Human Bladder Cancer Cell Lines Research Articles

Glucosinolate Profiles of Capparis spp. and Maerua baillonii (Capparaceae) and Cytotoxicity of Methyl Isothiocyanate-Rich Isolates From Capparis spinosa subsp. rupestris.

This study investigates the glucosinolate profiles of various Capparis spp. and Maerua baillonii, from the Capparaceae family, and evaluates the cytotoxic potential of volatile isolates from Capparis spinosa subsp. rupestris. Using UHPLC-DAD-MS/MS, GSLs were identified and quantified across different plant tissues. Glucocapparin, predominant glucosinolate in C. spinosa subsp. spinosa, C. spinosa subsp. rupestris, and M. baillonii, were isolated and analyzed by NMR in its desulfo-form. Notably, glucosinolates were absent in C. richardii and specific tissues of M. baillonii (e.g., leaves and stems), underscoring species and/or tissue-specific variability within Capparaceae. Less common glucosinolates for Capparaceae plants, such as glucocochlearin, glucohirsutin, and glucoarabin were also detected. Methyl isothiocyanate was found to be the main volatile in all isolates obtained through various isolation methods, comprising ca. 80%-90% of the total volatiles. Methyl isothiocyanate-rich volatile isolates were tested for cytotoxicity against human breast cancer (MDA-MB-231) and bladder cancer (T24) cell lines using the MTT assay, showing significant activity with IC50 values of 3.81 and 5.95µg/mL, respectively. These findings underscore the anticancer potential of glucocapparin-bearing plants from the Capparaceae family.

Hyperthermia reduces cancer cell invasion and combats chemoresistance and immune evasion in human bladder cancer.

Bladder cancer (BC) is a common malignancy and its most prevalent type is urothelial carcinoma, which accounts for ~90% of all cases of BC. The current treatment options for BC are limited, which necessitates the development of alternative treatment strategies. Hyperthermia (HT), as an adjuvant cancer therapy, is known to improve the efficacy of chemotherapy or radiotherapy. The present study aimed to investigate the anti‑tumor effects of HT on cell survival, invasiveness, chemoresistance and immune evasion in human BC cell lines (5637, T24 and UMUC3). Calcein AM staining was performed to analyze the cytotoxicity of natural killer (NK) cells against human BC cells following HT treatment. Cell migration and invasion affected by HT were analyzed using Transwell migration and invasion assays. It was found that HT inhibited the proliferation of BC cells by downregulating the phosphorylation of protein kinase B. Moreover, HT effectively enhanced the sensitivity of BC cells to the chemotherapy drug cisplatin (DDP) and reduced the chemoresistance of DDP‑resistant cells by downregulating the expression of cadherin‑11. It was further demonstrated that HT inhibited the migration and invasion of BC cells and enhanced the cytotoxic effects of NK cells. In summary, the antineoplastic effects of HT were mediated through three main mechanisms: Enhancement of the chemosensitivity of BC cells and mitigation of DDP‑induced chemoresistance, suppression of the invasive potential of BC cells and reinforcement of the anticancer response of NK cells. Thus, HT appears to be a promising adjunctive therapy for human BC.

Synergistic Combination of Quercetin and Mafosfamide in Treatment of Bladder Cancer Cells.

Bladder cancer, which has a rising incidence, is the 10th most common cancer. The transitional cell carcinoma histotype is aggressive and often current therapies are ineffective. We investigated the anti-proliferative effect of quercetin, a natural flavonoid, in combination with the alkylating agent mafosfamide (MFA) on two human bladder cancer cell lines, namely RT112 and J82, representing the progression from low-grade to high-grade tumors, respectively. In both cell types, the combined treatment led to a synergic reduction in cell viability confirmed by a combination index of less than one, though different biological responses were noted. In J82 cells, MFA alone and, to a lesser extent, with quercetin caused cell cycle arrest in the G2/M phase, but only the combined treatment triggered apoptotic cell death. In contrast, in RT112 cells, quercetin induced autophagy, evidenced by the autophagosome formation and the increase in LC-3 lipidation. Interestingly, the synergistic effect was observed only when cells were pre-treated with MFA for 24 h before adding quercetin, not in the reverse order. This suggests that quercetin may help overcome MFA resistance to apoptosis. Although further studies are needed, investigating the combined effects of quercetin and MFA could help elucidate the mechanisms of drug resistance in bladder cancer treatment.

