Effect of LDHA Inhibition on TNF-α-Induced Cell Migration in Esophageal Cancers.

Cell migration is an essential part of the complex and multistep process that is the development of cancer, a disease that is the second most common cause of death in humans. An important factor promoting the migration of cancer cells is TNF-α, a pro-inflammatory cytokine that, among its many biological functions, also plays a major role in mediating the expression of MMP9, one of the key regulators of cancer cell migration. It is also known that TNF-α is able to induce the Warburg effect in some cells by increasing glucose uptake and enhancing the expression and activity of lactate dehydrogenase subunit A (LDHA). Therefore, the aim of the present study was to investigate the interrelationship between the TNF-α-induced promigratory activity of cancer cells and their glucose metabolism status, using esophageal cancer cells as an example. By inhibiting LDHA activity with sodium oxamate (SO, also known as aminooxoacetic acid sodium salt or oxamic acid sodium salt) or siRNA-mediated gene silencing, we found using wound healing assay and gelatin zymography that LDHA downregulation impairs TNF-α-dependent tumor cell migration and significantly reduces TNF-α-induced MMP9 expression. These effects were associated with disturbances in the activation of the ERK1/2 signaling pathway, as we observed by Western blotting. We also reveal that in esophageal cancer cells, SO effectively reduces the production of lactic acid, which, as we have shown, synergizes the stimulating effect of TNF-α on MMP9 expression. In conclusion, our findings identified LDHA as a regulator of TNF-α-induced cell migration in esophageal cancer cells by the ERK1/2 signaling pathway, suggesting that LDHA inhibitors that limit the migration of cancer cells caused by the inflammatory process may be considered as an adjunct to standard therapy in esophageal cancer patients.

The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma.

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin-proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.

The usefulness of lactate dehydrogenase measurements in current oncological practice.

One of the hallmarks of cancer cells is increased energy requirements associated with the higher rate of cellular proliferative activity. Metabolic changes in rapidly dividing cancer cells are closely associated with increased uptake of glucose and abnormal activity of lactate dehydrogenase (LDH), which regulates the processing of glucose to lactic acid. As serum LDH levels were found to be commonly increased in cancer patients and correlated with poor clinical outcome and resistance to therapy, the determination of LDH has become a standard supportive tool in diagnosing cancers or monitoring the effects of cancer treatment. The aim of this review is to summarize the current knowledge about methods and the practical utility for measuring both the total LDH and LDH isoenzymatic activities in the diagnosis, prognosis and prediction of cancer diseases.

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC.

Membrane rafts as a novel target in cancer therapy.

Membrane rafts are distinct plasma membrane microdomains that are enriched in sphingolipids and cholesterol. They organize receptors and their downstream molecules and regulate a number of intracellular signaling pathways. This review presents information on the dependence of several growth factor receptor signaling pathways on membrane rafts. It also discusses the involvement of rafts in the regulation of differentiation, apoptosis and cell migration connected with invasiveness and metastasis. Examples of known synthetic and naturally occurring substances that are known to affect lateral membrane organization in tumor cell growth are discussed as potential or actual therapeutics.

The role of MPP1/p55 and its palmitoylation in resting state raft organization in HEL cells.

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Lach et al., J. Biol. Chem., 2012, 287, 18974-18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.

miRNA-548c: a specific signature in circulating PBMCs from dilated cardiomyopathy patients.

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells.

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Achalasia--balloon dilation or surgery?

BACKGROUND: The optimal therapeutic schedule in patients with achalasia is still under discussion. The aim of this study was to review our institution's experience with myotomy and dilation in patients with achalasia. MATERIAL AND METHODS: Clinical data were available for 59 patients who had ever had myotomy (n=38), dilation (n=21), or both procedures (n=8) between 2000 and 2007. Patients were followed prospectively with objective (a barium esophagogram) and subjective (a simple survey that scored dysphagia and overall patient satisfaction with the procedure) diagnostic tools. In the group of patients after pneumatic dilatations, frequency of interventions was higher (1, 2) than in the myotomy group (0, 2) at 2-year follow-up. Patients after myotomy with recurrence of dysphagia were treated with dilations. RESULTS: Mean time of dysphagia occurrence was similar in both groups (10 months). The statistically significant differences in treatment outcomes in both groups were in favor of myotomy during 2-year follow-up. CONCLUSIONS: The data indicates that both methods of treatment might be useful in dysphagia control, but better results are obtained after myotomy. Repeat interventions are more frequent after endoscopic dilation. One method of treatment does not exclude the other. A short period of symptom relief after myotomy may suggest the myotomy was incomplete.

miR-31 and its host gene lncRNA LOC554202 are regulated by promoter hypermethylation in triple-negative breast cancer.

