Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.

• MeSH
• anthrachinony MeSH
• barvicí látky MeSH
• buněčná diferenciace MeSH
• extracelulární matrix metabolismus   MeSH
• imunohistochemie MeSH
• mezenchymální kmenové buňky cytologie   metabolismus   MeSH
• osteokalcin fyziologie   metabolismus   MeSH
• osteonektin metabolismus   MeSH
• osteopontin metabolismus   MeSH
• Sus scrofa MeSH
• tkáňové podpůrné struktury MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

AIM: This study aimed to investigate the phytochemical composition of Psychotria montana extract (PME) and evaluate its inhibitory effects on MCF7 breast cancer cells. METHODS: The chemical composition of PME was analyzed using UPLC-QToF-MS. The effects of PME on cell proliferation were evaluated using the MTT assay. Flow cytometry was used for cell cycle and apoptosis analysis. The effects of PME on the transcription of cell cycle control genes were assessed using real-time PCR. RESULTS: UPLC-QToF-MS analysis revealed major compounds of PME, including terpenoids and flavonoids, with the potential to inhibit proliferation, migration, and induce apoptosis in MCF7 cancer cells. PME effectively suppressed MCF7 cell proliferation under 2D culture, with a low IC50 value of 34.7 μg/ml. PME also hindered cell migration (p < 0.01) and reduced spheroid number (p < 0.001) and size (p < 0.001) in serum-free 3D culture. Apoptosis analysis via nuclear staining with DAPI and flow cytometry revealed an increase in the number of apoptotic cells after PME treatment (p < 0.001). Additionally, the PME induced cell cycle arrest at the G0/G1 phase (p < 0.05). PME altered the expression of cell cycle control genes (cyclins and CDKs) as well as cancer suppressor genes including p16, p27, and p53 at the transcriptional level (mRNA). The results of molecular docking suggest that the compounds present in PME exhibit a high binding affinity for CDK3, CDK4, CDK6, and CDK8 proteins, which are essential regulators of the cell cycle. CONCLUSION: Psychotria montana has the potential to inhibit cancer cells by inducing apoptosis and halting the cell cycle of MCF7 breast cancer cells.

• MeSH
• apoptóza * účinky léků   MeSH
• buněčný cyklus účinky léků   MeSH
• fytogenní protinádorové látky farmakologie   chemie   MeSH
• lidé MeSH
• MFC-7 buňky MeSH
• nádory prsu * farmakoterapie   patologie   genetika   metabolismus   MeSH
• počítačová simulace MeSH
• pohyb buněk účinky léků   MeSH
• proliferace buněk * účinky léků   MeSH
• Psychotria * chemie   MeSH
• rostlinné extrakty * farmakologie   chemie   MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Aberrant DNA methylation that results in silencing of genes has remained a significant interest in cancer research. Despite major advances, the success of epigenetic therapy is elusive due to narrow therapeutic window. A wide variety of naturally occurring epigenetic agents and synthetic molecules that can alter methylation patterns exist, however, their usefulness in epigenetic therapy remains unknown. This underlines the need for effective tumor models for large-scale screening of drug candidates with potent hypomethylation activity. In this study, we present the development of a cell-based DNA demethylation detection system, which is amenable for high content screening of epigenetic drugs in two-dimensional and three-dimensional cell culture models. Additionally, the detection system also supports the in vivo monitoring of demethylation efficacy of potential lead compounds from in vitro screens in tumor xenografts. The described detection system not only permits the continuous monitoring of demethylation but also of the induced cytostatic/cytotoxic drug effects in live cells, as a function of time. The detection system is fluorescence based and exploits the dominant ability of DNA methylation to inhibit gene transcription, and utilizes FLJ32130 gene, which is silenced on account of promoter hypermethylation in human colorectal cancer. The described work will provide the researchers with an efficient tool for epigenetic drug screens on a high throughput platform and would therefore benefit academic and industrial drug discovery. © 2016 International Society for Advancement of Cytometry.