Mechanistic study of NUPR1 in bladder cancer development through transcriptional regulation of CCR2.

Nuclear protein-1 (NUPR1) (also known as p8) is one of the genes associated with transcription factors that participate in various aspects of cancer initiation and development. However, the molecular mechanisms of NUPR1 in bladder cancer (BLCA) remain unclear. We conducted an analysis of the correlation between NUPR1 expression and related genes using the Gene Expression Omnibus (GEO) online database. We employed lentivirus-mediated small interfering RNA (siRNA) to knockdown the expression of NUPR1 in two human BLCA cell lines. In vitro experiments were conducted to validate the impact of NUPR1 interference on BLCA and the influence of NUPR1 on the transcription of chemokine receptor-2 (CCR2). Furthermore, transcription factors for CCR2 were predicted using the PROMO database. Co-immunoprecipitation (Co-IP) and immunofluorescence double staining were used to detect the binding between NUPR1 and CCAAT/enhancer binding protein γ (CEBPG). In vivo and in vitro experiments were conducted to validate that NUPR1 regulates CCR2 transcription through CEBPG. In vitro experiments indicate that the suppression of NUPR1 inhibited BLCA growth. Analysis of the GEO database revealed a positive correlation between the expression of NUPR1 and CCR2. Luciferase experiments confirmed that NUPR1 influences the transcription of CCR2. Online data indicates that CEBPG is a transcription factor for CCR2. Co-IP and immunofluorescence double staining confirmed binding between NUPR1 and CEBPG. Luciferase assays and chromatin immunoprecipitation (ChIP) demonstrate that CEBPG regulates the transcription of CCR2. Additionally, rescue experiments at the cellular level and animal experiments validated the aforementioned mechanism. NUPR1 promotes a promotional role in BLCA, and interference with NUPR1 can inhibit the proliferation and invasive abilities of BLCA. There was a correlation between the expressions of NUPR1 and CCR2, and NUPR1 binds with CEBPG in the cell nucleus. Transcriptional regulation of CCR2 by NUPR1 may be achieved through the involvement of CEBPG.

Mechanisms of the Antineoplastic Effects of New Fluoroquinolones in 2D and 3D Human Breast and Bladder Cancer Cell Lines.

Antibacterial fluoroquinolones have emerged as potential anticancer drugs, thus prompting the synthesis of novel molecules with improved cytotoxic characteristics. Ciprofloxacin and norfloxacin derivatives, previously synthesized by our group, showed higher anticancer potency than their progenitors. However, no information about their mechanisms of action was reported. In this study, we selected the most active among these promising molecules and evaluated, on a panel of breast (including those triple-negative) and bladder cancer cell lines, their ability to induce cell cycle alterations and apoptotic and necrotic cell death through cytofluorimetric studies. Furthermore, inhibitory effects on cellular migration, metalloproteinase, and/or acetylated histone protein levels were also evaluated by the scratch/wound healing assay and Western blot analyses, respectively. Finally, the DNA relaxation assay was performed to confirm topoisomerase inhibition. Our results indicate that the highest potency previously observed for the derivatives could be related to their ability to induce G2/M cell cycle arrest and apoptotic and/or necrotic cell death. Moreover, they inhibited cellular migration, probably by reducing metalloproteinase levels and histone deacetylases. Finally, topoisomerase inhibition, previously observed in silico, was confirmed. In conclusion, structural modifications of progenitor fluoroquinolones resulted in potent anticancer derivatives possessing multiple mechanisms of action, potentially exploitable for the treatment of aggressive/resistant cancers.

Targeting Chemoresistance in Advanced Bladder Cancers with a Novel Adjuvant Strategy.

