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1.
Ann Surg Oncol ; 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38969859

ABSTRACT

BACKGROUND: Analysis of temporal trends of urinary diversion (UD) and identification of predictive factors for continent urinary diversion (CUD) in patients with bladder cancer (BC) is scarce and data on large cohorts are missing. We aimed to describe longitudinal temporal trends and predictive factors for UD among patients with BC receiving radical cystectomy (RC). PATIENTS AND METHODS: We retrospectively analysed institutional data collected from patients undergoing RC from 1986 to 2022 to describe changes in patients' characteristics and UD. Primary end points were patients' characteristics associated with type of UD. Logistic regression analysis was used to determine predictive factors for CUD. RESULTS: In total, 2224 patients (77.16% male, 22.84% female) with a mean age of 66 years [standard deviation (SD), 10.64 years] were included. We observed an increase in mean age from 59.86 (10.8) years (1986-1990) to 69.85 (9.99) years (2016-2022) (p < 0.001). The proportion of CUD gradually declined from 43.72% (94/215; 1986-1990) to 18.38% (86/468; 2016-2022). Patients who were male [odds ratio (OR): 1.92, 95% confidence interval (CI): 1.43-2.57, p < 0.001), younger (OR: 0.88, 95% CI: 0.87-0.89, p < 0.001) and had no hydronephrosis prior to RC (OR: 2.2, 95% CI: 1.66-2.92, p < 0.001) were more likely to receive CUD. CONCLUSIONS: We report the largest European single-center cohort of UD after RC, demonstrating a significant shift from CUD to IUD, accompanied by an increasing age. Finally, our data mirrors the development and extensive experience with the Mainz Pouch-I in the 1980's and 1990's together with other colon pouches.

2.
Int J Mol Sci ; 24(15)2023 Aug 05.
Article in English | MEDLINE | ID: mdl-37569837

ABSTRACT

While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases.


Subject(s)
Toll-Like Receptor 1 , Toll-Like Receptor 2 , Humans , Toll-Like Receptor 2/metabolism , Dimerization , Toll-Like Receptor 1/metabolism , Toll-Like Receptors , Anti-Inflammatory Agents , Toll-Like Receptor 6/metabolism , Toll-Like Receptor 10
3.
Int J Mol Sci ; 22(17)2021 Aug 26.
Article in English | MEDLINE | ID: mdl-34502140

ABSTRACT

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.


Subject(s)
Genes, Reporter , Light , NF-kappa B/metabolism , Optogenetics/methods , Toll-Like Receptor 4/metabolism , Cell Adhesion , Cell Movement , HEK293 Cells , HeLa Cells , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Oxygen/metabolism , Pancreas/cytology , Podosomes/metabolism , Signal Transduction , Toll-Like Receptor 4/genetics
4.
Molecules ; 26(3)2021 Jan 30.
Article in English | MEDLINE | ID: mdl-33573155

ABSTRACT

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Melanoma/drug therapy , Quercetin/pharmacology , Antineoplastic Agents/chemistry , Carboxylic Ester Hydrolases/genetics , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , MAP Kinase Signaling System/drug effects , Melanoma/genetics , Melanoma/pathology , NF-E2-Related Factor 2/genetics , NF-kappa B/genetics , Phosphorylation/drug effects , Quercetin/chemistry , Spheroids, Cellular/drug effects
5.
Int J Mol Sci ; 21(13)2020 Jun 28.
Article in English | MEDLINE | ID: mdl-32605217

ABSTRACT

Specific gene promoter DNA methylation is becoming a powerful epigenetic biomarker in cancer diagnostics. Five genes (CDH1, CDKN2Ap16, RASSF1A, TERT, and WT1) were selected based on their frequently published potential as epigenetic markers. Diagnostic promoter methylation assays were generated based on bisulfite-converted DNA pyrosequencing. The methylation patterns of 144 non-small-cell lung cancer (NSCLC) and 7 healthy control formalin-fixed paraffin-embedded (FFPE) samples were analyzed to evaluate the applicability of the putative diagnostic markers. Statistically significant changes in methylation levels are shown for TERT and WT1. Furthermore, 12 NSCLC and two benign lung cell lines were characterized for promoter methylation. The in vitro tests involved a comparison of promoter methylation in 2D and 3D cultures, as well as therapeutic tests investigating the impact of CDH1/CDKN2Ap16/RASSF1A/TERT/WT1 promoter methylation on sensitivity to tyrosine kinase inhibitor (TKI) and DNA methyl-transferase inhibitor (DNMTI) treatments. We conclude that the selected markers have potential and putative impacts as diagnostic or even predictive marker genes, although a closer examination of the resulting protein expression and pathway regulation is needed.


