Metastatic Potential of the Combination of Fisetin with Paclitaxel on A549 Non-Small Cell Lung Cancer Cells.

The identification and development of new agents with a therapeutic potential as well as novel drug combinations are gaining the attention of scientists and clinicians as a plausible approach to improve therapeutic regimens for chemoresistant tumors. We have recently reported that the flavonoid fisetin (FIS), at physiologically attainable concentrations, acts synergistically with clinically achievable doses of paclitaxel (PTX) to produce growth inhibitory and pro-death effects on A549 human non-small cell lung cancer (NSCLC) cells. To further investigate a potential therapeutic efficacy of the combination of fisetin with paclitaxel, we decided to assess its impact on metastatic capability of A549 cells as well as its toxicity toward normal human lung fibroblast. Cell viability, cell migration, and invasion were measured by thiazolyl blue tetrazolium bromide (MTT) assay, wound healing assay, and Transwell chamber assay, respectively. The expression of metastasis-related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). Actin and vimentin filaments were examined under the fluorescence microscope. The combination of FIS and PTX significantly reduced cancer cell migration and invasion, at least partially, through a marked rearrangement of actin and vimentin cytoskeleton and the modulation of metastasis-related genes. Most of these effects of the combination treatment were significantly greater than those of individual agents. Paclitaxel alone was even more toxic to normal cells than the combination of this drug with the flavonoid, suggesting that FIS may provide some protection against PTX-mediated cytotoxicity. The combination of FIS and PTX is expected to have a synergistic anticancer efficacy and a significant potential for the treatment of NSCLC, however, further in vitro and in vivo studies are required to confirm this preliminary evidence.