Expression of BCL2, TP53, FOXA1, and GATA3 in pTa bladder cancer recurrence.

OBJECTIVES: In this study, we analyzed pTa bladder cancer (BC) for molecular markers BCL2, TP53, FOXA1, and GATA3 in relation to cancer recurrence. METHODS: We analyzed samples of 79 patients with the pTa stage of BC using a real-time polymerase chain reaction (real-time PCR) between September 2018 and September 2020. The expression levels of BCL2, TP53, FOXA1, and GATA3 were compared with homologous non-tumor bladder tissue. RESULTS: Expression of FOXA1, GATA3, and TP53 was significantly higher (p<0.01) in NMIBC samples compared to homologous non-tumor tissue. The expression of TP53 and FOXA1 in pTa was significantly lower (p<0.01) in the high-grade (HG) tumor when compared to the low-grade (LG) tumor. In contrast, the relative quantification (RQ) of GATA3 was significantly higher (p<0.01) in HG pTa. Patients with recurrence (pTa=33) had significantly higher expression of TP53, and GATA3 (p<0.01), and the gene of FOXA1 (p<0.01) had a significantly lower expression when compared to pTa tumors without recurrence. The expression of Bcl-2 was not statistically significant. CONCLUSION: Our results, indicate, that comparing expression levels of these genes in cancer and cancer-free tissue could provide valuable data, as patients with pTa BC recurrence within up to 54 months of follow-up had a significantly higher RQ of TP53, GATA3, and FOXA1 when compared to pTa BC patients without recurrence (Tab. 2, Fig. 8, Ref. 54). Text in PDF www.elis.sk Keywords: bladder cancer, gene expression, recurrence, GATA3, FOXA1, TP53, BCL2.

The third dimension of tumor microenvironment-The importance of tumor stroma in 3D cancer models.

Recent advances in the three-dimensional (3D) cancer models give rise to a plethora of new possibilities in the development of anti-cancer drug therapies and bring us closer to personalized medicine. Three-dimensional models are undoubtedly more authentic than traditional two-dimensional (2D) cell cultures. Nowadays, they are becoming preferentially used in most cancer research fields due to their more accurate biomimetic characteristics. On the contrary, they still lack the cellular and matrix complexity of the native tumor microenvironment (TME). This review focuses on the description of existing 3D models, the incorporation of TME and fluidics into these models, and their perspective in the future research. It is clear that such an improvement would need not only biological but also technical progress. Therefore, the modern approach to anti-cancer drug discovery should involve various fields.

Decellularization of the human urethra for tissue engineering applications.

Recently, several scaffolds have been introduced for urethral tissue engineering. However, acellular human urethral scaffold harvested from deceased donors may provide significant advantages compared to synthetic, composite, or other biological scaffolds. This study aims to develop the protocol for decellularization of the human urethra that preserves substantial extracellular matrix (ECM) components, which are essential for subsequent recellularization mimicking the natural environment of the native ECM. A total of 12 human urethras were harvested from deceased donors. An equal part of every harvested urethra was used as a control sample for analyses. The protocol design was based on the enzyme-detergent-enzyme method. Trypsin and Triton X-100 were used to remove cells, followed by DNase treatment to remove DNA residues. Subsequently, the specimens were continually rinsed in deionized water for seven days. The efficiency of decellularization was determined by histochemistry, immunohistochemical staining, scanning electron microscopy (SEM), and DNA quantification. Histological analysis confirmed cell removal and preservation of urethral structure after decellularization. The preservation of collagen IV and fibronectin was confirmed by histologic examination and immunohistochemical staining. SEM confirmed the maintenance of the ultrastructural architecture of ECM and fibers. DNA content in decellularized urethra was significantly lower compared to the native sample (P < 0.001), and so the criteria for decellularized tissue were met. Cytotoxicity analysis data showed that the matrix-conditioned medium did not contain soluble toxins and had no significant inhibitory effect on cell proliferation, providing evidence that the decellularized samples are not toxic. This study demonstrates the feasibility of the enzyme-detergent-enzyme-based decellularization protocol for removing cellular components and maintaining urethral ECM and its ultrastructure. Moreover, obtained results provide solid ground for recellularization and urethral tissue engineering, which will follow.

