Bladder Cancer Cell Line T24 Research Articles

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.

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.

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.

Abstract 5196: Stepwise molecular mechanisms involved in chemoresistance in bladder cancer cells

Abstract Bladder cancer (BC) is one of the most prevalent genitourinary tumors worldwide, with approximately 573,000 new cases reported. Approximately 80% of BC patients are diagnosed with non-muscle-invasive bladder cancer (NMIBC) with a high five-year survival rate, while the remaining 20% are diagnosed with muscle-invasive bladder cancer (MIBC). Although the surgical operation has been utilized to BC, patients with NMIBC frequently experience disease recurrence, of which about 20% progress to MIBC. However, the response rate of MIBC patients to gemcitabine treatment has been shown to be limited to less than 40% efficacy, and only a small number of patients have potential benefits.Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23-genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. We suggest that our findings improve prognosis by predicting to acquiring of chemotherapy resistance in bladder cancer patients. Citation Format: Jae-Jun Kim, Gi-Eun Yang, Tae Hwan Shin, Sun-Hee Leem. Stepwise molecular mechanisms involved in chemoresistance in bladder cancer cells [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 5196.

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.

Biological function and regulatory mechanism of mta2 expression in bladder cancerous cells

Purpose: To study the function, regulation, and potential clinical use of mta2 gene in carcinoma of the bladder.
 Methods: Viral vector and si-mta2 were transfected into both T24 and EJ bladder cancer cells, for induction of overexpression and knockdown of metastasis-associated gene 2 (mta2), respectively. In vitro invasiveness and migratory potential of cell lines were assayed using Matrigel and Transwell procedures, respectively, under the two mta2 expression conditions.
 Results: Transwell migration data revealed that overexpressed mta2 protein significantly enhanced the migratory capacity of T24 bladder cancer cell line, while knockdown of mta2 significantly reduced the migration of EJ bladder cancer cell line (p < 0.01). Matrigel invasion assay showed that overexpression of mta2 protein significantly enhanced the invasive potential of T24 bladder cancer cell line, but knockdown of mta2 significantly (p < 0.01) reduced the invasive capacity of EJ bladder cancer cell line. There was a lower amount of E-cadherin protein in T24 bladder cancer cell line overexpressing mta2 than in cd511b-transfected and un-transfected cells, but N-cadherin protein level was significantly higher. Furthermore, there was a significantly higher amount of E-cadherin protein expression in EJ bladder cancer cell line with mta2 knockdown than in si-NC transfection and un-transfected cells (p < 0.01).
 Conclusion: Knockdown of mta2 inhibits the proliferation, migration and invasion of bladder cancer cell lines through a mechanism related to the inhibition of epithelial-mesenchymal transition-related process proteins.

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.

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.

Preliminary study on the role of the CSMD2 gene in bladder cancer

BackgroundCSMD2 has been reported as a potential prognostic factor in several cancers. However, whether CSMD2 affects bladder cancer (BC) remains unclear. MethodsPublic data were obtained from the TCGA (https://cancergenome.nih.gov) databases. CSMD2expression and its prognostic value were analyzed using bioinformatics methods. CSMD2 mRNA level in patients with BC and BC cell lines was evaluated via quantitative reverse transcriptase polymerase chain reaction. CSMD2 protein level in patients with BC was evaluated via immunohistochemistry. BC cell lines T24 and UMUC-3 were selected for loss-of-function assays targeting CSMD2. Cell viability was determined by CCK8 and clone formation experiments. Cell migration and invasion were evaluated using Transwell assays. Furthermore, the transcriptome of UMUC-3 with CSMD2 knockdown was sequenced to analyze potential signaling network pathways. Finally, the TIMER2.0 database was employed to identify the correlation between CSMD2 and immune cells in the tumor microenvironment. ResultsCSMD2 expression was up-regulated in BC tissues compared to adjacent tissues. High CSMD2 expression was associated with poor survival and could serve as an independent predictor for survival in patients with BC. Furthermore, down-regulation of CSMD2 notably restrained the viability, migration, and invasion abilities of T24 and UMUC-3 cells. Moreover, transcriptomic sequencing after CSMD2 knockdown in UMUC-3 cells revealed its involvement in the regulation of the malignant phenotype in BC. Finally, public databases suggest a connection between CSMD2 and immune cell infiltration in BC. ConclusionsThese findings suggest that CSMD2 may promote proliferation and tumorigenicity, and could represent a potential target for improving the prognosis of BC.

