The Antipsychotic Aripiprazole Induces Cytotoxicity in Bladder Cancer Cells While Preserving Urothelial and Bladder Function.

Objective To investigate the effects of glucose in formation of tunneling nanotubes (TNTs) between bladder cancer cells and the potential mechanism. Methods The highly invasive bladder cancer cells T24 were labeled with Mito-Tracker Deep Red, and then co-cultured with the less invasive bladder cancer cells RT4 for 24 hours. Actin-Tracker Green was used to label the cytoskeletal protein to detect the TNTs between T24 and RT4 cells. T24 and RT4 cells were cultured separately for 12 hours and 24 hours respectively, and the medium was collected and analyzed by liquid chromatography and mass spectrometry to screened out the difference factors between the micro-environment. T24 cells were cultured with the factors to confirm whether they could induce and enhance TNTs formation. The expression level of Fyn, Rho-associated kinase (ROCK) and p-paxillin proteins were detected and compared by Western blotting analysis. Results The formation of TNTs was found between T24 and RT4 cells. And the main component of TNTs was F-actin. Metabolomics analysis revealed that glucose was a differential factor between the micro-environments of T24 and RT4 cells, which could induce the formation of TNTs between T24 and RT4 cells (P=0.005). Concentration gradient of glucose between T24 and RT4 cells can up-regulate the expression of Fyn/ROCK and p-paxillin proteins (P=0.001, P=0.004, P=0.010). Conclusion Concentration gradient of glucose in the micro-environment of bladder cancer cells can promote the formation of TNTs between highly invasive and less invasive bladder cancer cells. The potential mechanism of the formation of TNTs induced by glucose might be explained by concentration gradient of glucose in the micro-environment of bladder cancer cells which can up-regulate the activity of Fyn/ROCK signaling pathway. Key words: Tunneling nanotubes; Bladder cancer; Cell conjunction; Micro-environment

The epidermal growth factor receptor (EGFR) is associated with aggressive phenotypes and is an independent predictor of stage progression and mortality in bladder cancer. Gefitinib ('Iressa,' ZD1839) is an orally active EGFR-tyrosine kinase inhibitor. The objective of this study was to evaluate the in vitro and in vivo effects of gefitinib in the EGFR-expressing human bladder cancer cell lines 253J B-V, RT-112, and T24. EGFR expression was 3- and 2-fold higher in 253J B-V and RT-112, respectively, compared with T24 cells. Ten microm gefitinib inhibited EGFR, p42/44 extracellular signal-regulated kinase (ERK), and Akt/protein kinase B phosphorylation in all three of the cell lines. Inhibition of ERK by gefitinib was significantly greater in 253J B-V compared with RT-112 and T24 cells (9:2:1 in 253J B-V:RT-112:T24), whereas inhibition of Akt phosphorylation was less in 253J B-V compared with RT-112 and T24 cells (1:9:30 in 253J B-V:RT-112:T24). When cultured in serum-free medium supplemented with epidermal growth factor, 10 microm gefitinib inhibited DNA synthesis in T24 and RT-112 cells, whereas 1 microm gefitinib was sufficient to inhibit DNA synthesis in 253J B-V cells. Similarly, in the presence of serum, 10 microm gefitinib induced a significant reduction in S-phase and viable cell number in T24 and RT-112 cells, whereas 1-10 microm gefitinib caused a dose-dependent effect on these phenotypes in 253J B-V cells. Gefitinib significantly enhanced the ability of ionizing radiation to reduce colony forming ability in 253J B-V and RT-112 cells. In nude mice, a daily oral dose of 150 mg/kg gefitinib induced regression of tumors produced by 253J B-V cells growing at s.c. sites and suppression of tumors produced by these cells at orthotopic sites but had no effect on tumors produced by RT-112 cells growing at s.c. sites. The data indicates that gefitinib has potential therapeutic value, alone or in combination with ionizing radiation, in a subset of EGFR-expressing bladder cancers. However, there is a differential response to gefitinib in these EGFR-expressing bladder cancer cell lines. Although gefitinib can inhibit phosphorylation of EGFR, ERK, and Akt, and inhibit growth of bladder cancer cells in vitro, it does not necessarily inhibit growth of bladder cancer cells in vivo. It is likely that optimized therapy approaches will require an accurate "molecular" diagnosis allowing effective, selective, tailored therapeutic strategies to be designed.

