RT4 Human Bladder Cancer Cells Research Articles

High P2X6 receptor expression in human bladder cancer predicts good survival prognosis

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.

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening.

Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better.Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation.Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43).Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

Abstract 5941: Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture

Abstract Introduction: Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in missense mutations in the integrin cell surface interactions pathways in advanced chemotherapy-resistant urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in human bladder cancer cells and demonstrate a potential therapeutic utility for this approach. Methods: RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib (VS-6063), an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single-cell suspensions and formed organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression. Results: Both RT4 and PM748 bladder cancer cell lines demonstrated consistent organoid growth in three-dimensional culturing conditions. Addition of defactinib to cultured cells showed a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells in the presence of defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p<0.0001 versus DMSO for all dilutions). Established bladder cancer organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p<0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in mouse xenograft models are currently underway. Conclusions: Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment. Citation Format: LaMont Barlow, Rebecca Meyer, Ethan Shelkey, Bishoy Faltas, Mark Rubin. Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5941. doi:10.1158/1538-7445.AM2017-5941

Inhibition of T24 and RT4 Human Bladder Cancer Cell Lines by Heterocyclic Molecules.

BackgroundBladder cancer is a major widespread tumor of the genitourinary tract. Around 30% of patients with superficial cancers develop invasive and metastatic pathology.Material/MethodsSome new heterocyclic 4-methyl coumarin derivatives were designed using molecular modeling studies to evaluate their potential against bladder cancer lines T24 and RT-4. The designed compounds that showed good binding affinity to T24 and RT4 were synthesized, with excellent yield. The synthesized compounds after structural evaluation were further evaluated for their antiproliferative activity by cell viability assay, cell cycle analysis, and apoptosis assay.ResultsThe compound BC-14 exhibited the best cytotoxicity against T24 cells, but were not highly active against RT4 cells.ConclusionsThe results of the present study may suggest the selectivity pattern of the synthesized compounds. These results should be explored further with chemical modification for other cancer types.

4-Methylcoumarins with cytotoxic activity against T24 and RT4 human bladder cancer cell lines

4-Methylcoumarins were synthesized by microwave-assisted synthesis via Pechmann condensation and their cytotoxic activity against human bladder cancer cell lines was investigated.

Catʼs Claw Oxindole Alkaloid Isomerization Induced by Cell Incubation and Cytotoxic Activity against T24 and RT4 Human Bladder Cancer Cell Lines

The antitumor activity of Uncaria tomentosa, a native vine from the Amazonian rainforest, has been ascribed to pentacyclic oxindole alkaloids occurring in its bark. Former studies have shown that this activity, as well as its intensity, depends on whether cat's claw alkaloids occur as original compounds or isomerized derivatives. This work addresses this aspect, using T24 and RT4 human bladder cancer cell lines for that purpose. Bark samples were extracted by dynamic maceration, prepurified with cross-linked polyvinylpyrrolidone and properly fractioned by an ion exchange process to obtain an oxindole alkaloid purified fraction. Alkaloid isomerization was induced by heating it under reflux at 85 °C. Samples collected after 5, 15, and 45 min of heating were analyzed by HPLC-PDA, freeze-dried at once, and separately assayed using the non-isomerized purified fraction for comparison purposes. The latter showed significant and dose-dependent cytotoxic activity against both T24 and RT4 cancer cell lines (IC50: 164.13 and 137.23 µg/mL, respectively). However, results for both cell lines were equivalent to those observed for isomerized samples (p > 0.05). The alkaloid isomerization induced by the incubation conditions (buffered medium pH 7.4 and temperature 37 °C) helps to explain the similar results obtained from non-isomerized and isomerized samples. Mitraphylline, speciophylline, uncarine F, and, to a lesser degree, pteropodine were more susceptible to isomerization under the incubation conditions. Thus, the alkaloid profile of all fractions and their cytotoxic activities against T24 and RT4 human bladder cancer cell lines are determined to a large extent by the incubation conditions.