Subject(s)
Antigens, CD/genetics , Biomarkers, Tumor/genetics , Cadherins/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA Methylation , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , Promoter Regions, Genetic , Aged , Antigens, CD/metabolism , Biomarkers, Tumor/metabolism , Cadherins/metabolism , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Middle Aged , Prognosis , Tumor Cells, Cultured
6.
J Nat Prod ; 79(1): 106-15, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26684177

ABSTRACT

A rapid and exhaustive one-step biomass extraction as well as an enrichment and cleanup procedure has been developed for HPLC-UV detection and quantification of closely related [7.7]paracyclophanes and structural derivatives based on a two-phase solvent system. The procedure has been validated using the biomass of the carbamidocyclophane- and cylindrocyclophane-producing cyanobacterium Nostoc sp. CAVN2 and was utilized to perform a screening comprising 102 cyanobacterial strains. As a result, three new cylindrocyclophane-related alkylresorcinols, cylindrofridins A-C (1-3), and known cylindrocyclophanes (4-6) were detected and isolated from Cylindrospermum stagnale PCC 7417. Structures of 1-3 were elucidated by a combination of 1D and 2D NMR experiments, HRMS, and ECD spectroscopy. Cylindrofridin A (1) is the first naturally occurring [7.7]paracyclophane-related monomeric derivative. In contrast, cylindrofridins B (2) and C (3) represent dimers related to 1. Due to chlorination at the alkyl carbon atom in 1-3, the site of [7.7]paracyclophane macrocycle formation, the cylindrofridins represent linearized congeners of the cylindrocyclophanes. Compounds 1-3 were not toxic against nontumorigenic HaCaT cells (IC50 values >25 µM) compared to the respective cylindrocyclophanes, but 1 was the only cylindrofridin showing moderate activity against methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae with MIC values of 9 and 17 µM, respectively.


Subject(s)
Cyanobacteria/chemistry , Resorcinols/isolation & purification , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Resorcinols/chemistry , Resorcinols/pharmacology , Streptococcus pneumoniae/drug effects , Structure-Activity Relationship
7.
Mar Drugs ; 14(1): 21, 2016 Jan 20.
Article in English | MEDLINE | ID: mdl-26805858

ABSTRACT

In this study, the influence of halide ions on [7.7]paracyclophane biosynthesis in the cyanobacterium Nostoc sp. CAVN2 was investigated. In contrast to KI and KF, supplementation of the culture medium with KCl or KBr resulted not only in an increase of growth but also in an up-regulation of carbamidocyclophane production. LC-MS analysis indicated the presence of chlorinated, brominated, but also non-halogenated derivatives. In addition to 22 known cylindrocyclophanes and carbamidocyclophanes, 27 putative congeners have been detected. Nine compounds, carbamidocyclophanes M-U, were isolated, and their structural elucidation by 1D and 2D NMR experiments in combination with HRMS and ECD analysis revealed that they are brominated analogues of chlorinated carbamidocyclophanes. Quantification of the carbamidocyclophanes showed that chloride is the preferably utilized halide, but incorporation is reduced in the presence of bromide. Evaluation of the antibacterial activity of 30 [7.7]paracyclophanes and related derivatives against selected pathogenic Gram-positive and Gram-negative bacteria exhibited remarkable effects especially against methicillin- and vancomycin-resistant staphylococci and Mycobacterium tuberculosis. For deeper insights into the mechanisms of biosynthesis, the carbamidocyclophane biosynthetic gene cluster in Nostoc sp. CAVN2 was studied. The gene putatively coding for the carbamoyltransferase has been identified. Based on bioinformatic analyses, a possible biosynthetic assembly is discussed.