• MeSH
• epigeneze genetická * MeSH
• kolorektální nádory farmakoterapie   genetika   MeSH
• lidé MeSH
• metylace DNA účinky léků   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk účinky léků   MeSH
• promotorové oblasti (genetika) MeSH
• protinádorové látky terapeutické užití   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

• MeSH
• aflatoxin B1 toxicita   MeSH
• biologické modely MeSH
• buněčné kultury metody   MeSH
• buněčné linie MeSH
• buněčné sféroidy * MeSH
• buňky Hep G2 MeSH
• hepatocyty účinky léků   MeSH
• játra účinky léků   MeSH
• lidé MeSH
• methylmethansulfonát toxicita   MeSH
• mikrojaderné testy metody   MeSH
• mutageny toxicita   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The alarming rate of failure of clinical trials is a major hurdle in cancer therapy that partly results from the inadequate use of in vitro tumor models for the screening of promising hits and leads in preclinical studies. 2D cultures of cancer cell lines that are primarily used for drug screening do not adequately recapitulate tumor microenvironment (TME) complexities compared with 3D cancer cell cultures and tumor-derived primary cell cultures. In this review, we focus on the potential use of in vitro tumor models that reproduce in vivo tumor complexities for effective drug selection in the preclinical stages of drug development.

• MeSH
• alternativy testů na zvířatech MeSH
• buněčné kultury MeSH
• lidé MeSH
• mezibuněčná komunikace účinky léků   MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí účinky léků   MeSH
• nádory farmakoterapie   metabolismus   patologie   patofyziologie   MeSH
• objevování léků metody   MeSH
• protinádorové látky farmakologie   MeSH
• reprodukovatelnost výsledků MeSH
• rychlé screeningové testy MeSH
• screeningové testy protinádorových léčiv metody   MeSH
• signální transdukce účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Androgen receptor targeted therapies have emerged as an effective tool to manage advanced prostate cancer (PCa). Nevertheless, frequent occurrence of therapy resistance represents a major challenge in the clinical management of patients, also because the molecular mechanisms behind therapy resistance are not yet fully understood. In the present study, we therefore aimed to identify novel targets to intervene with therapy resistance using gene expression analysis of PCa co-culture spheroids where PCa cells are grown in the presence of cancer-associated fibroblasts (CAFs) and which have been previously shown to be a reliable model for antiandrogen resistance. METHODS: Gene expression changes of co-culture spheroids (LNCaP and DuCaP seeded together with CAFs) were identified by Illumina microarray profiling. Real-time PCR, Western blotting, immunohistochemistry and cell viability assays in 2D and 3D culture were performed to validate the expression of selected targets in vitro and in vivo. Cytokine profiling was conducted to analyze CAF-conditioned medium. RESULTS: Gene expression analysis of co-culture spheroids revealed that CAFs induced a significant upregulation of cholesterol and steroid biosynthesis pathways in PCa cells. Cytokine profiling revealed high amounts of pro-inflammatory, pro-migratory and pro-angiogenic factors in the CAF supernatant. In particular, two genes, 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (HMGCS2) and aldo-keto reductase family 1 member C3 (AKR1C3), were significantly upregulated in PCa cells upon co-culture with CAFs. Both enzymes were also significantly increased in human PCa compared to benign tissue with AKR1C3 expression even being associated with Gleason score and metastatic status. Inhibiting HMGCS2 and AKR1C3 resulted in significant growth retardation of co-culture spheroids as well as of various castration and enzalutamide resistant cell lines in 2D and 3D culture, underscoring their putative role in PCa. Importantly, dual targeting of cholesterol and steroid biosynthesis with simvastatin, a commonly prescribed cholesterol synthesis inhibitor, and an inhibitor against AKR1C3 had the strongest growth inhibitory effect. CONCLUSIONS: From our results we conclude that CAFs induce an upregulation of cholesterol and steroid biosynthesis in PCa cells, driving them into AR targeted therapy resistance. Blocking both pathways with simvastatin and an AKR1C3 inhibitor may therefore be a promising approach to overcome resistances to AR targeted therapies in PCa. Video abstract.