Objective: <br/>Bladder cancer, one of the ten most common cancers diagnosed worldwide, presents as a common, complex, and costly disease. Current treatments, including intravesical chemotherapy and immunotherapy, are associated with frequent recurrences and local side effects that significantly affect quality of life.1 These side effects, including urinary frequency, urgency, dysuria and hematuria, contribute to or intensify urinary incontinence, posing significant challenges in patient care.2 Consequently, there is an important need for novel therapeutic approaches that offer effective cancer treatment with reduced side effects. In this context, α1-ADR antagonists, known for their cytotoxic effects on prostate cancers, emerge as promising candidates. This study aims to investigate the cytotoxicity of the α1-ADR antagonist prazosin (Minipress) in bladder cancer cell lines, considering their potential as a bladder cancer treatment with minimal side effects. Additionally, the research explores the impact of luminal prazosin treatment on bladder function using a porcine model, to provide insights <br/>relevant to the management of urinary incontinence in bladder cancer patients.<br/><br/>Methods: <br/>Human malignant urothelial cells lines (invasive T24 cells and non-invasive RT4 cells) were incubated with prazosin (1-300 μM) or vehicle control (DMSO) for either a 30-minute or 2-hour period (mimicking the duration of intravesical treatment). Cell viability was then assessed 24, 48, and 72 hours after prazosin incubation using the resazurin reduction assay. To examine the effects of prazosin on bladder function, the luminal surface of female pig bladders were treated with prazosin (300 μM) or vehicle control in modified Ussing chambers for 2 hours, followed by organ bath studies to assess functional responses to agonists carbachol, isoprenaline, adenosine triphosphate (ATP), electrical field stimulation (EFS) and high potassium Krebs.<br/><br/>Results: <br/>Incubation of T24 and RT4 cells with prazosin (300µM) resulted in a statistically significant concentration-dependent decrease in cell viability at 72 hours (Figure 1A). In bladder functional experiments, pre-treatment with 300 μM prazosin did not significantly affect nerve evoked contractile bladder responses (Figure 1B). Acetylcholine (ACh) remained the dominant neurotransmitter in control and treated tissues. Responses to purinergic stimulation, <br/>β-adrenoceptor relaxation to isoprenaline and general bladder contractility to high potassium were similarly unchanged. However, the maximal response of intact bladder tissues to carbachol was significantly enhanced after prazosin pre-treatment (P&lt;0.001) (Figure 1C), while responses of denuded detrusor and urothelium/lamina propria to muscarinic stimulation were unchanged.<br/><br/>Conclusions: <br/>The study highlights prazosin’s concentration-dependent cytotoxicity against both invasive T24 and non-invasive RT4 bladder cancer cells, highlighting this drug as a promising candidate for targeted bladder cancer therapy. Significantly, prazosin demonstrates minimal impact on normal bladder function in the porcine model, suggesting a lower risk of exacerbating urinary incontinence; a major concern with current intravesical treatments. Future research should investigate prazosin’s long-term effects and potential integration into existing treatment regimes, offering a holistic approach to bladder cancer treatment that priorities both tumor suppression and quality of life.<br/>

MicroRNA-221 silencing predisposed human bladder cancer cells to undergo apoptosis induced by TRAIL

TRPV2 Activation Induces Apoptotic Cell Death in Human T24 Bladder Cancer Cells: A Potential Therapeutic Target for Bladder Cancer

N-Acetylcysteine Augments The Cellular Redox Changes and Cytotoxic Activity of Internalized Mycobacterium Bovis in Human Bladder Cancer Cells