BACKGROUND: microRNAs have been established as powerful regulators of gene expression in normal physiological as well as in pathological conditions, including cancer progression and metastasis. Recent studies have demonstrated a key role of miR-31 in the progression and metastasis of breast cancer. Downregulation of miR-31 enhances several steps of the invasion-metastasis cascade in breast cancer, i.e., local invasion, extravasation and survival in the circulation system, and metastatic colonization of distant sites. miR-31 exerts its metastasis-suppressor activity by targeting a cohort of pro-metastatic genes, including RhoA and WAVE3. The molecular mechanisms that lead to the loss of miR-31 and the activation of its pro-metastatic target genes during these specific steps of the invasion-metastasis cascade are however unknown. RESULTS: In the present report, we identify promoter hypermethylation as one of the major mechanisms for silencing miR-31 in breast cancer, and in the triple-negative breast cancer (TNBC) cell lines of basal subtype, in particular. miR-31 maps to the intronic sequence of a novel long non-coding (lnc)RNA, LOC554202 and the regulation of its transcriptional activity is under control of LOC554202. Both miR-31 and the host gene LOC554202 are down-regulated in the TNBC cell lines of basal subtype and over-expressed in the luminal counterparts. Treatment of the TNBC cell lines with either a de-methylating agent alone or in combination with a de-acetylating agent resulted in a significant increase of both miR-31 and its host gene, suggesting an epigenetic mechanism for the silencing of these two genes by promoter hypermethylation. Finally, both methylation-specific PCR and sequencing of bisulfite-converted DNA demonstrated that the LOC554202 promoter-associated CpG island is heavily methylated in the TNBC cell lines and hypomethylated in the luminal subtypes. CONCLUSION: Loss of miR-31 expression in TNBC cell lines is attributed to hypermethylation of its promoter-associated CpG island. Together, our results provide the initial evidence for a mechanism by which miR-31, an important determinant of the invasion metastasis cascade, is regulated in breast cancer.

Expression of MMP-2, TIMP-2, TGF-ß1, and decorin in Dupuytren's contracture.

To investigate the mechanisms underlying matrix deposition in Dupuytren's disease, the expression of gelatinase A (MMP-2), the tissue inhibitor of metalloproteinase-2 (TIMP-2), transforming growth factor beta 1 (TGF-ß1), decorin (DCN), and periostin was studied. The level of relative MMP-2 activation was investigated using zymography. The mRNA expression of MMP-2, TIMP-2, TGF-ß1, and DCN was detected using reverse transcription polymerase chain reaction (RT-PCR), while the presence of protein was detected using immunohistochemical (IHC) and Western blot techniques. The level of MMP-2 activation was significantly elevated in tissues with Dupuytren's contracture. RT-PCR demonstrated significantly higher expression of MMP-2, TIMP-2, TGF-ß1, and DCN mRNA in the pathological tissues; and the IHC and immunoblotting studies revealed elevated expression of TGF-ß1, DCN, and periostin. The balance between MMP-2 and TIMP-2 was disrupted in patients with Dupuytren's disease. TGF-ß1, DCN, and periostin are involved in extracellular matrix (ECM) homeostasis in Dupuytren's contracture.

MMP9: A Tough Target for Targeted Therapy for Cancer.

Having the capability to proteolyze diverse structural and signaling proteins, matrix metalloproteinase 9 (MMP9), one of the best-studied secretory endopeptidases, has been identified as a crucial mediator of processes closely associated with tumorigenesis, such as the extracellular matrix reorganization, epithelial to mesenchymal transition, cell migration, new blood vessel formation, and immune response. In this review, we present the current state of knowledge on MMP9 and its role in cancer growth in the context of cell adhesion/migration, cancer-related inflammation, and tumor microenvironment formation. We also summarize recent achievements in the development of selective MMP9 inhibitors and the limitations of using them as anticancer drugs.

Vitamin B6 in Health and Disease.

Vitamin B6 is a fascinating molecule involved in the vast majority of changes in the human body because it is a coenzyme involved in over 150 biochemical reactions. It is active in the metabolism of carbohydrates, lipids, amino acids, and nucleic acids, and participates in cellular signaling. It is an antioxidant and a compound with the ability to lower the advanced glycation end products (AGE) level. In this review, we briefly summarize its involvement in biochemical pathways and consider whether its deficiency may be associated with various diseases such as diabetes, heart disease, cancer, or the prognosis of COVID-19.

Invadopodia: clearing the way for cancer cell invasion.

The invasive nature of many cancer cells involves the formation of F-actin-based, lipid-raft-enriched membrane protrusions known as invadopodia or, more broadly, invadosomes. Invadopodia are specialized adhesive structures arising from ventral cell surface within cell-extracellular matrix (ECM) contacts and concentrate high proteolytic activities that allow cells to overcome the dense scaffold of local microenvironment, comprising a natural barrier to cell spreading. This degradative activity distinguishes invadopodia from other adhesive structures like focal adhesions, lamellipodia or filopodia, and is believed to drive cancer progression.

