Selective targeting of lectins and their macropinocytosis in urothelial tumours: translation from in vitro to ex vivo.

Urinary bladder cancer can be treated by intravesical application of therapeutic agents, but the specific targeting of cancer urothelial cells and the endocytotic pathways of the agents are not known. During carcinogenesis, the superficial urothelial cells exhibit changes in sugar residues on the apical plasma membranes. This can be exploited for selective targeting from the luminal side of the bladder. Here we show that the plant lectins Jacalin (from Artocarpus integrifolia), ACA (from Amaranthus caudatus) and DSA (from Datura stramonium) selectively bind to the apical plasma membrane of low- (RT4) and high-grade (T24) cancer urothelial cells in vitro and urothelial tumours ex vivo. The amount of lectin binding was significantly different between RT4 and T24 cells. Endocytosis of lectins was observed only in cancer urothelial cells and not in normal urothelial cells. Transmission electron microscopy analysis showed macropinosomes, endosome-like vesicles and multivesicular bodies filled with lectins in RT4 and T24 cells and also in cells of urothelial tumours ex vivo. Endocytosis of Jacalin and ACA in cancer cells was decreased in vitro after addition of inhibitor of macropinocytosis 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and increased after stimulation of macropinocytosis with epidermal growth factor (EGF). Clathrin, caveolin and flotillin did not colocalise with lectins. These results confirm that the predominant mechanism of lectin endocytosis in cancer urothelial cells is macropinocytosis. Therefore, we propose that lectins in combination with conjugated therapeutic agents are promising tools for improved intravesical therapy by targeting cancer cells.

Cannabidiol as a Promising Therapeutic Option in IC/BPS: In Vitro Evaluation of Its Protective Effects against Inflammation and Oxidative Stress.

Several animal studies have described the potential effect of cannabidiol (CBD) in alleviating the symptoms of interstitial cystitis/bladder pain syndrome (IC/BPS), a chronic inflammatory disease of the urinary bladder. However, the effects of CBD, its mechanism of action, and modulation of downstream signaling pathways in urothelial cells, the main effector cells in IC/BPS, have not been fully elucidated yet. Here, we investigated the effect of CBD against inflammation and oxidative stress in an in vitro model of IC/BPS comprised of TNFα-stimulated human urothelial cells SV-HUC1. Our results show that CBD treatment of urothelial cells significantly decreased TNFα-upregulated mRNA and protein expression of IL1α, IL8, CXCL1, and CXCL10, as well as attenuated NFκB phosphorylation. In addition, CBD treatment also diminished TNFα-driven cellular reactive oxygen species generation (ROS), by increasing the expression of the redox-sensitive transcription factor Nrf2, the antioxidant enzymes superoxide dismutase 1 and 2, and hem oxygenase 1. CBD-mediated effects in urothelial cells may occur by the activation of the PPARγ receptor since inhibition of PPARγ resulted in significantly diminished anti-inflammatory and antioxidant effects of CBD. Our observations provide new insights into the therapeutic potential of CBD through modulation of PPARγ/Nrf2/NFκB signaling pathways, which could be further exploited in the treatment of IC/BPS.

The Golgi complex: An organelle that determines urothelial cell biology in health and disease.

The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood-urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell-cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood-urine barrier in normal UCs and how it may be involved in the loss of the blood-urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.

Astrocytes in stress accumulate lipid droplets.

When the brain is in a pathological state, the content of lipid droplets (LDs), the lipid storage organelles, is increased, particularly in glial cells, but rarely in neurons. The biology and mechanisms leading to LD accumulation in astrocytes, glial cells with key homeostatic functions, are poorly understood. We imaged fluorescently labeled LDs by microscopy in isolated and brain tissue rat astrocytes and in glia-like cells in Drosophila brain to determine the (sub)cellular localization, mobility, and content of LDs under various stress conditions characteristic for brain pathologies. LDs exhibited confined mobility proximal to mitochondria and endoplasmic reticulum that was attenuated by metabolic stress and by increased intracellular Ca2+ , likely to enhance the LD-organelle interaction imaged by electron microscopy. When de novo biogenesis of LDs was attenuated by inhibition of DGAT1 and DGAT2 enzymes, the astrocyte cell number was reduced by ~40%, suggesting that in astrocytes LD turnover is important for cell survival and/or proliferative cycle. Exposure to noradrenaline, a brain stress response system neuromodulator, and metabolic and hypoxic stress strongly facilitated LD accumulation in astrocytes. The observed response of stressed astrocytes may be viewed as a support for energy provision, but also to be neuroprotective against the stress-induced lipotoxicity.