REIC/Dkk-3-encoding adenoviral vector as a potentially effective therapeutic agent for bladder cancer

Bladder cancer is one of the most common urogenital malignancies. The intravesical instillation of anticancer agents is an attractive strategy to treat a superficial lesion or floating/disseminated cancer cells after transurethral operation. An adenovirus carrying REIC/Dkk-3, a tumor suppressor gene (Ad-REIC), exhibits cancer-specific apoptotic effects in various types of cancer cells. The aim of the present study was to examine the potential of Ad-REIC as a therapeutic agent for bladder cancer. KK47 and RT4 human bladder cancer cells were sensitive to the Ad-REIC treatment for apoptosis induction, but some human bladder cancer cell lines (T24, J82 and TccSup) were resistant. Significant cell growth inhibition was observed when these resistant cancer cell lines were treated with Ad-REIC in a condition of floating cells, which is clinically observed after transurethral operation and becomes a cause of intravesical cancer dissemination. The therapeutic potential of Ad-REIC for the treatment of multidrug-resistant bladder cancer was investigated. The adriamycin-resistant KK47 bladder cancer cells (KK47/ADM), which also present multidrug resistance, showed induction of significant apoptosis following Ad-REIC treatment. The Ad-REIC treatment induced downregulation of P-glycoprotein in KK47/ADM cells and restored the sensitivity to doxorubicin (adriamycin). Ad-REIC suppressed P-glycoprotein expression in a c-Jun-NH2-kinase (JNK)-dependent manner. Therefore, the current study indicated two therapeutic aspects of the Ad-REIC agent against human bladder cancer cells, as an apoptosis inducer/cell growth inhibitor and as a sensitizer of chemotherapeutic agents in multidrug-resistant cancer cells. The intravesical instillation of Ad-REIC could be an attractive therapeutic method in human bladder cancer where the treatment of superficial lesions and floating/disseminated or multidrug-resistant cancer cells is necessary.

Microfluidic micropipette aspiration for measuring the deformability of single cells

We present a microfluidic technique for measuring the deformability of single cells using the pressure required to deform such cells through micrometre-scale tapered constrictions. Our technique is equivalent to whole-cell micropipette aspiration, but involves considerably simpler operation, less specialized equipment, and less technical skill. Single cells are infused into a microfluidic channel, and then deformed through a series of funnel-shaped constrictions. The constriction openings are sized to create a temporary seal with each cell as it passes through the constriction, replicating the interaction with the orifice of a micropipette. Precisely controlled deformation pressures are generated using an external source and then attenuated 100 : 1 using an on-chip microfluidic circuit. Our apparatus is capable of generating precisely controlled pressures as small as 0.3 Pa in a closed microchannel network, which is impervious to evaporative losses that normally limit the precision of such equipment. Intrinsic cell deformability, expressed as cortical tension, is determined from the threshold deformation pressure using the liquid-drop model. We measured the deformability of several types of nucleated cells and determined the optimal range of constriction openings. The cortical tension of passive human neutrophils was measured to be 37.0 ± 4.8 pN μm(-1), which is consistent with previous micropipette aspiration studies. The cortical tensions of human lymphocytes, RT4 human bladder cancer cells, and L1210 mouse lymphoma cells were measured to be 74.7 ± 9.8, 185.4 ± 25.3, and 235.4 ± 31.0 pN μm(-1) respectively. The precision and usability of our technique demonstrates its potential as a biomechanical assay for wide-spread use in biological and clinical laboratories.

Src and Caveolin-1 Reciprocally Regulate Metastasis via a Common Downstream Signaling Pathway in Bladder Cancer

In bladder cancer, increased caveolin-1 (Cav-1) expression and decreased Src expression and kinase activity correlate with tumor aggressiveness. Here, we investigate the clinical and functional significance, if any, of this reciprocal expression in bladder cancer metastasis. We evaluated the ability of tumor Cav-1 and Src RNA and protein expression to predict outcome following cystectomy in 257 patients enrolled in two independent clinical studies. In both, high Cav-1 and low Src levels were associated with metastasis development. We overexpressed or depleted Cav-1 and Src protein levels in UMUC-3 and RT4 human bladder cancer cells and evaluated the effect of this on actin stress fibers, migration using Transwells, and lung metastasis following tail vein inoculation. Cav-1 depletion or expression of active Src in metastatic UMUC-3 cells decreases actin stress fibers, cell migration, and metastasis, while Cav-1 overexpression or Src depletion increased the migration of nonmetastatic RT4 cells. Biochemical studies indicated that Cav-1 mediates these effects via its phosphorylated form (pY14), whereas Src effects are mediated through phosphorylation of p190RhoGAP and these pathways converge to reduce activity of RhoA, RhoC, and Rho effector ROCK1. Treatment with a ROCK inhibitor reduced UMUC-3 lung metastasis in vivo, phenocopying the effect of Cav-1 depletion or expression of active Src. Src suppresses whereas Cav-1 promotes metastasis of bladder cancer through a pharmacologically tractable common downstream signaling pathway. Clinical evaluation of personalized therapy to suppress metastasis development based on Cav-1 and Src profiles seems warranted.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Expression Contributes to Bladder Cancer Progression