CUDC‑101 is a potential target inhibitor for the EGFR‑overexpression bladder cancer cells.

Bladder cancer is one of the most common urological malignancies worldwide. The molecular mechanism underlying its development is complex, but its carcinogenesis has been proposed to occur with cell proliferation and resistance to apoptosis, driven by the signaling activity of abundant EGFR and receptor tyrosine‑protein kinase erbB‑2. In the present study, T24 bladder cancer cell lines with EGFR‑overexpression were constructed, before the multi‑target inhibitor CUDC‑101 was used to investigate its potential as a targeted therapeutic agent for bladder cancer using chemosensitivity methods. The results showed that CUDC‑101 induced cytotoxic effects, inhibited growth vitality and proliferation in a dose‑dependent manner. CUDC‑101 also altered the skeletal morphology and microfilament structure, while blocking cell cycle progression and causing apoptosis. These results supported the proposed cytotoxic effects of CUDC‑101, in addition to its inhibitory effects on cell division and proliferation in EGFR‑overexpressing bladder cancer cells. Therefore CUDC‑101 may to be a potential therapeutic option for the treatment of bladder cancer.

Isolation of Volatile Compounds by Microwave-Assisted Extraction from Six Veronica Species and Testing of Their Antiproliferative and Apoptotic Activities.

This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different Veronica species (V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, Veronica cymbalaria, and V. officinalis) and to test their antiproliferative and apoptotic activities, according to the authors' knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34-6.49%), linalool (0.34-6.61%), (E)-caryophyllene (0.97-7.55%), allo-aromadendrene (0.18-2.21%), caryophyllene oxide (1.42-23.83%), benzene acetaldehyde (0.26-13.34%), and β-ionone (1.08-16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41-42.05%) compared to EOs (IC50 158.1-970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line (10.47 ± 0.53% and 9.06 ± 0.74% of early/late apoptosis, compared with control 3.61 ± 0.62% and 0.80 ± 0.17% of early/late apoptosis, respectively) and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control (for EO was 7.45 ± 1.01% and 0.54 ± 0.25%, and for HY was 4.91 ± 1.97% and 0.70 ± 0.09% of early/late apoptosis, respectively) on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.

TGF-β1 dominates stromal fibroblast-mediated EMT via the FAP/VCAN axis in bladder cancer cells

BackgroundBladder cancer is one of the most common malignant tumors of the urinary system and is associated with a poor prognosis once invasion and distant metastases occur. Epithelial-mesenchymal transition (EMT) drives metastasis and invasion in bladder cancer. Transforming growth factor β1 (TGF-β1) and stromal fibroblasts, especially cancer-associated fibroblasts (CAFs), are positive regulators of EMT in bladder cancer. However, it remains unclear how TGF-β1 mediates crosstalk between bladder cancer cells and CAFs and how it induces stromal fibroblast-mediated EMT in bladder cancer. We aimed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.MethodsPrimary CAFs with high expression of fibroblast activation protein (FAP) were isolated from bladder cancer tissue samples. Subsequently, different conditioned media were used to stimulate the bladder cancer cell line T24 in a co-culture system. Gene set enrichment analysis, a human cytokine antibody array, and cytological assays were performed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.ResultsAmong the TGF-β family, TGF-β1 was the most highly expressed factor in bladder cancer tissue and primary stromal fibroblast supernatant. In the tumor microenvironment, TGF-β1 was mainly derived from stromal fibroblasts, especially CAFs. In stimulated bladder cells, stromal fibroblast-derived TGF-β1 promoted bladder cancer cell migration, invasion, and EMT. Furthermore, TGF-β1 promoted the activation of stromal fibroblasts, inducing CAF-like features, by upregulating FAP in primary normal fibroblasts and a normal fibroblast cell line. Stromal fibroblast-mediated EMT was induced in bladder cancer cells by TGF-β1/FAP. Versican (VCAN), a downstream molecule of FAP, plays an essential role in TGF-β1/FAP axis-induced EMT in bladder cancer cells. VCAN may also function through the PI3K/AKT1 signaling pathway.ConclusionsTGF-β1 is a critical mediator of crosstalk between stromal fibroblasts and bladder cancer cells. We revealed a new mechanism whereby TGF-β1 dominated stromal fibroblast-mediated EMT of bladder cancer cells via the FAP/VCAN axis and identified potential biomarkers (FAP, VCAN, N-cadherin, and Vimentin) of bladder cancer. These results enhance our understanding of bladder cancer invasion and metastasis and provide potential strategies for diagnosis, treatment, and prognosis.