Induced Pluripotent Stem Cell-Derived Organoids: Their Implication in COVID-19 Modeling.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a significant global health issue. This novel virus's high morbidity and mortality rates have prompted the scientific community to quickly find the best COVID-19 model to investigate all pathological processes underlining its activity and, more importantly, search for optimal drug therapy with minimal toxicity risk. The gold standard in disease modeling involves animal and monolayer culture models; however, these models do not fully reflect the response to human tissues affected by the virus. However, more physiological 3D in vitro culture models, such as spheroids and organoids derived from induced pluripotent stem cells (iPSCs), could serve as promising alternatives. Different iPSC-derived organoids, such as lung, cardiac, brain, intestinal, kidney, liver, nasal, retinal, skin, and pancreatic organoids, have already shown immense potential in COVID-19 modeling. In the present comprehensive review article, we summarize the current knowledge on COVID-19 modeling and drug screening using selected iPSC-derived 3D culture models, including lung, brain, intestinal, cardiac, blood vessels, liver, kidney, and inner ear organoids. Undoubtedly, according to reviewed studies, organoids are the state-of-the-art approach to COVID-19 modeling.

The Role of BRCA1/2-Mutated Tumor Microenvironment in Breast Cancer.

Taking into account the factors of high incidence rate, prevalence and mortality, breast cancer represents a crucial social and economic burden. Most cases of breast cancer develop as a consequence of somatic mutations accumulating in mammary epithelial cells throughout lifetime and approximately 5-10% can be ascribed to monogenic predispositions. Even though the role of genetic predispositions in breast cancer is well described in the context of genetics, very little is known about the role of the microenvironment carrying the same aberrant cells impaired by the germline mutation in the breast cancer development and progression. Based on the clinical observations, carcinomas carrying mutations in hereditary tumor-suppressor genes involved in maintaining genome integrity such as BRCA1/2 have worse prognosis and aggressive behavior. One of the mechanisms clarifying the aggressive nature of BRCA-associated tumors implies alterations within the surrounding adipose tissue itself. The objective of this review is to look at the role of BRCA1/2 mutations in the context of breast tumor microenvironment and plausible mechanisms by which it contributes to the aggressive behavior of the tumor cells.

Somatic stem cell aging and malignant transformation--impact on therapeutic application.

Somatic stem cells possess unique properties of self-renewal and plasticity which make them promising candidates for use in tissue engineering and regenerative medicine, in addition to serving as efficient delivery vehicles in site-specific therapy. In the case of therapeutic application, it is essential to isolate and culture stem cells in vitro, to obtain them in sufficient quantities. Although long-term cultivation provides an adequate number of cells, it has been shown that this approach is associated with increased risk of transformation of cultured cells, which presents a significant biological hazard. This article reviews information about biological features and cellular events which occur during long-term cultivation of somatic stem cells, with respect to their safe utilization in potential clinical practice.

Modulation of BV-2 microglia functions by novel quercetin pivaloyl ester.

Chronic inflammation in brain plays a critical role in major neurodegenerative diseases such as Alzheimer's, Parkinson's disease, stroke or multiple sclerosis. Microglia, resident macrophages and intristinc components of CNS, appear to be main effectors in this pathological process. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3'-O-(3-chloropivaloyl) quercetin (CPQ), is assumed to possess better biological availability and enhanced antioxidant properties. In the present study, antineuroinflammatory capability of the novel compound CPQ was assessed in BV-2 microglial cells. Our data show that treatment with CPQ attenuated the production of the inflammatory mediators, nitric oxide (NO) and tumour necrosis factor-α (TNF-α), in LPS-stimulated microglia somewhat more efficiently than did quercetin (p > 0.05 for CPQ vs. quercetin-treated group). Also, protein level of inducible NO synthase (iNOS) in LPS-activated BV-2 microglia was to some extent more effectively supressed by CPQ than by unmodified flavonoid. In consistence with the extent of their effects on pro-inflammatory markers, CPQ and quercetin showed down-regulation of NFκB activation. This quercetin analogue caused also a decline in BV-2 microglia proliferation with interfering with cell cycle progression (p < 0.001 for CPQ vs. quercetin-treated group). However, CPQ did not remarkably affect cell viability. In addition, CPQ showed a minor better suppression of PMA-induced generation of superoxide than did quercetin. Neither CPQ nor quercetin influenced phagocytosis of BV-2 cells. These results point to the therapeutic potential of 3'-O-(3-chloropivaloyl)quercetin (CPQ) as a novel antiinflammatory drug in neurodegenerative diseases, mediating favourable modulation of pro-inflammatory functions of microglia.