Advanced urinary bladder cancer is characterized by rapid progression and development of therapy resistance. About 30% of the patients are diagnosed with high-grade tumors (grade > T2a). A typical nonsurgical treatment is systemic chemotherapy using cisplatin (C) and gemcitabine (G). However, treatment failure and subsequent disease progression are common in treated patients, and adjuvant therapies are not significantly effective. The therapeutic potential of a molecular hybrid of ursolic acid (UA), a pentacyclic-triterpene conjugated to N-methyl piperazine (UA4), was tested on both naïve (WT) and gemcitabine-resistant (GemR) variants of two human invasive bladder cancer cell lines, 5637 and T24. UA4 killed 5637 (4 µmol/L), T24 (4 µmol/L) WT, and GemR cells in vitro at equal potency. Pretreatment with UA4 followed by G synergistically killed WT and GemR cells by >50% compared with G followed by UA4. Oral gavage of UA4 (100 mg/kg) inhibited WT and GemR tumor growth in athymic mice. UA4 + G was more effective against GemR tumors than either drug alone. Studies revealed cytotoxic autophagy as a mechanism of UA4 cytotoxicity. UA4 induced moderate apoptosis in T24 but not in 5637 cells. Mitochondrial integrity and function were most affected by UA4 because of high levels of reactive oxygen species, disruption of mitochondrial membrane, and cell cycle arrest. These effects were enhanced in the UA4 + G combination. UA4 was well-tolerated in mice, and oral gavage led to a serum level >1 µmol/L with no systemic toxicity. These results show the potential of UA4 as a nontoxic alternative treatment for high-grade bladder cancer.

Abstract B019: Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer

Abstract Introduction: Interferon α gene therapy (Ad-IFNα/Syn3) is FDA approved for treatment of BCG-unresponsive non-muscle invasive bladder cancer. Ad-IFNα/Syn3 is a non-replicating adenovirus that when transduced into target tissues, produces IFNa2b protein with anti-tumor activity. In the phase 3 multicenter trial, 45.5% of patients with carcinoma-in-situ and 60% of those with Ta/T1 disease who had a complete response to Ad-IFNα/Syn3 at 3 months remained recurrence free at 12 months. Targeting mechanisms of resistance and identifying predictive biomarkers to improve patient selection and overall response rates is a top priority. Towards this, we treated 29 human bladder cancer cell lines with Ad-IFNα/Syn3 and assessed their responses to therapy. We identified sensitive and resistant cell lines and gene signatures predictive of response. Methods: Human bladder cancer cell lines were grown in MEM supplemented with 10% FBS in 96-well plate. After overnight attachment, these were treated with Ad-IFNα/Syn3 (MOI 1000) and cell titre glow assay was performed 72h post-treatment. Cells were also seeded in 6-well plates. and after 24h cell-free supernatants were collected for ELISA and cells were collected for RNA isolation using RNAqueous Total RNA Isolation kit (Thermo Fisher). RNA quality was assessed using Tapestation. RNAseq analysis was carried out using Ion proton Sequencer. Ingenuity pathway analysis (IPA) and gene set enrichment were performed on the sequencing data for the cell lines. ELISA was used to measure IFNα and IL6 protein levels in cell free supernatants. We also tested organoids developed from bladder cancer patients to test drug efficacy and identify gene signatures. Results: Out of the 29 cell lines, we identified that sensitivity to Ad-IFNα/Syn3 was pronounced in luminal cells (8/13 luminal cell lines). Most basal cells were either resistant (>70% viability) or only moderately sensitive to Ad-IFNα/Syn3 (between 70-40% viability) when compared to control. We selected 7 cell lines for sequencing and ELISA measurements: luminal cell lines BV, UC6, Rt4V6 and UC5, and basal cell lines ScaBER, HT1197, and T24. All cell lines mentioned above could be successfully transduced by the adenoviral vector as measured by increased expression of IFNα protein in the cell-free supernatants. Ad-IFNα/Syn3-sensitive luminal cell lines at baseline had lower IFNα and IFNγ scores when compared to resistant basal cell lines. We identified several cell-death pathways enriched in treated cells that were sensitive to Ad-IFNα/Syn3. IL6 measurements in cell free supernatants revealed increased expression of IL6 after treatment in sensitive cells. However, in resistant cells IL6 expression was either high at baseline (ScaBER) or both undetectable at baseline and after treatment (UC5). Conclusion: Our results suggest that the luminal cell lines are more sensitive to Ad-IFNα/Syn3 and that high baseline expression of IFNα and IFNγ in basal cell lines contributes to resistance. IL6 levels are also predictive of response to Ad-IFNα/Syn3. Citation Format: Morgan E. Pierce, Alexis R. Steinmetz, Aruna Nannapaneni, Tanner S. Miest, David J. McConkey, Sharada Mokkapati, Colin P.N. Dinney. Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B019.