• MeSH
• androgenní receptory metabolismus   MeSH
• anotace sekvence MeSH
• benzamidy farmakologie   MeSH
• biologické modely MeSH
• biosyntetické dráhy genetika   MeSH
• buněčné sféroidy metabolismus   patologie   MeSH
• buněčný cyklus genetika   MeSH
• chemorezistence účinky léků   genetika   MeSH
• cholesterol biosyntéza   MeSH
• extracelulární matrix metabolismus   MeSH
• fenotyp MeSH
• fenylthiohydantoin farmakologie   MeSH
• fibroblasty asociované s nádorem metabolismus   patologie   MeSH
• kultivační média speciální farmakologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• nádory prostaty rezistentní na kastraci genetika   patologie   MeSH
• nádory prostaty genetika   metabolismus   patologie   MeSH
• nitrily farmakologie   MeSH
• progrese nemoci * MeSH
• proliferace buněk genetika   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• senioři MeSH
• simvastatin farmakologie   MeSH
• stanovení celkové genové exprese MeSH
• upregulace * MeSH
• viabilita buněk účinky léků   genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Currently used vascular prostheses are hydrophobic and do not allow endothelial cell (EC) adhesion and growth. The aim of this study was to prepare fibrin (Fb)-based two-dimensional (2D) and three-dimensional (3D) assemblies coated with extracellular matrix (ECM) proteins and to evaluate the EC adhesion, proliferation and differentiation on these assemblies in vitro. Coating of Fb with collagen, laminin (LM), and fibronectin (FN) was proved using the surface plasmon resonance technique. On all Fb assemblies, ECs reached higher cell densities than on polystyrene after 3 and 7 days of culture. Immunoflurescence staining showed better assembly of talin and vinculin into focal adhesion plaques, and also more apparent staining of vascular endothelial cadherin on surface-attached 3D Fb and protein-coated Fb assemblies. On these samples, ECs also contained a lower concentration of intercellular adhesion molecule-1, measured by enzyme-linked immunosorbent assay. Higher concentrations of CD31 (platelet-endothelial cell adhesion molecule-1) were found on 3D Fb coated with LM, and higher concentrations of von Willebrand factor were found on 3D Fb coated with type I collagen or LM in comparison to 2D Fb layers. The results indicate that ECM protein-coated 2D and 3D Fb assemblies can be used for versatile applications in various tissue replacements where endothelialization is desirable, for example, vascular prostheses and heart valves.