Subject(s)
Anti-Bacterial Agents/biosynthesis , Cyanobacteria/metabolism , Ethers, Cyclic/metabolism , Culture Media , Fluorides/pharmacology , Humans , Potassium Compounds/pharmacology , Potassium Iodide/pharmacology , Up-Regulation/drug effects
8.
Urol Int ; 95(3): 329-35, 2015.
Article in English | MEDLINE | ID: mdl-26397097

ABSTRACT

INTRODUCTION: To analyze the primary stone free rate (pSFR) of flexible ureterorenoscopy (fURS) in the treatment of renal stones and to identify clinical predictors for the primary freedom from renal stones. MATERIALS AND METHODS: Two hundred and seventy five patients, who underwent fURS for kidney stones were analyzed. RESULTS: Index stone size was 6 mm. The stone was located in the lower calyx in 48%. Ureteral access sheath was used in 97%. Operation time was 35 min and primary stone clearance was 83%. pSFR increased from 74% in 2012 to 83% in 2013 and 90% in 2014 (p = 0.001). Preoperative stenting, index stone size, cumulative stone size, lithotripsy, ureteral access sheath and operation time were significantly correlated with the pSFR by univariate analysis. Multivariate regression analysis showed index stone size, cumulative stone size, ureteral access sheath and operation time as independent parameters for pSFR. CONCLUSIONS: fURS for kidney stones is safe with a high pSFR. Clinical parameters for pSFR are stone size, use of ureteral access sheath and operation time. In future, the effective use of fURS for the removal of kidney stones needs to be checked by prospective randomized trials.


Subject(s)
Kidney Calculi/surgery , Standard of Care , Ureteroscopy/standards , Adult , Female , Humans , Male , Middle Aged , Tertiary Care Centers , Ureteroscopes
9.
BJUI Compass ; 5(1): 90-100, 2024 Jan.
Article in English | MEDLINE | ID: mdl-38179024

ABSTRACT

Objectives: Most renal tumours can be treated with a partial nephrectomy, with robot-assisted partial nephrectomy becoming the new gold standard. This procedure is challenging to learn in a live setting, especially the enucleation and renorraphy phases. In this study, we attempted to evaluate face, content, and preliminary construct validity of a 3D-printed silicone renal tumour model in robotic training for robot-assisted partial nephrectomy. Materials and Methods: We compared the operative results of three groups of surgeons with different experience levels (>20 partial nephrectomies, 1-20 partial nephrectomies and no experience at all) performing a robotic tumour excision of a newly developed silicone model with four embedded 3D-printed renal tumours. We evaluated the participants' performance using surgical margins, excision time, total preserved parenchyma, tumour injury and GEARS score (as assessed by two blinded experts) for construct validity. Postoperatively, the participants were asked to complete a survey to evaluate the usefulness, realism and difficulty of the model as a training and/or evaluation model. NASA-TLX scores were used to evaluate the operative workload. Results: Thirty-six participants were recruited, each group consisting of 10-14 participants. The operative performance was significantly better in the expert group as compared to the beginner group. NASA-TLX scores proved the model to be of an acceptable difficulty level.Expert group survey results showed an average score of 6.3/10 on realism of the model, 8.2/10 on the usefulness as training model and 6.9/10 score on the usefulness as an evaluation tool. GEARS scores showed a non-significant tendency to improve between trials, emphasizing its potential as a training model. Conclusion: Face and content validity of our 3D renal tumour model were demonstrated. The vast majority of participants found the model realistic and useful for training and for evaluation. To evaluate construct and predictive validity, we require further research, aiming to compare the results of 3D-model trained surgeons with those of untrained surgeons in real-life surgery.