Elemene inhibits the growth and promotes apoptosis of bladder cancer cells through PTEN-Akt signaling pathway

Purpose: To investigate the influence of elemene on growth and apoptotic changes in bladder cancer cells, and the involvement of the phosphatase and tensin homologous protein (PTEN)-serine/threonine kinase (Akt) signal pathway in the process.
 Methods: Human bladder cell line SV-HUC-1 and human bladder cancer cell line T24 were randomly assigned to blank control group and elemene groups. Protein expression levels of apoptosis-related factors (Bcl-2, Bax, and caspase-3] and PTEN, Akt, and p-Akt in both cells were determined using western blot assay.
 Results: In T24 cells, the protein level of Bcl-2 was significantly higher in elemene group than in blank control, while the protein expression levels of Bax and caspase-3 in elemene group were significantly lower than the corresponding levels in blank control group (p < 0.05). The protein expression of PTEN level in T24 cells was significantly higher in elemene group than in blank control group, while protein expression level of p-Akt was significantly lower in elemene group than in blank control (p < 0.05). The Akt protein expression was comparable in elemene and blank control groups.
 Conclusion: Elemene inhibits the growth and enhances apoptosis of bladder cancer cells through a mechanism involving up-regulation of the expression of PTEN and suppression of the expression of pAkt.

Preclinical development of RAG1-40-31L: A novel small activating RNA-lipid conjugate targeting tumor suppressor gene p21 for treatment of non-muscle invasive bladder cancer.

e16620 Background: Loss-of-function of tumor suppressor genes are the dominant driving force in tumorigenesis. Restoration in function and/or expression of these genes holds tremendous therapeutic potential in cancer treatment. Bladder cancer is the 9th leading cause of cancer-related deaths in the United States in which ~75% of all newly diagnosed cases are non-muscle invasive bladder cancer (NMIBC). Standard-of-care includes intravesical installation of Bacillus Calmette-Guerin following tumor resection with an expected failure rate of ~50% within 6 months for patients with high-grade tumors. RNA activation (RNAa) is a mechanism in which short RNA duplexes termed small activating RNA (saRNA) induce gene transcription by targeting non-coding regulatory sequence ( i.e., promoters) embedded in chromatin. Thus, saRNA-induced overexpression of p21WAF1/CIP1 (p21) – a tumor suppressor of several anti-growth pathways – may hold therapeutic potential in bladder cancer. Through conjugation of a novel lipid ( i.e., C5X5), we have created a p21-targeting saRNA drug candidate referred to as RAG01-40-31L for delivery to bladder cancer cells in vivo. The aim of this work was to characterize the intravesical administration of RAG01-40-31L on p21 induction and its anti-tumor potential in NMBIC models. Methods: In vitro expression analyses were performed to investigate p21 induction via saRNA in KU7 and T24 bladder cancer cell lines by RT-qPCR and immunoblot analysis. Cell cycle analysis and tumorigenicity assays examined impact of RAG1-40-31L on cancer cell growth. Orthotopic human bladder tumors were established in female BALB/c nude mice using the recombinant cell line KU7 engineered to overexpress luciferase reporter. Tumor growth was monitored in live animals via luciferase bioluminescence using an in vivo imaging system. Plasma and tissue were harvested for drug exposure and expression analytics. Results: In vitro analyses indicated RAG1-40-31L activated p21 expression by approximately 6-fold and inhibited bladder cancer cell growth ( i.e., cell cycle arrest, apoptosis, and senescence) in a dose-dependent manner with low nanomolar potency. Intravesical administration of RAG1-40-31L (3 times per week for 2 weeks) inhibited growth of orthotopic tumors by 65% in mouse bladder models. Single-dose pharmacokinetics demonstrated a half-life (t1/2) of ~114 h with minimal systemic exposure (~0.05% of that of bladder tissue). Conclusions: These data constitute a preclinical proof-of-concept for saRNA as a novel p21-targeting modality in the treatment of cancer. The C5X5 lipid conjugate appears to be a viable approach to enhance oligonucleotide delivery and activity in bladder cancer. Preclinical safety assessment studies to enable clinical trials of RAG01-40-31L are currently underway.