Only 20% of patients with muscle-invasive bladder carcinoma respond to cisplatin-based chemotherapy. Since the natural phytochemical sulforaphane (SFN) exhibits antitumor properties, its influence on the adhesive and migratory properties of cisplatin- and gemcitabine-sensitive and cisplatin- and gemcitabine-resistant RT4, RT112, T24, and TCCSUP bladder cancer cells was evaluated. Mechanisms behind the SFN influence were explored by assessing levels of the integrin adhesion receptors β1 (total and activated) and β4 and their functional relevance. To evaluate cell differentiation processes, E- and N-cadherin, vimentin and cytokeratin (CK) 8/18 expression were examined. SFN down-regulated bladder cancer cell adhesion with cell line and resistance-specific differences. Different responses to SFN were reflected in integrin expression that depended on the cell line and presence of resistance. Chemotactic movement of RT112, T24, and TCCSUP (RT4 did not migrate) was markedly blocked by SFN in both chemo-sensitive and chemo-resistant cells. Integrin-blocking studies indicated β1 and β4 as chemotaxis regulators. N-cadherin was diminished by SFN, particularly in sensitive and resistant T24 and RT112 cells, whereas E-cadherin was increased in RT112 cells (not detectable in RT4 and TCCSup cells). Alterations in vimentin and CK8/18 were also apparent, though not the same in all cell lines. SFN exposure resulted in translocation of E-cadherin (RT112), N-cadherin (RT112, T24), and vimentin (T24). SFN down-regulated adhesion and migration in chemo-sensitive and chemo-resistant bladder cancer cells by acting on integrin β1 and β4 expression and inducing the mesenchymal–epithelial translocation of cadherins and vimentin. SFN does, therefore, possess potential to improve bladder cancer therapy.

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.

Krüppel-like factor 8 (KLF8) belongs to the Sp/KLF family of transcription factors. Recently, it is affirmed that KLF8 plays an important role in the regulation of epithelial-mesenchymal transition, which is a key process that occurs during cancer metastasis. Although the overexpression of KLF8 has been observed in several types of human cancers, the functional role of KLF8 in human bladder cancer remains unknown. Here, we investigated the effects of KLF8 knockdown on bladder cancer cell proliferation and migration in vitro. Lentivirus-mediated small interfering RNA (siRNA) targeting KLF8 specifically downregulated its expression in T24 and BT5637 bladder cancer cells. Knockdown of KLF8 significantly inhibit cell proliferation and colony formation. Cell cycle analysis showed that knockdown of KLF8 arrested T24 cells in the G0/G1 phase. Moreover, cell migration was attenuated in T24 cells after KLF8 knockdown. Furthermore, knockdown of KLF8 resulted in a reduction in vimentin and N-cadherin expression and an increase in β-catenin expression. These results indicate that KLF8 plays a crucial role in proliferation and migration of bladder cancer cells, and inhibition of KLF8 by siRNA may provide a potential therapeutic approach for gene therapy in bladder cancer.

Objective To investigate the expression of long-stranded non-coding RNA HOXA transcript at the distal tip (HOTTIP) (lncRNA HOPTTIP) in bladder cancer cells (BCC) and its effect on proliferation, migration and invasion of BCC and potential mechanisms. Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression differences of HOTTIP between normal tissue and bladder cancer tissues, normal cell and bladder cancer cell. Cell counting kit-8 (CCK-8) was used to observe the proliferation levels of BCC. Cell cycle progression of BCC was detected by flow cytometry. Cell invasion and metastasis were observed by transwell and wound healing experiments. Western blotting assay was used to detect the protein expressions of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase (TIMPs). Results The expression level of HOTTIP in BCC (1.943±0.251) was significantly higher than that of paracancerous tissue (1.002±0.091), and the difference was statistically significant (P<0.05); The expression of HOTTIP in human normal epithelial cells (SV-HUC-1) was significantly lower than that of 5637 and T24 (1.003±0.092 vs. 2.241±0.306 and 1.692±0.203), with significant difference (P<0.05). After si-HOTTIP and si-NC intervention 48h on 5637 and T24 cells, the proliferation of 5637 (0.881±0.182 vs. 1.824±0.193) and T24 (0.623±0.094 vs. 1.332±0.153) cells decreased significantly (P<0.05), the cell cycle were stuck into G0/G1 phase, rate of invasion and migration were reduced, MMP-2 and MMP-9 protein expression levels decreased, TIMP1 and TIMP2 protein expression levels increased. Conclusion Long-stranded non-coding RNA (LncRNA) HOTTIP is down-regulated in bladder cancer tissues and cells, and promotes the proliferation, migration and invasion of BCC by decreasing MMPs and increasing TIMPs. Key words: Long non-coding RNAHOXA transcript at the distal tip; Bladder cancer; Proliferation; Migration; Invasion

Bladder cancer cell viability inhibition and apoptosis induction by baicalein through targeting the expression of anti-apoptotic genes.