Cytotoxic Activity of LLO Y406A Is Targeted to the Plasma Membrane of Cancer Urothelial Cells.

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.

Attachment of Cancer Urothelial Cells to the Bladder Epithelium Occurs on Uroplakin-Negative Cells and Is Mediated by Desmosomal and Not by Classical Cadherins.

Urinary bladder cancer is often multifocal; however, the intraluminal dissemination of the urothelial cancer cells is poorly understood. The involvement of N-cadherin in the adhesion of the cancer urothelial cells to the urothelium had not previously been studied. Therefore, we herein explore the possibility of the intraluminal dissemination of the urothelial cancer cells by evaluating the role of classical cadherins in the adhesion of urothelial cancer cells to the urothelium. We used E-cadherin negative T24 cells and established a T24 Ncadlow cell line with an additionally decreased expression of N-cadherin in the plasma membrane and a decreased secretion of proform of metalloproteinase 2. The labelled T24 and T24 Ncadlow cells were seeded onto urothelial in vitro models. After 24 h in co-culture, unattached cancer cells were rinsed and urothelia with attached cancer urothelial cells were processed for fluorescence and electron microscopy. Both the T24 and T24 Ncadlow cells attached to the urothelium, yet only to the uroplakin-negative urothelial cells. The ultrastructural analysis showed that T24 and T24 Ncadlow cells adhere to poorly differentiated urothelial cells by desmosomes. To achieve this, they first disrupt tight junctions of superficial urothelial cells. This study indicates that the lack of E-cadherin expression and decreased expression of N-cadherin in the plasma membrane of T24 cells does not interfere with their adhesion to the urothelium; therefore, our results suggest that intraluminal dissemination of cancer urothelial cells along the urothelium occurs on uroplakin-negative cells and is desmosome-mediated.

Different Culture Conditions Affect Drug Transporter Gene Expression, Ultrastructure, and Permeability of Primary Human Nasal Epithelial Cells.

PURPOSE: This study aimed to characterize a commercially available primary human nasal epithelial cell culture and its gene expression of a wide range of drug transporters under different culture conditions. METHODS: Human nasal cells were cultured in three different types of culture media at the air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 wks. The effects of the different cell culture conditions were evaluated using light and electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran, and gene expression profiling of 84 drug transporters. RESULTS: The type of culture medium affected cell ultrastructure, TEER, and dextran permeation across epithelia. The expression of 20 drug transporter genes depended on the culture interface and/or time in culture; the A-L interface and longer time in culture favored higher expression levels of five ABC and seven SLC transporters. CONCLUSIONS: Culture conditions influence the morphology, barrier formation, permeation properties, and drug transporter expression of human nasal epithelial cells, and this must be taken into consideration during the establishment and validation of in vitro models. A thorough characterization of a nasal epithelial model and its permeability properties is necessary to obtain an appropriate standardized model for the design of aerosol therapeutics and drug transport studies.

Triple labelling of actin filaments, intermediate filaments and microtubules for broad application in cell biology: uncovering the cytoskeletal composition in tunneling nanotubes.

We report a protocol for simultaneous triple labelling of intermediate filaments, microtubules and actin filaments. The described procedure offers an optimal preservation of the structure and antigenicity of individual representatives of cytoskeletal elements and is applicable for labelling of tissue samples and cultured cells. Namely, we demonstrate that using this protocol the cytoskeletal elements are well-preserved and detectable in the whole mount urinary bladder tissue pieces, cryosections of the urinary bladder, and in cultured normal and cancer urothelial cells including their delicate intercellular connections such as tunneling nanotubes (TnTs). The protocol uncovers for the first time the co-distribution of actin filaments, intermediate filaments and microtubules in TnTs, which were up to now known as mono- or bi-cytoskeletal structures. Presented triple labelling protocol provides an efficient tool for studying co-distribution of actin filaments, intermediate filaments, and microtubules and therefore offers new insights into their cellular and tissue distribution.

Intracellular Activation of a Prostate Specific Antigen-Cleavable Doxorubicin Prodrug: A Key Feature Toward Prodrug-Nanomedicine Design.