10.
J Immunother Cancer ; 12(5)2024 May 03.
Article in English | MEDLINE | ID: mdl-38702145

ABSTRACT

BACKGROUND: Skeletal morbidity in patients with cancer has a major impact on the quality of life, and preserving bone health while improving outcomes is an important goal of modern antitumor treatment strategies. Despite their widespread use in early disease stages, the effects of immune checkpoint inhibitors (ICIs) on the skeleton are still poorly defined. Here, we initiated a comprehensive investigation of the impact of ICIs on bone health by longitudinal assessment of bone turnover markers in patients with cancer and by validation in a novel bioengineered 3D model of bone remodeling. METHODS: An exploratory longitudinal study was conducted to assess serum markers of bone resorption (C-terminal telopeptide, CTX) and formation (procollagen type I N-terminal propeptide, PINP, and osteocalcin, OCN) before each ICI application (programmed cell death 1 (PD1) inhibitor or programmed death-ligand 1 (PD-L1) inhibitor) for 6 months or until disease progression in patients with advanced cancer and no evidence of bone metastases. To validate the in vivo results, we evaluated osteoclast (OC) and osteoblast (OB) differentiation on treatment with ICIs. In addition, their effect on bone remodeling was assessed by immunohistochemistry, confocal microscopy, and proteomics analysis in a dynamic 3D bone model. RESULTS: During the first month of treatment, CTX levels decreased sharply but transiently. In contrast, we observed a delayed increase of serum levels of PINP and OCN after 4 months of therapy. In vitro, ICIs impaired the maturation of preosteoclasts by inhibiting STAT3/NFATc1 signaling but not JNK, ERK, and AKT while lacking any direct effect on osteogenesis. However, using our bioengineered 3D bone model, which enables the simultaneous differentiation of OB and OC precursor cells, we confirmed the uncoupling of the OC/OB activity on exposure to ICIs by demonstrating impaired OC maturation along with increased OB differentiation. CONCLUSION: Our study indicates that the inhibition of the PD1/PD-L1 signaling axis interferes with bone turnover and may exert a protective effect on bone by indirectly promoting osteogenesis.


Subject(s)
Bone Remodeling , Immune Checkpoint Inhibitors , Humans , Bone Remodeling/drug effects , Male , Female , Prospective Studies , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Middle Aged , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/metabolism , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/metabolism , Aged , Longitudinal Studies , Neoplasms/drug therapy , Adult
11.
Int J Cancer ; 132(3): 521-30, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-22733455

ABSTRACT

Metastasis is associated with poor prognosis for melanoma responsible for about 90% of skin cancer-related mortality. To metastasize, melanoma cells must escape keratinocyte control, invade across the basement membrane and survive in the dermis by resisting apoptosis before they can intravasate into the circulation. α-Catulin (CTNNAL1) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, binds to ß-catenin and increases the level of both α-catenin and ß-catenin and therefore has potential effects on inflammation, apoptosis and cytoskeletal reorganization. Here, we show that α-catulin is highly expressed in melanoma cells. Expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. Knockdown of α-catulin promoted adhesion to and inhibited migration away from keratinocytes in an E-cadherin-dependent manner and decreased the transmigration through a keratinocyte monolayer, as well as in Transwell assays using collagens, laminin and fibronectin coating. Moreover, knockdown promoted homotypic spheroid formation and concomitantly increased E-cadherin expression along with downregulation of transcription factors implicated in its repression (Snail/Slug, Twist and ZEB). Consistent with the molecular changes, α-catulin provoked invasion of melanoma cells in a three-dimensional culture assay by the upregulation of matrix metalloproteinases 2 and 9 and the activation of ROCK/Rho. As such, α-catulin may represent a key driver of the metastatic process, implicating potential for therapeutic interference.


Subject(s)
Cadherins/genetics , Cadherins/metabolism , Melanoma/metabolism , Melanoma/pathology , alpha Catenin/metabolism , Cadherins/biosynthesis , Cell Adhesion/genetics , Cell Line, Tumor , Disease Progression , Down-Regulation , Epidermis/metabolism , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Humans , Keratinocytes/metabolism , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Melanocytes/metabolism , Melanoma/genetics , Melanoma/secondary , NF-kappa B/genetics , NF-kappa B/metabolism , Neoplasm Invasiveness , Neoplasm Metastasis , Snail Family Transcription Factors , Spheroids, Cellular , Transcription Factors/biosynthesis , Transcriptional Activation , Up-Regulation , alpha Catenin/genetics , beta Catenin/metabolism , rho-Associated Kinases/metabolism
12.
Front Immunol ; 14: 1264889, 2023.
Article in English | MEDLINE | ID: mdl-38077393

ABSTRACT

Toll-like receptor 4 (TLR4) are part of the innate immune system. They are capable of recognizing pathogen-associated molecular patterns (PAMPS) of microbes, and damage-associated molecular patterns (DAMPs) of damaged tissues. Activation of TLR4 initiates downstream signaling pathways that trigger the secretion of cytokines, type I interferons, and other pro-inflammatory mediators that are necessary for an immediate immune response. However, the systemic release of pro-inflammatory proteins is a powerful driver of acute and chronic inflammatory responses. Over the past decades, immense progress has been made in clarifying the molecular and regulatory mechanisms of TLR4 signaling in inflammation. However, the most common strategies used to study TLR4 signaling rely on genetic manipulation of the TLR4 or the treatment with agonists such as lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, which are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner. This review provides an overview of the structure, function and signaling pathways of the TLR4 and its fundamental role in endothelial cells under physiological and inflammatory conditions, as well as the advances in TLR4 modulation strategies.


Subject(s)
Endothelial Cells , Toll-Like Receptor 4 , Humans , Toll-Like Receptor 4/metabolism , Endothelial Cells/metabolism , Signal Transduction , Lipopolysaccharides , Cytokines/metabolism , Inflammation
13.
Cells ; 12(5)2023 02 22.
Article in English | MEDLINE | ID: mdl-36899833

ABSTRACT

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.


Subject(s)
Lipopolysaccharides , Toll-Like Receptor 4 , Endothelial Cells/metabolism , Gene Expression , Lipopolysaccharides/pharmacology , Signal Transduction , Toll-Like Receptor 4/metabolism , Human Umbilical Vein Endothelial Cells , Humans
14.
Cells ; 12(10)2023 05 19.
Article in English | MEDLINE | ID: mdl-37408259

ABSTRACT

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.


Subject(s)
Chemotaxis , Toll-Like Receptor 2 , Humans , Cell Adhesion , Endothelial Cells/metabolism , Integrins , THP-1 Cells , Toll-Like Receptor 2/metabolism , Cell Movement
15.
Prostate ; 72(16): 1719-35, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22473339

ABSTRACT

We evaluated whether low-dosed interferon alpha (IFNa) may augment the anti-tumor potential of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer cells in vitro and in vivo. PC-3, DU-145, or LNCaP prostate cancer cells were treated with VPA (1 mM), IFNa (200 U/ml), or with the VPA-IFNa combination. Tumor cell growth, cell cycle progression, and cell cycle regulating proteins were then investigated by the MTT assay, flow cytometry, and western blotting. Tumor cell adhesion to endothelium or to immobilized extracellular matrix proteins, as well as migratory properties of the cells, were evaluated. Integrin α and ß adhesion molecules and alterations of cell signaling pathways were analyzed. Finally, effects of the drug treatment on prostate cancer growth in vivo were determined in the NOD/SCID mouse model. VPA reduced tumor cell adhesion, migration, and growth in vitro. A much stronger anti-cancer potential was evoked by the VPA-IFNa combination, although IFNa in itself did not block growth or adhesion. The same effect was seen when tumor growth was evaluated in vivo. Molecular analysis revealed distinct elevation of histone H3 acetylation caused by VPA which was further up-regulated by VPA-IFNa, whereas IFNa alone did not alter H3 acetylation. The combinatorial benefit became obvious in Akt phosphorylation, p21 and p27 and integrin α1, α3, and ß1 expression. Application of low-dosed IFNa to a VPA based regimen profoundly boosts the anti-tumor properties of VPA. The combined use of VPA and low-dosed IFNa may therefore be an innovative option in treating advanced prostate cancer.


Subject(s)
Cell Movement/drug effects , Cell Proliferation/drug effects , Histone Deacetylase Inhibitors/pharmacology , Interferon-alpha/pharmacology , Prostate/drug effects , Prostatic Neoplasms/drug therapy , Valproic Acid/pharmacology , Animals , Cell Adhesion/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Histone Deacetylase Inhibitors/therapeutic use , Human Umbilical Vein Endothelial Cells , Humans , Interferon-alpha/therapeutic use , Male , Mice , Prostate/pathology , Prostatic Neoplasms/pathology , Signal Transduction/drug effects , Valproic Acid/therapeutic use
16.
BJU Int ; 109(2): 214-9, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21592293