Acidotropic properties of synthetic hexahydropyridoindole antioxidants.

In acidic intracellular organelles, sequestration via a proton-trapping mechanism is observed for many amine-containing drugs. It may be related to several adverse effects of a drug, yet accumulation of amines bearing antioxidant functionality may provide efficient protection of these compartments. In the present study, a possible proton-trapping mechanism of the novel antioxidant reference stobadine (STO) and its selected derivatives was investigated also with regard to their antioxidant properties, using BV-2 microglia. Unlike its 2-ethoxycarbonyl-8-methoxy derivative EC-STO (pKa1 4.95, pKa2 -3.58), STO, bearing weakly basic piperidine moiety (pKa2 9.03), induced vacuolar response in the cells. EC-STO, compared to STO, failed to provide better protection against oxidative damage induced by tert-butyl hydroperoxide (BHP), and that in spite of its predicted improved bioavailability and antioxidant properties. However, disruption of the lysosomal proton gradient abolished the efficacy of STO in suppressing oxidants generation and injury of the cells. NT-STO, the 6-nitro derivative of stobadine, lacking antiradical efficacy, showed a lower effect in protecting the cells against BHP. In conclusion, our study suggests that weakly basic hexahydropyridoindoles may act as lysosomotropic compounds. Furthermore, their weakly basic characteristics may contribute to their improved efficacy in suppressing peroxidative processes within lysosomes, and thus possibly combating ageing-related pathologies.

The effect of salivary gland extract of Lucilia sericata maggots on human dermal fibroblast proliferation within collagen/hyaluronan membrane in vitro: transmission electron microscopy study.

BACKGROUND: Lucilia sericata maggots are applied to chronic wounds to aid healing when conventional treatments have failed. After their application into a necrotic wound, they potentially influence wound healing with a combination of specific proteinases that are involved in the remodeling of extracellular matrix. These proteases cause changes in fibroblast adhesion and spread upon extracellular matrix protein surfaces, affecting integrity of the protein surfaces-especially fibronectin-while maintaining cell viability. OBJECTIVE: This study focused on in vitro monitoring of the effect of homogenate substances prepared from maggot salivary gland of L sericata on the ultrastructure of human neonatal fibroblasts. METHODS: Collagen/hyaluronan membrane was used as the synthetic substitute of extracellular matrix. The cultured human neonatal fibroblasts B-HNF-1 were seeded on the surface of the collagen/hyaluronan membrane and cultured with maggot salivary gland extract (SGE) at a concentration of 2.4 glands/1 mL. RESULTS: The authors observed increased cell metabolism and protein production (euchromatic nucleus, voluminous nuclear membrane, large reticular nuclei, distended and filled cisterns of rough endoplasmic reticulum, Golgi apparatus with saccules, and vesicles packed with fine fibrillar material) after incubating the cells in culture medium with SGE. CONCLUSION: The authors believe that increased cell metabolism and protein production corresponded with formation of microfibrillar net used for migration of fibroblasts in culture, but mainly for proper production of extracellular matrix. The authors suggest that their results may help explain the effect of SGE on wound healing and support implementation of maggot therapy into human medicine.

Modulation of microglial function by the antidepressant drug venlafaxine.

An increasing amount of data suggests that depression is an inflammatory disease. Depressed patients have higher peripheral blood levels of inflammatory markers which have been shown to access the brain and interact with the pathophysiological domain known to be involved in depression. Furthermore, microglia activation may play an important role in the inflammatory pathophysiology of depression. In BV-2 microglia cell line, the present study investigated the potential anti-inflammatory effects of venlafaxine, along with its potential influence on injury of lipopolysaccharide (LPS)-stimulated cells. Although venlafaxine showed only marginal influence on the majority of the pro-inflammatory parameters assessed (in particular NO release, phagocytosis and proliferation), it significantly suppressed superoxide production by the stimulated cells. In addition, venlafaxine exerted also a protective effect on mitochondrial membrane potential and lysosomes of the stimulated microglia. In conclusion, our results suggest that although VEN might have only a marginal effect on major pro-inflammatory parameters of microglia, its inhibitory effect on superoxide generation can contribute to the prevention of harmful effects of oxidative and nitrosative stress involved in the pathogenesis of depression. Moreover, the protective effect of VEN on viability of microglia can prevent a rapid reduction of these cells, thus avoiding limitations of several physiological processes in the brain and possibly also the progression of depression.