Medicarpin induces G1 arrest and mitochondria-mediated intrinsic apoptotic pathway in bladder cancer cells.

Bladder cancer (BC) is the tenth most commonly diagnosed cancer. High recurrence, chemoresistance, and low response rate hinder the effective treatment of BC. Hence, a novel therapeutic strategy in the clinical management of BC is urgently needed. Medicarpin (MED), an isoflavone from Dalbergia odorifera, can promote bone mass gain and kill tumor cells, but its anti-BC effect remains obscure. This study reve aled that MED effectively inhibited the proliferation and arrested the cell cycle at the G1 phase of BC cell lines T24 and EJ-1 in vitro. In addition, MED could significantly suppress the tumor growth of BC cells in vivo. Mechanically, MED induced cell apoptosis by upregulating pro-apoptotic proteins BAK1, Bcl2-L-11, and caspase-3. Our data suggest that MED suppresses BC cell growth in vitro and in vivo via regulating mitochondria-mediated intrinsic apoptotic pathways, which can serve as a promising candidate for BC therapy.

Identification of the KIF and MCM protein families as novel targets for combination therapy with CDK4/6 inhibitors in bladder cancer.

CDK4/6 inhibitors have proven their potency for the treatment of cancer but only in combination with hormone or targeted therapies. The aim of this study was the identification of molecules that are involved in response mechanisms to CDK4/6 inhibitors and the development of novel combination therapies with corresponding inhibitors in bladder cancer. Genes of response to therapy and genes that confer resistance to the CDK4/6 inhibitor palbociclib were identified by performing an analysis of published literature and own published data using a CRISPR-dCas9 genome wide gain of function screen. Genes that were down-regulated upon treatment were compared with genes that confer resistance when up-regulated. Two of the top 5 genes were validated by quantitative PCR and western blotting upon treatment with palbociclib in the bladder cancer cell lines T24, RT112 and UMUC3. As inhibitors for combination therapy, we used ciprofloxacin, paprotrain, ispinesib and SR31527. Analysis of synergy was done using the "zero interaction potency" model. Cell growth was examined using sulforhodamine B staining. A list of genes that met the requirements for inclusion in the study was generated from 7 publications. Of the 5 most relevant genes, MCM6 and KIFC1 were chosen and their down-regulation upon treatment with palbociclib was confirmed by qPCR and immunoblotting. The combination of inhibitors against both, KIFC1 and MCM6 with PD resulted in a synergistic inhibition of cell growth. We have identified 2 molecular targets whose inhibition has promising potential for effective combination therapies with the CDK4/6 inhibitor palbociclib.

Differential Occupancy and Regulatory Interactions of KDM6A in Bladder Cell Lines.

Epigenetic deregulation is a critical theme which needs further investigation in bladder cancer research. One of the most highly mutated genes in bladder cancer is KDM6A, which functions as an H3K27 demethylase and is one of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor settings, we identified the genomic landscape of KDM6A in normal, immortalized, and cancerous bladder cells. Our results showed differential KDM6A occupancy in the genes involved in cell differentiation, chromatin organization, and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified in the T24 bladder cancer cell line; moreover, it has a truncating mutation in KDM6A and lacks a demethylase domain. Our co-immunoprecipitation experiments revealed TLE co-repressors and HES1 as potential truncated and wild-type KDM6A interactors. With the aid of structural modeling, we explored how truncated KDM6A could interact with TLE and HES1, as well as RUNX and HHEX transcription factors. These structures provide a solid means of studying the functions of KDM6A independently of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.

RNA-seq reveals novel mechanistic targets of Livin in bladder cancer

BackgroundBladder cancer is a very common malignancy with a high recurrence rate. The survival of patients with muscle-invasive bladder cancer is poor, and new therapies are needed. Livin has been reported to be upregulated in bladder cancer and influence the proliferation of cancer cells.Materials and methodsThe Livin gene in human bladder cancer cell line T24 was knocked out, and the differentially expressed genes were identified by RNA-seq and qPCR.ResultsLivin knockdown affects gene expression and has strong negative effects on some cancer-promoting pathways. Furthermore, combined with bladder cancer clinical sample data downloaded from TCGA and GEO, 2 co-up-regulated genes and 58 co-down-regulated genes were identified and validated, which were associated with cancer proliferation and invasion.ConclusionAll these results suggest that Livin plays an important role in bladder cancer and could be a potential anticancer target in clinical therapy.