L-377,202 prodrug (Dox-PSA) was in phase I clinical trials for patients with metastatic castration-resistant prostate cancer (mCRPC). It consists of doxorubicin (Dox) conjugated to a prostate specific antigen (PSA)-cleavable peptide that can be selectively activated by secreted PSA at the tumor site. However, despite the initial promising results, further clinical testing with Dox-PSA was halted due to toxicity concerns emerging from non-PSA-specific cleavage, following systemic administration. In the present study, we have reported, for the first time, the intracellular activation of Dox-PSA, where Dox nuclear uptake was specific to C4-2B (PSA-expressing) cells, which agreed with the cytotoxicity studies. This finding was confirmed by encapsulating Dox-PSA prodrug into pH-sensitive liposomes to enable prodrug intracellular release, followed by its enzymatic activation. Interestingly, our results demonstrated that Dox-PSA loaded into pH-responsive nanoparticles exhibited cytotoxicity comparable to free prodrug in C4-2B monolayers, with superior activity in tumor spheroids, due to deeper penetration within tumor spheroids. Our approach could open the doors for novel Dox-PSA nanomedicines with higher safety and efficacy to treat advanced and metastatic prostate cancer.

Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia.

Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.

Increased endocytosis of magnetic nanoparticles into cancerous urothelial cells versus normal urothelial cells.

The blood-urine barrier is the tightest and most impermeable barrier in the body and as such represents a problem for intravesical drug delivery applications. Differentiation-dependent low endocytotic rate of urothelial cells has already been noted; however, the differences in endocytosis of normal and cancer urothelial cells have not been exploited yet. Here we analysed the endocytosis of rhodamine B isothiocyanate-labelled polyacrylic acid-coated cobalt ferrite nanoparticles (NPs) in biomimetic urothelial in vitro models, i.e., in highly and partially differentiated normal urothelial cells, and in cancer cells of the papillary and invasive urothelial neoplasm. We demonstrated that NPs enter papillary and invasive urothelial neoplasm cells by ruffling of the plasma membrane and engulfment of NP aggregates by macropinocytotic mechanism. Transmission electron microscopy (TEM) and spectrophotometric analyses showed that the efficacy of NPs delivery into normal urothelial cells and intercellular space is largely restricted, while it is significantly higher in cancer urothelial cells. Moreover, we showed that the quantification of fluorescent NP internalization in cells or tissues based on fluorescence detection could be misleading and overestimated without TEM analysis. Our findings contribute to the understanding of endocytosis-mediated cellular uptake of NPs in cancer urothelial cells and reveal a highly selective mechanism to distinguish cancer and normal urothelial cells.

Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research.

New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), claudin 4, claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca2+ - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca2+ - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1µm, and larger vesicles of around 10-µm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Properties of the Urothelium that Establish the Blood-Urine Barrier and Their Implications for Drug Delivery.

The primary function of the urinary bladder is to store and periodically release urine. How the urothelium prevents permeation of water, ions, solutes, and noxious agents back into the bloodstream and underlying tissues as well as serving as a sensor and transducer of physiological and nociceptive stimuli is still not completely understood, and thus its unique functional complexity remains to be fully elucidated. This article reviews the permeation routes across urothelium as demonstrated in extensive morphological and electrophysiological studies on in vivo and in vitro urothelia. We consider the molecular and morphological structures of urothelium and how they contribute to the impermeability of the blood-urine barrier. Based on the available data, the extremely low permeability properties of urothelium can be postulated. This remarkable impermeability is necessary for the normal functioning of all mammals, but at the same time represents limitations regarding the uptake of drugs. Therefore, the current progress to overcome this most resilient barrier in our body for drug therapy purposes is also summarized in this review.

Differentiation-dependent rearrangements of actin filaments and microtubules hinder apical endocytosis in urothelial cells.

During differentiation, superficial urothelial cells (UCs) of the urinary bladder form the apical surface, which is almost entirely covered by urothelial plaques containing densely packed uroplakin particles. These urothelial plaques are the main structural components of the blood-urine permeability barrier in the urinary bladder. We have shown previously that endocytosis from the apical plasma membrane decreases during urothelial cell differentiation. Here, we investigated the role of actin filament and microtubule rearrangements in apical endocytosis of differentiating UCs cells using hyperplastic and normoplastic porcine urothelial models. Partially differentiated normal porcine UCs contained actin filaments in the subapical cytoplasm, while microtubules had a net-like appearance. In highly differentiated UCs, actin filaments mostly disappeared from the subapical cytoplasm and microtubules remained as a thin layer close to the apical plasma membrane. Inhibition of actin filament formation with cytochalasin-D in partially differentiated UCs caused a decrease in apical endocytosis. Depolymerisation of microtubules with nocodazole did not prevent endocytosis of the endocytotic marker WGA into the subapical cytoplasm; however, it abolished WGA transport to endolysosomal compartments in the central cytoplasm. Cytochalasin-D or nocodazole treatment did not significantly change apical endocytosis in highly differentiated UCs. In conclusion, we showed that the physiological differentiation-dependent or chemically induced redistribution and reorganization of actin filaments and microtubules impair apical endocytosis in UCs. Importantly, reduced apical endocytosis due to cytoskeletal rearrangements in highly differentiated UCs, together with the formation of rigid urothelial plaques, reinforces the barrier function of the urothelium.