ABSTRACT

OBJECTIVE: To evaluate clinical predictors for Gleason score upgrade (GSU) in radical prostatectomy (RP) specimen, especially in patients with 'very' low risk PCA (T1c and biopsy Gleason score ≤6 and PSA <10 ng/ml and ≤2 positive biopsy cores and PSA density <0.15). PATIENTS AND METHODS: 402 consecutive patients undergoing RP between 2004 and 2006, including a subgroup of 62 patients with 'very' low risk PCA, were examined. Patients were categorized for clinically relevant GSU (defined as upgrade into a higher PCA risk category). Parameters including number of biopsy cores obtained, positive biopsy cores, prostate weight, PSA, DRE and pathology department were evaluated for their role as predictors. Furthermore, GSU in RP specimen was analyzed for its impact on pT-stage. RESULTS: Clinically relevant GSU occurred in 38.1% in the whole cohort and in 32.3% in the 'very' low risk PCA subgroup. Gleason score downgrade (GSD) occurred in 4.7%. Number of biopsy cores obtained and prostate weight were independent negative predictors of GSU in all 402 patients (P = 0.02 and P = 0.03, respectively). In the 'very' low risk group, only number of biopsy cores obtained revealed as an independent negative predictor of GSU (P = 0.02). PSA, DRE, number of positive cores or pathology department were not associated to GSU. In the 'very' low risk group, GSU was related with extracapsular tumor extension (P = 0.05). CONCLUSIONS: Clinically relevant GSU in RP specimen is still a challenging problem. Increasing the number of biopsy cores lower this risk significantly. GSD is rare and thus of minor importance for treatment decisions.


Subject(s)
Neoplasm Grading/classification , Prostate/pathology , Prostatic Neoplasms/pathology , Aged , Aged, 80 and over , Biopsy, Needle , Disease Progression , Early Detection of Cancer/methods , Humans , Male , Middle Aged , Predictive Value of Tests , Prostate-Specific Antigen/blood , Prostatectomy , Prostatic Neoplasms/blood , Prostatic Neoplasms/surgery , Retrospective Studies
17.
Cancer Med ; 11(4): 956-967, 2022 02.
Article in English | MEDLINE | ID: mdl-34951143

ABSTRACT

Malignant melanoma is the deadliest form of skin cancer and NRF2 has been proposed as a main regulator of tumor cell malignancy. Still the mechanisms how NRF2 is contributing to melanoma progression are incompletely understood. Here we analyzed the effects of either NRF2 induction or depletion, and we also quantified changes on the whole cell proteome level. Our results showed that inhibition of NRF2 leads to a loss of reactive oxygen species protection, but at the same time to an induction of an epithelial mesenchymal transition (EMT) phenotype and an up-regulation of the stem cell marker CD44. Additionally, cells devoid of NRF2 showed increased cell viability after treatment with a MYC and a BRAF inhibitor. Importantly, survival upon vemurafenib treatment was dependent on CD44 expression. Finally, analysis of archival melanoma patient samples confirmed a vice versa relationship of NRF2 and CD44 expression. In summary, we recorded changes in the proteome after NRF2 modulation in melanoma cells. Surprisingly, we identified that NRF2 inhibition lead to induction of an EMT phenotype and an increase in survival of cells after apoptosis induction. Therefore, we propose that it is important for future therapies targeting NRF2 to consider blocking EMT promoting pathways in order to achieve efficient tumor therapy.


Subject(s)
Melanoma , Proteome , Apoptosis , Cell Line, Tumor , Humans , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Melanoma/drug therapy , Melanoma/genetics , Melanoma/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Proteome/metabolism , Proto-Oncogene Proteins B-raf/genetics , Up-Regulation , Vemurafenib/pharmacology
18.
Anticancer Drugs ; 22(10): 1002-9, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21822119

ABSTRACT

Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.


Subject(s)
Cell Cycle/drug effects , Histone Deacetylase Inhibitors/pharmacology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/pathology , Valproic Acid/pharmacology , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Cyclins/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Humans , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Urinary Bladder Neoplasms/metabolism
19.
BJU Int ; 108(8 Pt 2): E284-8, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21244611