TLR4- and TLR9-dependent effects on cytokines, cell viability, and invasion in human bladder cancer cells

BackgroundmiRNAs have been widely used in cancer treatment. Our study was designed to explore the effects of miR-325-3p in bladder cancer cells.Material/MethodsLevels ofd miR-325-3p and MT3 in bladder cancer tissues and cells were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). miR-325-3p mimics were transfected into bladder cancer T24 cells, and cell migration and invasion rates and cell proliferation were assessed by transwell assay and Cell Counting Kit-8 (CCK-8). The target mRNA for miR-325-3p was predicted by Targetscan7.2 and confirmed by dual-luciferase reporter assay. More experiments were performed to confirm the effects of miR-325-3p and MT3 in T24 cells. Additionally, the levels of TIMP-2, MMP9, and E-cadherin were assessed by Western blotting to identify the effects of miR-325-3p and MT3 on epithelial-mesenchymal transition (EMT).ResultsmiR-325-3p expression was reduced and MT3 was increased in bladder cancer tissues and bladder cancer cells. miR-325-3p mimics suppressed cell proliferation ability and invasion and migration rates of T24 cells. Moreover, miR-325-3p was confirmed to target MT3. Further experiments showed that the effects of increased cell proliferation, invasion, migration, and EMT promoted by MT3 overexpression were abolished by miR-325-3p mimics, proving that miR-325-3p is a tumor suppressor through targeting MT3 in bladder cancer cells.ConclusionsDownregulation of miR-325-3p in bladder cancer regulates cell proliferation, migration, invasion, and EMT by targeting MT3. Furthermore, miR-325-3p is a potential therapeutic target in treating bladder cancer.

Bladder cancer is a common malignancy worldwide. However, there is still no effective therapy for bladder cancer. In this study, we investigated the cytotoxic effects of cantharidin [a natural toxin produced (pure compound) from Chinese blister beetles (Mylabrisphalerata or Mylabriscichorii) and Spanish flies (Cantharis vesicatoria)] in human bladder cancer cell lines (including: T24 and RT4 cells). Treatment of human bladder cancer cells with cantharidin significantly decreased cell viability. The increase in the expressions of caspase-3 activity and cleaved form of caspase-9/-7/-3 were also increased in cantharidin-treated T24 cells. Furthermore, cantharidin increased the levels of phospho-eIF2α and Grp78 and decreased the protein expression of procaspase-12, which was accompanied by the increase in calpain activity in T24 cells. Cantharidin was capable of increasing the intracellular Ca (2+) and the phosphorylation of protein kinase C (PKC) in T24 cells. The addition of BAPTA/AM (a Ca (2+) chelator) and RO320432 (a selective cell-permeable PKC inhibitor) effectively reversed the increase in caspase-3 and calpain activity, the phosphorylation levels of PKC and eIF2α and Grp78 protein expression, and the decrease in procaspase-12 expression induced by cantharidin. Importantly, cantharidin significantly decreased the tumor volume (a dramatic 71% reduction after 21 days of treatment) in nude mice xenografted with T24 cells. Taken together, these results indicate cantharidin induced human bladder cancer cell apoptosis through a calcium/PKC-regulated ER stress pathway. These findings suggest that cantharidin may be a novel and potential anticancer agent targeting on bladder cancer cells.

As alterations in purinergic signaling have been observed in bladder diseases, we aimed to assess the potential prognostic role of purinergic receptors in bladder cancer in a translational approach based on clinical databases and in vitro data. The prognostic role of purinergic receptors in the survival of patients with bladder cancer and the expression profile of the altered P2 receptors in normal and in tumor samples were determined using The Cancer Genome Atlas databank. In T24 and RT4 human bladder cancer cell lines, the P2 purinergic receptors were characterized by RT-PCR and RT-qPCR analysis including radiotherapy exposure as treatment. The cell number and the cumulative population doubling were also assessed. The expression profile of P2X6 receptor in the cancer pathological stage and in the nodal metastasis status was in agreement with Kaplan-Meier analysis, indicating that high expression of this receptor was related to an increased survival rate in patients with bladder cancer. Of all the P2 receptors expressed on T24 cell line, P2X6 presented high expression after radiotherapy, while it was not altered in RT4 cells. In addition, irradiation promoted a decrease of T24 cell number, but did not change the cell number of RT4 after the same time and radiation dose. Along 7days after irradiation exposure, both cells regrew. However, while P2X6 receptor was downregulated in T24 cells, it was upregulated in RT4 cells. Our findings indicated that high P2X6 receptor expression induced by radiation in T24 cell line may predict a good survival prognostic factor.