MANNosylation of Mesoporous Silica Nanoparticles Modifies TLR4 Localization and NF-κB Translocation in T24 Bladder Cancer Cells.

D-mannose is widely used as non-antibiotic treatment for bacterial urinary tract infections. This application is based on a well-studied mechanism of binding to the type 1 bacterial pili and, therefore, blocking bacteria adhesion to the uroepithelial cells. To implement D-mannose into carrier systems, the mechanism of action of the sugar in the bladder environment is also relevant and requires investigation. Herein, two different MANNosylation strategies using mesoporous silica nanoparticles (MSNs) are described. The impact of different chemical linkers on bacterial adhesion and bladder cell response is studied via confocal microscopy imaging of the MSN interactions with the respective organisms. Cytotoxicity is assessed and the expression of Toll-like receptor 4 (TLR4) and caveolin-1 (CAV-1), in the presence or absence of simulated infection with bacterial lipopolysaccharide(LPS), is evaluated using the human urinary bladder cancer cell line T24. Further, localisation of the transcription factor NF-κB due to the MANNosylated materials is examined over time. The results show that MANNosylation modifies bacterial adhesion to the nanomaterials and significantly affects TLR4, caveolin-1, and NF-κB in bladder cells. These elements are essential components of the inflammatory cascade/pathogens response during urinary tract infections. These findings demonstrate that MANNosylation is a versatile tool to design hybrid nanocarriers for targeted biomedical applications.

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

Oncopig bladder cancer cells recapitulate human bladder cancer treatment responses in vitro.

Bladder cancer is a common neoplasia of the urinary tract that holds the highest cost of lifelong treatment per patient, highlighting the need for a continuous search for new therapies for the disease. Current bladder cancer models are either imperfect in their ability to translate results to clinical practice (mouse models), or rare and not inducible (canine models). Swine models are an attractive alternative to model the disease due to their similarities with humans on several levels. The Oncopig Cancer Model has been shown to develop tumors that closely resemble human tumors. However, urothelial carcinoma has not yet been studied in this platform. We aimed to develop novel Oncopig bladder cancer cell line (BCCL) and investigate whether these urothelial swine cells mimic human bladder cancer cell line (5637 and T24) treatment-responses to cisplatin, doxorubicin, and gemcitabine in vitro. Results demonstrated consistent treatment responses between Oncopig and human cells in most concentrations tested (p>0.05). Overall, Oncopig cells were more predictive of T24 than 5637 cell therapeutic responses. Microarray analysis also demonstrated similar alterations in expression of apoptotic (GADD45B and TP53INP1) and cytoskeleton-related genes (ZMYM6 and RND1) following gemcitabine exposure between 5637 (human) and Oncopig BCCL cells, indicating apoptosis may be triggered through similar signaling pathways. Molecular docking results indicated that swine and humans had similar Dg values between the chemotherapeutics and their target proteins. Taken together, these results suggest the Oncopig could be an attractive animal to model urothelial carcinoma due to similarities in in vitro therapeutic responses compared to human cells.

AIM high: Epigenetic modulation and immune stimulation in bladder cancer.

648 Background: Immune-checkpoint inhibitors (ICI) have emerged as valuable treatment options for patients with bladder cancer, particularly those who are ineligible for other therapies or have experienced disease progression on treatment. Response rates are only 20-30%, highlighting the need for biomarkers to predict treatment response and strategies to overcome resistance. Epigenetic modulators including the DNA methyltransferase (DNMT) inhibitor 5-azacitidine (AZA) and histone deacetylase inhibitors (HDACi) may represent co-targeting therapies in combination with ICI to enhance antitumor immunity. AZA has been demonstrated to increase the expression of a panel of immunomodulatory genes termed the “AZA Immune gene panel (AIM)” in breast, colorectal, and ovarian cancer cell lines. We hypothesized that bladder cancer with low AIM expression can be therapeutically converted by epigenetic modulation to “AIM-high” for future combination with ICI. Methods: Five human bladder cancer cell lines were selected: SCaBER, SW780, T24, RT4 and one developed at our institution (CoCaB1). The Cancer Cell Line Encyclopedia (CCLE) was used to screen the four included lines for intrinsic AIM expression, which were predicted to be AIM-high (n=1) and AIM-low (n=3). A cell viability assay was performed to screen for AZA and HDACi toxicity. Cells were treated with drug or vehicle for 72 hours and harvested at day 6, 14, and 21. RNA was extracted for RNA sequencing (RNA-seq) analyses. For analysis of AIM gene expression in clinical specimens, RNA-seq data was obtained from primary (n=6) and metastatic (n=83) bladder cancer specimens for 20 patients participating in the University of Washington Bladder Cancer Rapid Autopsy Program. Results: RNA-seq analyses of 5 cell lines demonstrated increased expression (>log 2-fold change, p<0.05) of a subset of AIM genes involved in inflammation, interferon, cytokine/chemokine signaling, and cancer testis antigens (CTA, a family of immunogenic proteins) with AZA treatment. Co-treatment with HDACi showed additional upregulation of AIM genes. Gene set enrichment analyses uncovered enrichment in T-cell pathway activation. In metastatic bladder cancer tissues, interferon, cytokine/chemokine, inflammation, and CTA AIM gene set categories were upregulated in a subset of patients. AIM gene enrichment displayed a patient-dependent pattern and was consistent across metastatic sites within a patient. Conclusions: Epigenetic priming therapy increases the expression of immunomodulatory and T-cell related genes in bladder cancer cell lines with low baseline AIM expression. Additional correlative studies in the rapid autopsy series will further determine clinical- and treatment-related factors contributing to intrinsic immune-related gene expression. Building on this paradigm, future in vivo and clinical studies could lead to novel combination therapies for patients with bladder cancer.

Near-infrared photoimmunotherapy targeting Nectin-4 in a preclinical model of bladder cancer

Enfortumab vedotin (EV), an antibody-drug conjugate (ADC) that targets Nectin-4, has shown promising results in the treatment of bladder cancer. However, multiple resistance mechanisms that are unique to ADCs limit the therapeutic potential of EV in clinical practice. Here, we developed and tested a Nectin-4-targeted near-infrared photoimmunotherapy (NIR-PIT) that utilizes the same target as EV but utilizes a distinct cytotoxic and immunotherapeutic pathway in preclinical models of bladder cancer. NIR-PIT was effective in vitro against luminal subtype human bladder cancer cell lines (RT4, RT112, MGH-U3, SW780, and HT1376-luc), but not against other subtype cell lines (UMUC3 and T24). In vivo, the tumor site was clearly visible by Nectin-4-IR700 fluorescence 24 h after its administration, suggesting the potential as an intraoperative imaging modality. NIR-PIT significantly suppressed tumor growth and prolonged survival in SW780 and RT112 xenograft models. Weekly treatment with NIR-PIT further improved tumor control in RT112 xenograft models. The effectiveness of NIR-PIT was also confirmed in HT1376-luc orthotopic xenograft models. Histological analysis verified that NIR-PIT induced a significant pathologic response. Taken together, Nectin-4-targeted NIR-PIT shows promise as a treatment for luminal subtype bladder cancers.

Molecular mechanism of anticancer effect of heat shock protein 90 inhibitor BIIB021 in human bladder cancer cell line

Bladder cancer is a type of urologic malignancy that exhibits significant morbidity, mortality, and treatment costs. Inhibition of heat shock protein 90 (HSP90) activity has been a promising pharmacological strategy for blocking of bladder cancer pathogenesis. BIIB021 is a next-generation HSP90 inhibitor which interrupts ATP hydrolysis process of HSP90 and inhibits the stabilization and correct folding of client proteins. In current study, we aimed to investigate the molecular mechanism of the anticancer activity of BIIB021 in human bladder cancer T24 cells. Our results revealed that nanomolar concentration of BIIB021 decreased viability of T24 cell. BIIB021 downregulated HSP90 expression in T24 cells and inhibited the refolding activity of luciferase in the presence of T24 cell lysate. PCR array data indicated a significant alteration in transcript levels of cancer-related genes involved in metastases, apoptotic cell death, cell cycle, cellular senescence, DNA damage and repair mechanisms, epithelial-to-mesenchymal transition, hypoxia, telomeres and telomerase, and cancer metabolism pathways in T24 cells. All findings hypothesize that BIIB021 could exhibit as effective HSP90 inhibitor in the future for treatment of bladder cancer patients.

Integrated bioinformatics analysis identifies a Ferroptosis-related gene signature as prognosis model and potential therapeutic target of bladder cancer.

Bladder cancer (BLCA) is one of the most prevalent cancers worldwide. Ferroptosis is a newly discovered form of non-apoptotic cell death that plays an important role in tumors. However, the prognostic value of ferroptosis-related genes (FRGs) in BLCA has not yet been well studied. In this study, we performed consensus clustering based on FRGS and categorized BLCA patients into 2 clusters (C1 and C2). Immune cell infiltration score and immune score for each sample were computed using the CIBERSORT and ESTIMATE methods. Functional annotation of differentially expressed genes were performed by Gene Ontology (GO) and KEGG pathway enrichment analysis. Protein expression validation were confirmed in Human Protein Atlas. Gene expression validation were performed by qPCR in human bladder cancer cell lines lysis samples. C2 had a significant survival advantage and higher immune infiltration levels than C1. Additionally, C2 showed substantially higher expression levels of immune checkpoint markers than C1. According to the Cox and LASSO regression analyses, a novel ferroptosis-related prognostic signature was developed to predict the prognosis of BLCA effectively. High-risk and low-risk groups were divided according to risk scores. Kaplan-Meier survival analyses showed that the high-risk group had a shorter overall survival than the low-risk group throughout the cohort. Furthermore, a nomogram combining risk score and clinical features was developed. Finally, SLC39A7 was identified as a potential target in bladder cancer. In conclusion, we identified two ferroptosis-clusters with different prognoses using consensus clustering in BLCA. We also developed a ferroptosis-related prognostic signature and nomogram, which could indicate the outcome.

Iberis sempervirens: Antiproliferative Potential from Our Garden

Glucosinolates (GSLs) extracted from various parts of Iberis sempervirens L., including seeds, stems, leaves, and flowers, were qualitatively and quantitatively assessed. The analyses of GSLs were performed by their desulfo counterparts using the UHPLC-DAD-MS/MS technique and by their volatile breakdown products, isothiocyanates, using the GC-MS technique. The GSL profile comprised various types, including those derived from: methionine, represented by methylsulfinylalkyl GSL (glucoiberin), and methylsulfanylalkyl GSL (glucoibervirin and glucoerucin); phenylalanine (glucotropaeolin); and tryptophan (4-methoxyglucobrassicin). Among these, the highest level of glucoiberin was detected in the leaves, reaching 35.37 µmol/g of dry weight (DW), while the highest level of glucoibervirin was detected in the seeds, reaching 18.51 µmol/g DW. To obtain GSL breakdown products, a variety of isolation methods were employed, including hydrodistillation in a Clevenger-type apparatus (HD), CH2Cl2 after myrosinase hydrolysis for 24 h (EXT), microwave-assisted distillation (MAD), and microwave hydrodiffusion and gravity (MHG). Volatile isolates were tested for their antiproliferative activity using an MTT assay against the human lung cancer cell line A549 and the human bladder cancer cell line T24 during an incubation period of 72 h. HD and MAD showed the best activity against T24, with IC50 values of 0.61 µg/mL and 0.62 µg/mL, respectively, while EXT was the most effective against the A549 cell line, with an IC50 of 1.46 µg/mL.