Immunohistochemical study of decorin expression in polyps and carcinomas of the colon.

BACKGROUND: Recent studies showed that a small leucine-rich proteoglycan, decorin, may suppress tumor progression as a natural anticancer agent negatively controlling cellular growth. It was hypothesized that physiological expression of decorin may be associated with cellular senescence of the colorectal mucosa and that its down-regulation, promoting an increase in cellular proliferation, could participate in the progression of adenoma to adenocarcinoma. Therefore the expression of decorin in hyperplastic and neoplastic polyps of the colorectum was examined and compared with normal colonic mucosa and colon cancer tissues. MATERIAL/METHODS: Tissue samples were obtained from 41 patients with different types of colonic polyps (6 hyperplastic adenomas, 34 neoplastic adenomas, and 1 adenomatous polyp with focal carcinoma) and 12 patients with colon cancer. Seven samples of normal colon tissue were used as controls. Paraffin-embedded samples were used for immunohistochemical study. RESULTS: Normal and hyperplastic tissues and the majority of tubular adenomas showed strong expression of decorin in the stroma. Adenomas with a villous component showed moderate and very low decorin immunoreactivity. The decrease in decorin reactivity in tubulo-villous adenomas was significant as compared with other polyps and controls. Weak decorin immunoreactivity in stroma adjacent to clusters of cancerous cells was also found in most cases of common types of adenocarcinoma, but not in adenocarcinoma mucinosum. CONCLUSIONS: The expression of decorin may be involved in the differentiation of colonic polyps and reduced expression of decorin may abrogate the defensive potential of stromal tissue and promote the development of common types of colon carcinoma.

Zenker diverticulum: Experience in surgical treatment of large diverticula.

The purpose of this retrospective study is to show that transcervical diverticulectomy (TD) in treatment of Zenker diverticulum (ZD) can still be a first choice procedure in selected patients and in experienced hands its safety might be compared to the minimally invasive endoscopic diverticulostomy.The study cohort consisted of 44 patients (18 male, 26 female) operated for (ZD). All the patients underwent open diverticulectomy. The decision to choose open surgical repair depended on surgical risk, age of the patient, size of the diverticular septum (the distance between the top of the diverticulum and its bottom on barium study), and patient's preference.Mean age of patients was 64.6 ±â€Š11.9 years; range: 26 to 88 years. A total of 36.4% out of them finished 70 years. Postoperative mortality was nil. Two major complications (4.5%) requiring surgical intervention occurred: leak and hematoma.Data were analyzed by t test for independent samples using Statistica 12.5 software. P value <0.05 was considered statistically significant.Surgical treatment of patients with ZD should be individualized. Large Zenker diverticula with the septum longer than 6 cm should preferably be resected through an open approach because it is not possible to remove the septum completely during one-step endoscopic procedure and diverticulostomy creates a weak and large common cavity in the esophagus. Surgical repair is effective for all sizes of diverticula, but its most serious complications such as leakage or laryngeal nerve injury should be considered, especially in elderly patients with comorbidities. However, age alone should not be the main criterion if choosing the treatment option.

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells.

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

Fibroblast activating protein-α expression in squamous cell carcinoma of the esophagus in primary and irradiated tumors: the use of archival FFPE material for molecular techniques.

There are numerous reports suggesting that fibroblast activating protein-α (FAP-α) plays an important role in invasion of various tumor types. We studied the expression pattern of FAP-α in esophageal squamous cell carcinoma (ESCC) patients who had not been treated primarily and those who had received neoadjuvant radiochemotherapy. Our goal was to establish whether readily available tissue specimens fixed in formalin and stored in paraffin blocks for years might still be a source of FAP-α RNA for PCR analysis. The study included 20 patients divided into two groups, 10 patients in each group. We evaluated the expression of FAP-α by PCR techniques in fresh frozen and in paraffin-embedded tissues, and compared it to the expression in non-cancer tissues. To detect the protein expression level of FAP-α in paraffin-embedded tissues we used chromogenic immunohistochemical (IHC) staining. Data were analyzed by t-test or the nonparametric Wilcoxon matched pair test using Statistica 12.5 software. We observed an increased level of the FAP-alpha gene and protein expression in cancer tissues when compared with their corresponding normal tissues. However, statistically significant differences were found only in the group of patients untreated before surgery. RNA extracted from paraffin-embedded tissue sections had very low quality, especially in the context of degradation. FAP-α remains a highly altered participant of a complex microenvironment in esophageal squamous cell carcinoma, and its role in cell signaling requires further study. In this paper, we conclude that the use of a regular RT-PCR method for diagnostic purposes, which we have presented in an earlier paper, can be as good as qRT-PCR. Also, immunohistochemistry proved to be very useful and the only reliable method that can be used on formalin-fixed, paraffin-embedded tissues stored long term.