• MeSH
• buněčná adheze MeSH
• buněčná diferenciace MeSH
• buněčné linie MeSH
• endoteliální buňky cytologie   MeSH
• fibrin chemie   MeSH
• kolagen chemie   MeSH
• laminin chemie   MeSH
• skot MeSH
• tkáňové podpůrné struktury chemie   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cell migration and invasiveness significantly contribute to desirable physiological processes, such as wound healing or embryogenesis, as well as to serious pathological processes such as the spread of cancer cells to form tumor metastasis. The availability of appropriate methods for studying these processes is essential for understanding the molecular basis of cancer metastasis and for identifying suitable therapeutic targets for anti-metastatic treatment. This review summarizes the current status of these methods: In vitro methods for studying cell migration involve two-dimensional (2D) assays (wound-healing/scratch assay), and methods based on chemotaxis (the Dunn chamber). The analysis of both cell migration and invasiveness in vitro require more complex systems based on the Boyden chamber principle (Transwell migration/invasive test, xCELLigence system), or microfluidic devices with three-dimensional (3D) microscopy visualization. 3D culture techniques are rapidly becoming routine and involve multicellular spheroid invasion assays or array chip-based, spherical approaches, multi-layer/multi-zone culture, or organoid non-spherical models, including multi-organ microfluidic chips. The in vivo methods are mostly based on mice, allowing genetically engineered mice models and transplant models (syngeneic mice, cell line-derived xenografts and patient-derived xenografts including humanized mice models). These methods currently represent a solid basis for the state-of-the art research that is focused on understanding metastatic fundamentals as well as the development of targeted anti-metastatic therapies, and stratified treatment in oncology.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: The choroid plexus (ChP) is the secretory epithelial structure located in the brain ventricles. Choroid plexus tumors (CPTs) are rare neoplasms predominantly occurring in young patients with intensified malignancy in children. CPT treatment is hindered by insufficient knowledge of tumor pathology and the limited availability of valid models. METHODS: Genomic and transcriptomic data from CPT patients were analyzed to identify the putative pathological pathway. Cellular and molecular techniques were employed to validate bioinformatic results in CPT patient samples. Pharmacologic inhibition of Wnt/β-catenin signaling was assessed in CPT cells. Cell-based assays of ChP cell lines were performed following CRISPR-Cas9-derived knockout and overexpression of Wnt/β-catenin pathway genes. A 3D CPT model was generated through CRISPR-Cas9-derived knockout of APC. RESULTS: We discovered that Wnt/β-catenin signaling is activated in human CPTs, likely as a consequence of large-scale chromosomal instability events of the CPT genomes. We demonstrated that CPT-derived cells depend on autocrine Wnt/β-catenin signaling for survival. Constitutive Wnt/β-catenin pathway activation, either through knockout of the negative regulator APC or overexpression of the ligand WNT3A, induced tumorigenic properties in ChP 2D in vitro models. Increased activation of the Wnt/β-catenin pathway in ChP organoids, through treatment with a potent GSK3β inhibitor, reduced the differentiation of mature ChP epithelial cells. Remarkably, the depletion of APC was sufficient to induce the oncogenic transformation of ChP organoids. CONCLUSIONS: Our research identifies Wnt/β-catenin signaling as a critical driver of CPT tumorigenesis and provides the first 3D in vitro model for future pathological and therapeutic studies of CPT.

• MeSH
• beta-katenin metabolismus   genetika   MeSH
• karcinogeneze metabolismus   MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• nádory plexus chorioideus * patologie   metabolismus   genetika   MeSH
• plexus chorioideus metabolismus   patologie   MeSH
• proliferace buněk MeSH
• regulace genové exprese u nádorů MeSH
• signální dráha Wnt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Magnetic polymer nanocomposites are inherently multifunctional and harbor assorted physiochemical actions for applications thereof as novel drug nanocarriers. Herein, Fe3O4-nanoparticles were supported on rice straw cellulose for 5-fluorouracil carrier abbreviated as MC/5-FU for potential colorectal cancer treatments. Several analyses indicated the multifunctional properties of MC/5-FU bionanocomposites. Transmission and scanning electron microscopy study demonstrated that Fe3O4 nanofillers covered the cellulose matrix. The drug release from MC/5-FU was evaluated under various pH and temperature conditions, showing the maximum release at pH 7.4 and 44.2 °C. In in vitro anticancer assay, MC/5-FU exhibited enhanced selectivity and anticancer actions against 2D monolayer and 3D tumour spheroid models colorectal cancer cells. The anticancer effects of MC/5-FU with magnetic targeting and heat induction were also examined. This easily synthesized MC/5-FU indicated the potential in application as a low-cost drug formulation for colorectal cancer treatments.

• MeSH
• buňky HT-29 MeSH
• celulosa chemie   MeSH
• fluoruracil chemie   farmakologie   MeSH
• HCT116 buňky MeSH
• kolorektální nádory farmakoterapie   metabolismus   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• magnetické jevy MeSH
• magnetické nanočástice oxidů železa chemie   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• nanokompozity chemie   MeSH
• nosiče léků chemie   MeSH
• polymery chemie   MeSH
• protinádorové látky farmakologie   MeSH
• uvolňování léčiv MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH