Cell Line T24 Research Articles

Dibenzolium induces apoptosis and inhibits epithelial-mesenchymal transition (EMT) in bladder cancer cell lines

Bladder cancer treatments are highly aggressive and have strong side effects. Safer and more effective treatments are needed. In this study, Dibenzolium (DIB), a potent NADPH oxidase inhibitor, was evaluated for its anti-tumor effects. KK-47 (non-invasive), T24 and 5637 (invasive) cells were used in experiments. Cell proliferation, apoptosis and wound healing assays and western blotting were conducted. In addition, DIB was intratumorally administered to mice bearing KK-47, T24 and 5637 tumors, and tumor size and weight were observed over time. After removing tumors, immunohistochemistry (IHC) staining was conducted. Cell proliferation was significantly suppressed in all cell lines, and apoptotic cells increased in the KK-47 and T24 cell lines after DIB. Wound healing was suppressed in all cell lines by DIB. In KK-47 and T24, DIB increased the protein expression of the epithelial marker E-cadherin. In vivo, DIB safely suppressed tumor growth in all cell lines-bearing mice. Cleaved-Caspase-3 and E-cadherin expression increased in KK-47 and T24 tumors after DIB. In conclusion, DIB inhibited tumor growth by inducing apoptosis through the Caspase-3 pathway and reduced migration and invasion by suppressing epithelial mesenchymal transition (EMT) in bladder cancer similarly shown as our previous study of prostate cancer.

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

Two-photon photodynamic therapy with curcumin nanocomposite

Two-photon photodynamic therapy (TP-PDT) offers an innovative approach to cancer treatment that utilizes near-infrared light to activate photosensitizers and generate reactive oxygen species (ROS) for targeted cancer cell elimination. TiO2-CUR-Sofast (TCS), which uses TiO2 nanoparticles and Sofast cationic polymer to modify curcumin (CUR), has demonstrated potential as a photosensitizer under visible light irradiation, addressing the limitations of CUR's narrow spectral range and low bioavailability. This study explores the utility of the two-photon technique to activate TCS within the infrared spectrum, aiming to enhance ROS production and penetration depth compared to traditional CUR. TCS exhibits a significantly higher ROS production at 900 nm excitation wavelength, approximately 6–7 times that of CUR, signifying a substantial increase in efficiency. In TP-PDT, TCS showed significant phototoxicity against HeLa and T24 cell lines compared to CUR. Furthermore, TCS's photodynamic efficacy is further confirmed by cell apoptosis and necrosis studies, where approximately 89 % of cells treated with TCS under 900 nm light irradiation were observed in an apoptosis/necrosis state. And the TP-PDT effect in deep tissue was simulated using pig skin. It shows that the two-photon excitation has a significant penetration depth advantage over the single-photon excitation. These results indicate that the two-photon PDT scheme of TCS has greater potential than the single-photon PDT scheme in the treatment of cancer, and provides an experimental foundation for the effective treatment of deep lesions.

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

Prognostic Significance and Immune Landscape of an Efferocytosis-Related Gene Signature in Bladder Cancer.

Bladder cancer poses a significant global health challenge, underscoring the imperative for precise prognostic instruments to advance patient care. Against the backdrop of efferocytosis's increasingly recognized role in cancer, this research endeavors to develop and authenticate a prognostic signature intricately linked to efferocytosis in bladder cancer. LASSO-COX regression analysis crafted an efferocytosis-related genes risk prognostic model, followed by the construction of a column chart. External validation sets confirmed the predictive accuracy of both the model and chart. Clinical, tumor microenvironment, drug sensitivity, and immunotherapy analyses were employed to comprehensively assess efferocytosis-related scores. The expression of TGFB3 key genes was validated via RT-PCR and western blotting. Further validation included Transwell, Wound healing, Colony formation, and EDU assays. We formulated and validated an efferocytosis-related genes risk model in bladder cancer, comprising 13 core genes. The risk model demonstrated autonomous prognostic significance in both univariate and multivariate Cox analyses. Following the multivariate analysis, we devised a nomogram. Moreover, by utilizing individual risk scores derived from this risk model, we successfully stratified patients into two discernible risk cohorts, unveiling noteworthy variances in immune infiltration profiles and responsiveness to immunotherapy. Notably, the model's key gene TGFB3 was validated through comprehensive experimental investigations, including Transwell assays for migration and invasion and Wound healing assays for motility on the T24 and BIU cell lines. This study has furnished innovative perspectives on an efferocytosis-related prognostic signature, elucidating the prognosis and immune milieu intricacies in patients with bladder cancer.

Discovery of a potential bladder cancer inhibitor CHNQD-01281 by regulating EGFR and promoting infiltration of cytotoxic T cells

As one of the common malignancies that threaten human life, bladder cancer occurs frequently with a high mortality rate in the world, due to its invasion, recurrence and drug resistance. Natural products from marine microorganisms are becoming the hotspots in discovery of new candidate drug entities, especially in the area of cancer. Brefeldin A (BFA) is a natural Arf-GEFs inhibitor, but due to the low aqueous solubility, strong toxicity, and poor bioavailability, it is urgent to conduct structural optimization research. Herein, a new BFA pyridine acrylate derivative CHNQD-01281 with improved solubility was prepared and found to exert moderate to strong antiproliferative activity on a variety of human cancer cell lines. It was noteworthy that CHNQD-01281 was most sensitive to two bladder cancer cell lines T24 and J82 (IC50 = 0.079 and 0.081 μmol/L) with high selectivity index (SI = 14.68 and 14.32), suggesting a superior safety to BFA. In vivo studies revealed that CHNQD-01281 remarkably suppressed tumor growth in a T24 nude mice xenograft model (TGI = 52.63%) and prolonged the survival time (ILS = 68.16%) in an MB49 allogeneic mouse model via inducing infiltration of cytotoxic T cells. Further mechanism exploration indicated that CHNQD-01281 regulated both EGFR/PI3K/AKT and EGFR/ERK pathways and mediated the chemotactic effect of chemokines on immune effector cells. Overall, CHNQD-01281 may serve as a potential therapeutic agent for bladder cancer through multiple mechanisms.

Knockdown of AMIGO2 suppresses proliferation and migration through regulating PPAR-γ in bladder cancer

PurposeThis study aims to reveal the relationship between AMIGO2 and proliferation, migration and tumorigenicity of bladder cancer, and explore the potential molecular mechanisms.MethodsThe expression level of AMIGO2 is measured by qRT-PCR and immunohistochemistry (IHC). Stable AMIGO2 knockdown cell lines T24 and 5637 were established by lentivirus transfection. Cell Counting Kit (CCK-8 assay) was produced to determine cell proliferation, flow cytometry analysis was utilized to detect cell cycle, and wound healing assay was proceeded to test migration ability of bladder cancer cells. Xenograft mouse model was established for investigating the effect of AMIGO2 on tumor formation in vivo. The RNA Sequencing technology was applied to explore the underlying mechanisms. The expression level of PPAR-γ was measured by Western Blot.ResultsAMIGO2 was upregulated in bladder cancer cells and tissues. Inhibited expression of AMIGO2 suppresses cell proliferation and migration. Low AMIGO2 expression inhibited tumorigenicity of 5637 in nude mice. According to RNA-Seq and bioinformatics analysis, 917 DEGs were identified. The DEGs were mainly enriched in cell–cell adhesion, peroxisome proliferators-activated receptors (PPARs) signaling pathway and some other pathways. PPAR-γ is highly expressed in bladder cancer cell lines T24 and 5637, but when AMIGO2 is knocked down in T24 and 5637, the expression level of PPAR-γ is also decreased, and overexpression of PPAR-γ could reverse the suppression effect of cell proliferation and migration caused by the inhibition of AMIGO2.ConclusionAMIGO2 is overexpressed in bladder cancer cells and tissues. Knockdown of AMIGO2 suppresses bladder cancer cell proliferation and migration. These processes might be regulated by PPAR-γ signaling pathway.

A four-gene model for prognostic prediction in bladder urothelial carcinoma

BackgroundBladder urothelial carcinoma (BLCA) is one of the most common malignant tumors in urinary system worldwide. High possibility of recurrence and progression leads to poor prognosis, revealing the significant role of long-term postoperative monitoring to the patients. However, effective noninvasive diagnosis is currently limited. Materials and methodsDifferentially expressed genes (DEGs) were analyzed using R-packages. Functional enrichment analyses were performed on Metascape. The prognostic model was established by multi-step cox regression and evaluated by survival plots and receiver operating characteristic (ROC) curves. The nomogram was then constructed based on three identified prognostic factors. C-index and calibration curves were calculated to testify the capacity for predicting survival possibility of BLCA patients. The transcription levels of model genes were further verified in a gemcitabine-resistant bladder cancer cell line T24 (TGR) by quantitative real-time PCR (qRT-PCR). Results360 genes were differentially expressed between BLCA and normal bladder mucosae simultaneously in three GEO datasets, of which 59 were up-regulated and 301 were down-regulated. 159 prognostic genes were obtained from DEGs. Lasso and multivariate cox regression were conducted in sequence and the prognostic model was eventually optimized to four genes (EHBP1, RHOJ, FASN, STXBP6). Survival analyses demonstrated that the overall survival (OS) of patients in high-risk group was significantly shorter than that in low-risk group. The area under the curve (AUC) values of 3–5 years survival were basically above 0.7. Moreover, cox regression analyses showed that age, T stage and risk score were independent indicators for BLCA prognosis. For further clinical application, a nomogram was then constructed by integrating these factors. The C-index (0.72, CI 95 %, 0.669–0.775) and calibration curves demonstrated the good performance of nomogram. Importantly, the mRNA level of model genes was significantly up-regulated in TGR compared to T24, indicating a better prediction for chemotherapy-resistant BLCA patients. ConclusionCollectively, our findings suggest a novel four-gene predictive model for BLCA prognosis. It is expected to provide a valuable reference for prognostic evaluation and treatment in BLCA patients.

EGFR/TKIs induce excessive apoptosis of bladder carcinoma cells by arresting cell cycle and promoting mitochondrial peroxidation damage.

In recent years, bladder carcinoma (BC) has shown an increasing incidence, with poor patient outcomes. In clinical practice, BC is still mainly treated by surgery combined with chemoradiotherapy. However, as chemotherapy resistance of tumor cells becomes more and more obvious, it is urgent to find more effective BC treatment regimes. With the increasing application and growing attention paid to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in various neoplastic diseases, EGFR-TKIs have been considered as a new treatment direction in the future. In this study, the research team used AG1478, an EGFR-TKI, to intervene with the BC cell line T24. It was found that the cell activity was statistically decreased, the apoptosis was enhanced, and the cells were dominantly arrested in the G0/G1 phase, confirming the future therapeutic potential of EGFR-TKIs in BC. Besides, the research team further observed that AG1478 also promoted pyroptosis in T24 cells, and its mechanism is related to the induction of mitochondrial oxidative stress damage. The findings lay a more reliable foundation for the future application of EGFR-TKIs in BC.

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.

Novel artesunate-metformin conjugate inhibits bladder cancer cell growth associated with Clusterin/SREBP1/FASN signaling pathway.

Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activity while high concentration of IC50 at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.

Abstract LB437: Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells

Abstract Goals: The primary goal of this study was to develop a 3D functional bioassay to assess the relative invasiveness of bladder cancer cells. This model will support the testing of migrastatic drugs and investigation of the molecular mechanisms which allow for bladder cancer cell invasion to occur. Methods: Our model uses an organ-on-a-chip platform to assess bladder cancer cell invasiveness. This platform supports 3D culture of cells as well as pharmacokinetic studies. Our chips were designed to have a center channel to hold cells supported in Matrigel®, 2 adjacent side channels filled with Matrigel® only, and 2 outer media channels. Total cell and Matrigel® side channel length is 3 cm, channel height is 150 µm, and each channel width is 1 mm. Four bladder cancer cell lines (MB49, T24, J82, RT4) and cells isolated from a mouse bladder were used to develop our model. Cells were mixed with 1:1 media and Matrigel® and a total of 5,000 cells/µl introduced into the center channel. The 2 adjacent side channels were then filled with Matrigel®. The upper media channel was filled with 10% FBS cell culture media while the lower media channel was filled with 1% charcoal stripped cell culture media to thereby create a nutrient and growth factor gradient. Invasion of cells into the Matrigel®-containing side channels was assessed every 24 hours. Using the ImageXpress machine (Molecular Dynamics), we measured the furthest distance travelled by the cells. The chips were then fixed in 4% paraformaldehyde and stained using FITC-phalloidin and DAPI. Results: J82 and T24 cell lines, derived from patients with muscle invasive bladder cancer, and the MB49 cell line, a highly invasive mouse bladder cancer cell line, started invading into the Matrigel® side channels within 24 hours. T24, J82, and MB49 cells moved a maximum of 110, 87, and 170 µm by 48 hours, respectively. In contrast, RT4 cells, derived from a patient with non-invasive bladder cancer, took 48 hours to invade the Matrigel® channels and only invaded a maximum of 3 µm. Cells isolated from a mouse bladder did not invade into the Matrigel® side channels but survived within the chips for over 20 days. Staining of chips with DAPI and FITC-phalloidin allowed for clear visualization of cells and the actin cytoskeleton and demonstrated that immunostaining of cells within chips is possible. Conclusions: Our chip model allows for studying the invasiveness of bladder cancer cells and immunostaining experiments which can be used to assess the molecular mechanisms which drive invasion. It can also support the growth and assessment of primary cells. This model has the potential to improve bladder cancer patient treatments and outcomes by providing a physiologically relevant system to test new and existing drug combinations and to better understand the molecular mechanisms which drive bladder cancer cell invasion. Citation Format: Desiree Ewell, Nita Vue, Sakib Moinuddin, Tanoy Sarkar, Fakhrul Ahsan, Ruth L. Vinall. Development of a bladder cancer-on-a-chip assay to assess the invasive potential of bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB437.

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.

Enhancing RECK Expression Through miR-21 Inhibition: A Promising Strategy for Bladder Carcinoma Control.

Bladder carcinoma (BC) is the tenth most frequent malignancy worldwide, with high morbidity and mortality rates. Despite recent treatment advances, high-grade BC and muscle-invasive BC present with significant progression and recurrence rates, urging the need for alternative treatments. The microRNA-21 (miR-21) has superexpression in many malignancies and is associated with cellular invasion and progression. One of its mechanisms of action is the regulation of RECK, a tumor suppressor gene responsible for inhibiting metalloproteinases, including MMP9. In a high-grade urothelial cancer cell line, we aimed to assess if miR-21 downregulation would promote RECK expression and decrease MMP9 expression. We also evaluated cellular migration and proliferation potential by inhibition of this pathway. In a T24 cell line, we inhibited miR-21 expression by transfection of a specific microRNA inhibitor (anti-miR-21). There were also control and scramble groups, the last with a negative microRNA transfected. After the procedure, we performed a genetic expression analysis of miR-21, RECK, and MMP9 through qPCR. Migration, proliferation, and protein expression were evaluated via wound healing assay, colony formation assay, flow cytometry, and immunofluorescence.After anti-miR-21 transfection, miR-21 expression decreased with RECK upregulation and MMP9 downregulation. The immunofluorescence assay showed a significant increase in RECK protein expression (p < 0.0001) and a decrease in MMP9 protein expression (p = 0.0101). The anti-miR-21 transfection significantly reduced cellular migration in the wound healing assay (p < 0.0001). Furthermore, in the colony formation assay, the anti-miR-21 group demonstrated reduced cellular proliferation (p = 0.0008), also revealed in the cell cycle analysis by flow cytometry (p = 0.0038). Our results corroborate the hypothesis that miR-21 is associated with BC cellular migration and proliferation, revealing its potential as a new effective treatment for this pathology.

Abstract 1783: Understanding lipogenesis in bladder cancer

Abstract Cancer cells are known to have different metabolic needs for growth and survival and by understanding these demands of individual cancers, targeted novel therapies may be developed. Lipogenesis and ß-oxidation serve to balance lipid levels in the cell. Lipogenesis is rate limited by a series of reactions that include enzymes fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC1) that facilitate lipid formation; whereas ß-oxidation is rate limited by the transporter carnitine palmitoyltransferase 1 (CPT1) and the enzyme thiolase that catalyzes the final step of the ß-oxidation cycle. Many cancers are dependent on lipogenesis, but the role of these factors in BCa is poorly defined. The Cancer Genome Atlas was used to assess levels of relevant metabolic proteins in normal and cancerous BCa tissue, and showed FASN levels were elevated in BCa tissue and were associated with worse survival. The purpose of this project is to better understand the metabolic demands of bladder cancer (BCa) cell lines, and we hypothesized lipogenesis is a major metabolic pathway for BCa cell growth. T24, HTB-5, and HTB-9 BCa cell lines were used to evaluate the role of metabolism. CPT1 was inhibited with etomoxir, thiolase with trimetazidine, FASN with cerulenin, and ACC1 with 5-(Tetradecyloxy)-2-furoic acid (TOFA). Cell viability was assessed at 48 and 72 hours with an enzymatic activity assay measuring ß-hexosaminidase activity as a surrogate for cell proliferation. The IC50 (concentration of a drug to reduce cell proliferation by 50%) was calculated from this data. At 48 hours, cerulenin-treated cells had an IC50 of 11.80 µM, 13.28 µM, 18.31 µM for HTB-9, T24, and HTB-5 respectively. At 48 hours, TOFA-treated cells had an IC50 of 64.25 µM, 59.29 µM, 32.03 µM for HTB-9, T24, and HTB-5 respectively. Cellular proliferation was not inhibited by CPT1 or trimetazidine at enzymatic IC50 concentrations consistent with their known levels of efficacy. When adding the lipid palmitate (20µM) to TOFA-treated cells, it appeared to mediate a rescue of cell proliferation almost to control, although statistical significance was not achieved. Crystal violet assays were performed to assess long-term cell viability. There was visible reduction in colony formation in a dose-dependent manner at 5 µM and 10 µM cerulenin in T24 cells at 14 days of culture, and there were similar results in HTB-9 cells treated with 20 µM and 40 µM TOFA at 17 days. In both cerulenin and TOFA-treated cells, there was elevated cleaved caspase-3 protein expression, indicating initiation of apoptosis. In conclusion, inhibition of lipogenesis in BCa cell lines reduces cell viability by reducing proliferation and inducing apoptosis whereas inhibitors of ß-oxidation did not produce the same effect at relevant concentrations. These data indicate lipogenesis is a key metabolic pathway for BCa cell growth. Citation Format: Juliann Leak, David Matye, Erika Abbott, Benjamin Woolbright, John A. Taylor. Understanding lipogenesis in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1783.

Alkaloids from Corydalis saxicola and their antiproliferative activity against cancer cells

Eight undescribed alkaloids named corydalisine D-K (1–7), including one isoquinoline benzopyranone alkaloid (1), one benzocyclopentanone alkaloid (2), four benzofuranone alkaloids (3, 4, and 5a/5b) and two protoberberine alkaloids (6 and 7), along with fourteen known ones, were isolated from the Corydalis saxicola. Their structures, including absolute configurations, were unambiguously identified using spectroscopic techniques, single-crystal X-ray diffraction and electron circular dichroism calculation. Compounds 2, 14 and 21 exhibit antiproliferative activity against five cancer cell lines. The aporphine alkaloid demethylsonodione (compound 14), which exhibited the best activity (IC50 = 3.68 ± 0.25 μM), was subjected to further investigation to determine its mechanism of action against the T24 cell line. The molecular mechanism was related to the arrest of cell cycle S-phase, inhibition of CDK2 expression, accumulation of reactive oxygen species (ROS), induction of cell apoptosis, inhibition of cell migration, and activation of p38 MAPK signaling pathway. The results indicated that 14 could be used as a potential candidate agent for further development of anti-bladder transitional cell carcinoma.

TFRC, associated with hypoxia and immune, is a prognostic factor and potential therapeutic target for bladder cancer

BackgroundBladder cancer is a common malignancy of the urinary system, and the survival rate and recurrence rate of patients with muscular aggressive (MIBC) bladder cancer are not ideal. Hypoxia is a pathological process in which cells acquire special characteristics to adapt to anoxic environment, which can directly affect the proliferation, invasion and immune response of bladder cancer cells. Understanding the exact effects of hypoxia and immune-related genes in BLCA is helpful for early assessment of the prognosis of BLCA. However, the prognostic model of BLCA based on hypoxia and immune-related genes has not been reported.PurposeHypoxia and immune cell have important role in the prognosis of bladder cancer (BLCA). The aim of this study was to investigate whether hypoxia and immune related genes could be a novel tools to predict the overall survival and immunotherapy of BLCA patients.MethodsFirst, we downloaded transcriptomic data and clinical information of BLCA patients from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. A combined hypoxia and immune signature was then constructed on the basis of the training cohort via least absolute shrinkage and selection operator (LASSO) analysis and validated in test cohort. Afterwards, Kaplan–Meier curves, univariate and multivariate Cox and subgroup analysis were employed to assess the accuracy of our signature. Immune cell infiltration, checkpoint and the Tumor Immune Dysfunction and Exclusion (TIDE) algorithm were used to investigate the immune environment and immunotherapy of BLCA patients. Furthermore, we confirmed the role of TFRC in bladder cancer cell lines T24 and UMUC-3 through cell experiments.ResultsA combined hypoxia and immune signature containing 8 genes were successfully established. High-risk group in both training and test cohorts had significantly poorer OS than low-risk group. Univariate and multivariate Cox analysis indicated our signature could be regarded as an independent prognostic factor. Different checkpoint was differently expressed between two groups, including CTLA4, HAVCR2, LAG3, PD-L1 and PDCD1. TIDE analysis indicated high-risk patients had poor response to immunotherapy and easier to have immune escape. The drug sensitivity analysis showed that high-risk group patients were more potentially sensitive to many drugs. Meanwhile, TFRC could inhibit the proliferation and invasion ability of T24 and UMUC-3 cells.ConclusionA combined hypoxia and immune-related gene could be a novel predictive model for OS and immunotherapy estimation of BLCA patients and TFRC could be used as a potential therapeutic target in the future.

Small Cajal Body-Specific RNA12 Promotes Carcinogenesis through Modulating Extracellular Matrix Signaling in Bladder Cancer

Background: Small Cajal body-specific RNAs (scaRNAs) are a specific subset of small nucleolar RNAs (snoRNAs) that have recently emerged as pivotal contributors in diverse physiological and pathological processes. However, their defined roles in carcinogenesis remain largely elusive. This study aims to explore the potential function and mechanism of SCARNA12 in bladder cancer (BLCA) and to provide a theoretical basis for further investigations into the biological functionalities of scaRNAs. Materials and Methods: TCGA, GEO and GTEx data sets were used to analyze the expression of SCARNA12 and its clinicopathological significance in BLCA. Quantitative real-time PCR (qPCR) and in situ hybridization were applied to validate the expression of SCARNA12 in both BLCA cell lines and tissues. RNA sequencing (RNA-seq) combined with bioinformatics analyses were conducted to reveal the changes in gene expression patterns and functional pathways in BLCA patients with different expressions of SCARNA12 and T24 cell lines upon SCARNA12 knockdown. Single-cell mass cytometry (CyTOF) was then used to evaluate the tumor-related cell cluster affected by SCARNA12. Moreover, SCARNA12 was stably knocked down in T24 and UMUC3 cell lines by lentivirus-mediated CRISPR/Cas9 approach. The biological effects of SCARNA12 on the proliferation, clonogenic, migration, invasion, cell apoptosis, cell cycle, and tumor growth were assessed by in vitro MTT, colony formation, wound healing, transwell, flow cytometry assays, and in vivo nude mice xenograft models, respectively. Finally, a chromatin isolation by RNA purification (ChIRP) experiment was further conducted to delineate the potential mechanisms of SCARNA12 in BLCA. Results: The expression of SCARNA12 was significantly up-regulated in both BLCA tissues and cell lines. RNA-seq data elucidated that SCARAN12 may play a potential role in cell adhesion and extracellular matrix (ECM) related signaling pathways. CyTOF results further showed that an ECM-related cell cluster with vimentin+, CD13+, CD44+, and CD47+ was enriched in BLCA patients with high SCARNA12 expression. Additionally, SCARNA12 knockdown significantly inhibited the proliferation, colony formation, migration, and invasion abilities in T24 and UMUC3 cell lines. SCARNA12 knockdown prompted cell arrest in the G0/G1 and G2/M phase and promoted apoptosis in T24 and UMUC3 cell lines. Furthermore, SCARNA12 knockdown could suppress the in vivo tumor growth in nude mice. A ChIRP experiment further suggested that SCARNA12 may combine transcription factors H2AFZ to modulate the transcription program and then affect BLCA progression. Conclusions: Our study is the first to propose aberrant alteration of SCARNA12 and elucidate its potential oncogenic roles in BLCA via the modulation of ECM signaling. The interaction of SCARNA12 with the transcriptional factor H2AFZ emerges as a key contributor to the carcinogenesis and progression of BLCA. These findings suggest SCARNA12 may serve as a diagnostic biomarker and potential therapeutic target for the treatment of BLCA.

Prediction of anticancer drug resistance using a 3D microfluidic bladder cancer model combined with convolutional neural network-based image analysis.

Bladder cancer is the most common urological malignancy worldwide, and its high recurrence rate leads to poor survival outcomes. The effect of anticancer drug treatment varies significantly depending on individual patients and the extent of drug resistance. In this study, we developed a validation system based on an organ-on-a-chip integrated with artificial intelligence technologies to predict resistance to anticancer drugs in bladder cancer. As a proof-of-concept, we utilized the gemcitabine-resistant bladder cancer cell line T24 with four distinct levels of drug resistance (parental, early, intermediate, and late). These cells were co-cultured with endothelial cells in a 3D microfluidic chip. A dataset comprising 2,674 cell images from the chips was analyzed using a convolutional neural network (CNN) to distinguish the extent of drug resistance among the four cell groups. The CNN achieved 95.2% accuracy upon employing data augmentation and a step decay learning rate with an initial value of 0.001. The average diagnostic sensitivity and specificity were 90.5% and 96.8%, respectively, and all area under the curve (AUC) values were over 0.988. Our proposed method demonstrated excellent performance in accurately identifying the extent of drug resistance, which can assist in the prediction of drug responses and in determining the appropriate treatment for bladder cancer patients.

Green synthesis and antitumor activity of (E)-diethyl 2-styrylquinoline-3,4-dicarboxylates.

In this work, a green, efficient and catalyst-free synthesis of a series of structurally novel (E)-diethyl 2-styrylquinoline-3,4-dicarboxylates via a direct olefination reaction between diethyl 2-methylquinoline-3,4-dicarboxylate and various aromatic aldehydes was successfully accomplished by employing eco-friendly 1,3-dimethylurea/l-(+)-tartaric acid (DMU/LTA) as an inexpensive, non-toxic and reusable reaction medium. This methodology has the attractive advantages of mild reaction conditions, simple experimental operation, and the absence of any dangerous catalysts or unsafe volatile organic solvents, with satisfactory to good yields. Subsequently, a primary in vitro evaluation for their anti-proliferative activity against human cancer cell lines A549, HT29 and T24 revealed that the compound with the 3,4,5-trimethoxystyryl moiety exhibited potent anti-tumor activity with IC50 values of 2.38, 4.52 and 9.86 μmol L-1, respectively, thereby being equipotent or even better than the reference cisplatin.