ABSTRACT

OBJECTIVE: •To analyze the impact of a delayed radical cystectomy (rCx) and clinical variables on cancer-specific survival (CSS) in patients presenting 'high-risk' carcinoma not invading bladder muscle (nmiBCA). PATIENTS AND METHODS: •Between 1989 and 2006, 278 patients who presented 'high-risk' nmiBCA finally underwent rCx in our institution. •CSS was correlated with clinical variables such as the number of transurethral resections of the bladder (TURBs), interval between first TURB and rCx, adjuvant therapies, tumour upstaging at rCx, tumour stage and lymph node (LN) status. RESULTS: •The overall 5- and 10-year CSS was 82% and 76%, respectively. Significant correlations were found between the 5-year CSS and categorized number of TURBs (≤2 vs >2: 88% vs 71%; P= 0.001), interval between first TURB and rCx (≤4 months vs >4 months: 86% vs 77%; P= 0.04), adjuvant therapies (no vs yes: 86% vs 66%; P= 0.001), tumour upstaging at rCx (no vs yes: 89% vs 67%; P < 0.001), tumour stage at rCx (bladder confined vs non-confined: 88% vs 56%; P < 0.001) and LN status (no vs yes: 88% vs 36%; P < 0.001). •Multivariate analysis identified categorized number of TURBs (hazard ratio, HR, 0.14; 95% CI, 0.07-0.44; P < 0.001), categorized interval between first TURB and rCx (HR, 3.27; 95% CI, 1.24-8.59; P= 0.017), LN status (HR, 0.13; 95% CI, 0.06-0.26; P < 0.001) and tumour stage at rCx (HR, 0.49; 95% CI, 0.26-0.92; P= 0.03) as independent risk factors for CSS. CONCLUSION: •Delay of rCx in 'high-risk' nmiBCA deteriorates CSS and should be avoided. The number of TURBs and the interval between first TURB and rCx are causative factors for delayed rCx and are independently correlated with CSS.


Subject(s)
Cystectomy , Urinary Bladder Neoplasms/mortality , Urinary Bladder Neoplasms/surgery , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Neoplasm Invasiveness , Retrospective Studies , Risk Factors , Survival Rate , Time Factors , Urinary Bladder Neoplasms/pathology
20.
BJU Int ; 107(5): 755-759, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20880193

ABSTRACT

OBJECTIVE: • To determine the value of systematic intraoperative peripheral frozen sections (FS) with or without extended resection during nerve-sparing radical prostatectomy for prediction of biochemical recurrence (BCR) compared with inked surgical margins. PATIENTS AND METHODS: • Between 1999 and 2003, in a prospective study, multiple peripheral FS (median 14; range 5-20) were taken from the urethral stump, circumferentially from the bladder neck, and from the lateral pedicles in 200 consecutive bilateral nerve-sparing radical prostatectomies for clinically localized prostate cancer by a single surgeon. • Patients with stage pT3b or more and/or positive lymph nodes were excluded. • Of the 188 patients, 178 (94.7%) were followed over a median of 82 months (62-124). • BCR, defined as prostate-specific antigen (PSA) ≥ 0.2 ng/mL, was related to status of both, inked specimen margins and FS. RESULTS: • Of all 188 prostatectomy specimens, 49 (26.1%) had positive surgical margins (PSM); these were found posterolaterally in 15 (30.6%), apically in 13 (26.5%), basally in 10 (20.4%) and at multiple sites in 11 (22.4%) specimens. • Intraoperative peripheral FS were positive in 19 (10.7%) patients, including 6.2% at urethral stump, 3.3% at lateral pedicles and 1.1% at bladder neck. • In organ-confined disease, BCR-free survival was 93.3% (111/119) for patients with negative surgical margins (NSM) and 72% (18/25) for patients with PSM (inked specimen), but negative peripheral FS (P < 0.001). • Five- and 10-year BCR-free survival for NSM was 94.9% and 92.8%, for PSM with negative peripheral FS it was 75.3% and 70.6%, and for PSM with positive peripheral FS it was 62.5% and 62.5%, respectively. CONCLUSIONS: • Frozen section biopsies of peripheral resection margins during nerve-sparing radical prostatectomy are not reliable in predicting PSM. • Intraoperative achievement of a locally disease-free status, as monitored by negative circumferential intraoperative FS of peripheral margins, is not associated with a statistically significant BCR-free survival benefit compared with patients with negative surgical margins on the prostatectomy specimen. • Based on these findings, we do not recommend a routine of systematically taking intraoperative FS biopsies during nerve-sparing radical prostatectomy.


Subject(s)
Frozen Sections , Neoplasm Recurrence, Local/pathology , Prostate/pathology , Prostatectomy/methods , Prostatic Neoplasms/pathology , Aged , Epidemiologic Methods , Humans , Male , Middle Aged , Prognosis , Prostate/innervation , Prostate/surgery , Prostatic Neoplasms/surgery , Trauma, Nervous System/prevention & control
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