The complete functional recovery of chitosan-treated biomimetic hyperplastic and normoplastic urothelial models.

The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05%) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.

Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout, and first description of erythrophore ultrastructure in salmonids.

Skin pigmentation in animals is an important trait with many functions. The present study focused on two closely related salmonid species, marble trout (Salmo marmoratus) and brown trout (S. trutta), which display an uncommon labyrinthine (marble-like) and spot skin pattern, respectively. To determine the role of chromatophore type in the different formation of skin pigment patterns in the two species, the distribution and ultrastructure of chromatophores was examined with light microscopy and transmission electron microscopy. The presence of three types of chromatophores in trout skin was confirmed: melanophores; xanthophores; and iridophores. In addition, using correlative microscopy, erythrophore ultrastructure in salmonids was described for the first time. Two types of erythrophores are distinguished, both located exclusively in the skin of brown trout: type 1 in black spot skin sections similar to xanthophores; and type 2 with a unique ultrastructure, located only in red spot skin sections. Morphologically, the difference between the light and dark pigmentation of trout skin depends primarily on the position and density of melanophores, in the dark region covering other chromatophores, and in the light region with the iridophores and xanthophores usually exposed. With larger amounts of melanophores, absence of xanthophores and presence of erythrophores type 1 and type L iridophores in the black spot compared with the light regions and the presence of erythrophores type 2 in the red spot, a higher level of pigment cell organisation in the skin of brown trout compared with that of marble trout was demonstrated. Even though the skin regions with chromatophores were well defined, not all the chromatophores were in direct contact, either homophilically or heterophilically, with each other. In addition to short-range interactions, an important role of the cellular environment and long-range interactions between chromatophores in promoting adult pigment pattern formation of trout are proposed.

The characterization of the human nasal epithelial cell line RPMI 2650 under different culture conditions and their optimization for an appropriate in vitro nasal model.

PURPOSE: The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa. METHODS: Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters. RESULTS: Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface. CONCLUSIONS: The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.

Combined cytotoxic effect of UV-irradiation and TiO2 microbeads in normal urothelial cells, low-grade and high-grade urothelial cancer cells.

The differentiation of urothelial cells results in normal terminally differentiated cells or by alternative pathways in low-grade or high-grade urothelial carcinomas. Treatments with traditional surgical and chemotherapeutical approaches are still inadequate and expensive, as bladder tumours are generally highly recurrent. In such situations, alternative approaches, using irradiation of the cells and nanoparticles, are promising. The ways in which urothelial cells, at different differentiation levels, respond to UV-irradiation (photolytic treatment) or to the combination of UV-irradiation and nanoparticles (photocatalytic treatment), are unknown. Here we tested cytotoxicity of UV-irradiation on (i) normal porcine urothelial cells (NPU), (ii) human low-grade urothelial cancer cells (RT4), and (iii) human high-grade urothelial cancer cells (T24). The results have shown that 1 minute of UV-irradiation is enough to kill 90% of the cells in NPU and RT4 cultures, as determined by the live/dead viability assay. On the other hand, the majority of T24 cells survived 1 minute of UV-irradiation. Moreover, even a prolonged UV-irradiation for 30 minutes killed <50% of T24 cells. When T24 cells were pre-supplemented with mesoporous TiO2 microbeads and then UV-irradiated, the viability of these high-grade urothelial cancer cells was reduced to <10%, which points to the highly efficient cytotoxic effects of TiO2 photocatalysis. Using electron microscopy, we confirmed that the mesoporous TiO2 microbeads were internalized into T24 cells, and that the cell's ultrastructure was heavily compromised after UV-irradiation. In conclusion, our results show major differences in the sensitivity to UV-irradiation among the urothelial cells with respect to cell differentiation. To achieve an increased cytotoxicity of urothelial cancer cells, the photocatalytic approach is recommended.