Can semi-synthetic flavonoids return old microglia to their youthful state?

A number of studies have indicated that brain inflammation may deteriorate during normal aging and that neuroinflammation is amplified in age-related neurodegenerative diseases. A pivotal role in age-related neuroinflammatory pathologies is attributed to amplified and prolonged activation of microglia. In addition, microglia from the aged brain were reported as senescent displaying many functional impairments. Flavonoids were shown to be promising molecules in modulation of neuroinflammation. Quercetin, a naturally occurring flavonoid, was proven to downregulate inflammatory genes in microglia. Synthetically modified quercetin, 3´-O-(3-chloropivaloyl)quercetin (CPQ), is assumed to posses better biological availability and enhanced antioxidant properties. In the present study, the antineuroinflammatory capacity of CPQ was assessed in BV-2 microglial cells and rat primary microglia. CPQ suppressed more efficiently than its precursor quercetin LPS-induced NO production and iNOS protein expression. However, neither of the compounds tested influenced significantly phagocytosis of BV-2 cells. In addition, CPQ showed a somewhat better suppression of PMA-induced generation of superoxide than did quercetin. Unlike quercetin, CPQ caused a decline in BV-2 microglia proliferation (without any impact on cell viability) along with interference with cell cycle progression. Both compounds tested at 10uM concentration notably enhanced viability of microglia-enriched cultures prepared from 22-month-old rat brains. This was followed by suppression of lipofuscin-like autofluorescence, improvement of lysosomal function and protection of mitochondria in the old microglia. These results can highlight the therapeutic potential of CPQ as a novel antiinflammatory drug in neurodegenerative diseases. In addition, our data suggest that both natural and semisynthetic flavonoids might protect functions of old microglia [VEGA2/0031/12,1/0076/13;APVV-0052-10;ITMS26240220040].

Pomegranate Seed Oil Modulates Functions and Survival of BV-2 Microglial Cells in vitro.

Current evidence has demonstrated the immunomodulatory efficacy of omega-3 polyunsaturated fatty acids (PUFAs) in glial cells, suggesting their therapeutic potential for diseases in the central nervous system (CNS). However, conjugated omega-5 PUFAs have also attracted considerable attention because of their suggested anti-inflammatory effects. In the present study, the effect of pomegranate (Punica granatum L.) seed oil (PSEO) (a rich source of omega-5 PUFAs) on the activation of cultured BV-2 microglia was investigated within a 24-hour incubation period. PSEO (25 µg/ml) showed only a slightly smaller inhibitory effect on LPS-stimulated NO production (243 ± 12.5 % of control, p<0.001 vs. 437 ± 9.2 % in stimulated cells) and TNF-α release (87.1 ± 5.62 pg/ml vs. 229 ± 24.4 pg/ml in stimulated cells), as well as iNOS expression (7.36-fold of control, p < 0.01, vs. 17.5-fold increase in stimulated cells) compared to a standardized omega-3 PUFAs mixture (25 µg/ml) and the flavonoid quercetin (25 µmol/l). Unlike quercetin and stobadine, only the PUFA preparations effectively prevented apoptosis of microglia (as confirmed by the suppression of caspase 3 activation) exposed to the toxic concentration of LPS. The PUFA preparations did not provide a notable suppression of the intracellular oxidant generation and did not influence the intracellular distribution of cholesterol (as confirmed by filipin staining). However, they appeared to affect the morphology of activated cells. In conclusion, our data point to the first evidence of immunomodulation and cytoprotection of BV-2 microglia by the pomegranate seed oil, indicating that it may be (comparably to omega-3 PUFAs) efficient against microglia-mediated neuroinflammation while preventing the premature depletion of these immune effector cells in the brain.