P190 - Studied on the effect of metformin combined with epirubicin on apoptosis of human bladder cancer cell line through PI3K/AKT/mTOR signaling pathway

P190 - Studied on the effect of metformin combined with epirubicin on apoptosis of human bladder cancer cell line through PI3K/AKT/mTOR signaling pathway

Cell Line-Based Human Bladder Organoids with Bladder-like Self-Organization-A New Standardized Approach in Bladder Cancer Research.

Three-dimensional tumor models have gained significant importance in bladder cancer (BCa) research. Organoids consisting of different cell types better mimic solid tumors in terms of 3D architecture, proliferation, cell-cell interaction and drug responses. We developed four organoids from human BCa cell lines with fibroblasts and smooth muscle cells of the bladder, aiming to find models for BCa research. The organoids were characterized in terms of cytokeratins, vimentin, α-actin and KI67 by immunoreactivity. Further, we studied ligand-dependent activation of the Wnt/β-catenin pathway and investigated the responses to anti-tumor therapies. The organoids mimicked the structure of an inverse bladder wall, with outside urothelial cells and a core of supportive cells. The cytokeratin staining patterns and proliferation rate were in conjunction with the origins of the BCa cells. RT-112 even showed stratification of the epithelium. Treatment with Wnt10B led to increased β-catenin (active) levels in high-grade organoids, but not in low-grade BCa cells. Doxorubicin treatment resulted in clearly reduced viability (10-30% vs. untreated). In contrast, the effectivity of radiotherapy depended on the proliferation status of BCa cells. In conclusion, cell-line-based organoids can form bladder-like structures and reproduce in vivo features such as urothelial differentiation and stratification. Thus, they can be useful tools for functional studies in BCa and anti-cancer drug development.

Cardamonin suppresses glycolysis and induces oxidative stress by inhibiting PI3K/AKT/mTOR pathway in bladder cancer cells

Purpose: To evaluate the effect and underlying mechanisms of action of cardamine on the progression of bladder cancer (BC).Methods: Human bladder epithelium immortalized cell line (SV-HUC-1) and human bladder cancer (BC) cell lines (T24 and UM-UC-3) were used in this investigation. They were treated with cardamine at concentrations of 0, 15, 30, 60 or 120 μmol/L. Cell viability was determined using cell counting kit 8(CCK-8) assay while 5-ethynyl-2'-deoxyuridine (Edu) assay was used to assess cell proliferation. Cell apoptosis as well as reactive oxygen species (ROS) accumulation were determined by flow cytometry whereas glucose uptake, adenosine triphosphate (ATP) level and lactate production were determined using their respective assay kits. Furthermore, the expression levels of nuclear factor level (erythroidderived 2)-like 2 (Nrf2), NAD(P)H, quinone oxidoreductase 1 (NQO1), protein kinase B (AKT), phosphorylated-AKT (p-AKT), phosphatidylinositol 3-kinase (PI3K), p-PI3K, mechanistic target of rapamycin kinase (mTOR) and p-mTOR were evaluated by western blot analysis.Results: Cardamine significantly reduced cell viability and inhibited cell proliferation in BC cells in a dose-dependent manner, but did not affect human normal cells. In addition, treatment with the compound induced apoptosis in BC cells; the higher the concentration, the higher the apoptosis level. Besides, cardamine administration suppressed aerobic glycolysis, and decreased the nuclear factor level (Nrf2) level, thereby increasing ROS production in a concentration-dependent manner.Furthermore, it blocked the activation of PI3K/AKT/mTOR signal cascade.Conclusion: Cardamine inhibits glycolysis and PI3K/AKT/mTOR pathway, and also promotes apoptosis as well as oxidative stress in BC cells. Thus, the compound is a potential therapeutic reagent for BC.

Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads

BackgroundHuman adipose tissue-derived stem cells (ASCs) are widely used in regenerative medicine and tissue engineering. Magnetic-activated cell sorting (MACS) with monoclonal antibodies (mAbs) that bind surface markers of stem cells such as CD105, CD73, and CD90 is currently applied for the enrichment and isolation of ASCs. Alternatives to mAbs are the variable domains of heavy chain antibodies (VHHs), which are naturally produced in camelids. We report the application of an anti-CD105 VHH conjugated to magnetic beads in the isolation of ASCs in vitro. ResultsTwo identical anti-CD105 VHHs (EngVHH17) were screened by phage display from a VHH cDNA library constructed from the PBMCs of an alpaca immunized with a lysate of human bladder cancer cell line (T24 cells). EngVHH17 was cloned in pET22b(+), expressed in Escherichia coli BL21 and purified in Ni-NTA chromatography resulting in a ∼15 kDa VHH antibody. EngVHH17 binds CD105 with high affinity (Kd = 3.9 × 10−10 M). The specific binding of EngVHH17 to CD105 was visualized by fluorescence inmunolabeling of phorbol-12-myristate-13-acetate (PMA)-differentiated THP1 cells using FITC labeled anti-6 × His Tag IgG1 mouse mAb. EngVHH17-magnetic beads selectively sorted human ASCs from osteoblasts in a mixed culture in vitro. The selective recovery of ASC cells using different ASC/Osteoblast ratios was higher than 85%. ConclusionsEngVHH17 coupled to magnetic beads binds CD105 expressed on the cell surface of ASCs and isolates them from osteoblasts in mixed cultures in vitro by application of an external magnetic field. EngVHH17 can be further evaluated for the isolation of MSCs by MACS.How to cite: Gushiken E, Quintana M, Perez L, et al. Isolation of adipose derived mesenchymal stem cells by using anti-CD105 VHH-magnetic beads. Electron J Biotechnol 2023;66. https://doi.org/10.1016/j.ejbt.2023.06.004.

Anticancer effects of cell-free culture supernatant of Escherichia coli in bladder cancer cell line: New insight into the regulation of inflammation

Bladder cancer (BC) is the 10th most common malignancy in worldwide, with substantial mortality and morbidity if not treated effectively. According to various research, inflammatory circumstances majorly impact the microenvironment of bladder cancer, and the chronic presence of cytokines and chemokines promotes tumor progression. In this investigation, we explored the impact of cell-free culture supernatant ofEscherichia colistrain 536 on inflammatory cytokines and chemokines in bladder cancer model microarray data (GSE162251). Then we examined in silico outcomes on human bladder cancer cell line 5637 to verify and extrapolate findings. This investigation revealed for the first time that this compound has potent suppressor effects on interleukin 1 beta (IL-1β), C–C motif chemokine ligand 2 (CCL2), and C-X3-C motif chemokine ligand 1 (CX3CL1) gene expression as well as increased NAD(P)H quinone dehydrogenase 1 (NQO1), as an anti-oxidant agent, gene expression in 4, 8, and 24 h. Moreover, we confirmed that c-MYC, a member of the MYC proto-oncogene family, gene expression reduced in 5637 cells in 4 h and then followed up its expression in 8 and 24 h. In addition, our investigation demonstrated that the supernatant raised the BCL2-Associated X Protein/B-cell lymphoma 2 (BAX/BCL2) ratio, and subsequent flow cytometry analysis demonstrated that the supernatant induction apoptosis and necrosis. In conclusion, our findings demonstrate that this compound is a potential candidate for the suppression of bladder cancer progression.