Prostaglandin E2, which is known to contribute to cancer progression, is inactivated by the catabolic enzyme, 15-hydroxyprostaglandin dehydrogenase (PGDH), which has tumor-suppressor activity in lung, colon, breast, and gastric cancers. Therefore, we evaluated the expression of PGDH in human bladder cancer tissue specimens and cell lines. Immunoperoxidase staining of bladder cancer tissues demonstrated that (1) PGDH is highly expressed by normal urothelial cells but (2) reduced in many low stage (Ta/Tis) bladder cancers, and (3) PGDH is completely lost in most invasive bladder cancers. Of eight cancer cell lines tested, only two relatively well-differentiated bladder cancer cell lines, RT4 and UM-UC9, expressed PGDH. Moreover, inhibition of PGDH expression in well-differentiated RT4 cells using small inhibitory RNA or short hairpin RNA resulted in a more aggressive phenotype with increased motility and anchorage-independent growth. Additionally, PGDH knockdown affected prostaglandin E2 signaling as measured by cAMP generation. These data indicate that loss of PGDH expression contributes to a more malignant bladder cancer phenotype and may be necessary for bladder cancer development and/or progression.

Loss of 15-Hydroxyprostaglandin Dehydrogenase Expression Disrupts Urothelial Differentiation

Urothelial differentiation is essential for the maintenance of urinary bladder function. We explored the expression and function of 15-hydroxyprostaglandin dehydrogenase (PGDH) during urothelial differentiation. We evaluated expression of PGDH by Northern and Western blotting and immunostaining in human urothelial cultures, cell lines, and tissues. We determined enzymatic function using enzyme-linked immunosorbent assay. Small inhibitory ribonucleic acids were used to inhibit PGDH expression in human bladder cancer cells. We found PGDH messenger ribonucleic acid was increased in an in vitro model of human urothelial differentiation by Northern blotting. Western blotting of human bladder cancer cell lines showed expression in the well-differentiated RT4 cells and no expression in poorly differentiated UC3 cells. Immunostaining showed that PGDH expression increased with differentiation in normal bladder urothelium. The enzyme was functional in the well-differentiated RT4 human bladder cancer cell line. Inhibition of PGDH expression resulted in disruption of E-cadherin expression at cell-cell contacts in well-differentiated RT4 bladder cancer cells. These studies indicate that PGDH expression is associated with urothelial differentiation, and loss of PGDH expression results in disruption of urothelial differentiation.

Modification of the alkaline Comet assay to allow simultaneous evaluation of mitomycin C-induced DNA cross-link damage and repair of specific DNA sequences in RT4 cells

The alkaline Comet assay is a simple, sensitive method for measuring the extent of DNA strand breaks in individual cells. Several modifications to the original assay have been developed to increase its applications. One such modification allows the measurement of DNA cross-links by assessing the relative reduction in DNA migration induced by a strand-breaking agent. Another modification includes the application of fluorescent in situ hybridisation (FISH) to investigate the localisation of specific gene domains within a cell. Although several studies have used these approaches separately, no report to date has combined these two versions of the Comet assay. The current study describes the modification of the Comet assay, to allow both measurement of mitomycin C (MMC)-induced cross-links and the subsequent application of FISH to study repair in the TP53 gene region. RT4 human bladder cancer cells were treated with 0, 5, 50 and 200 μg/ml MMC to study dose response, whilst for cross-link repair studies, they were treated with 50 μg/ml MMC and allowed to repair for up to 24 h. A clear dose response to MMC was displayed, demonstrable by a marked reduction in DNA migration, whilst repair studies showed that MMC-induced cross-links take at least 24 h to repair fully in RT4 cells. For Comet–FISH experiments, the number and location of TP53 hybridisation spots was also recorded for each cell. In dose response experiments, the number of spots per cell, and per Comet tail, decreased as MMC dose increased. In repair experiments, the number of spots, particularly in the Comet tail, increased as repair time increased. Furthermore, our results suggest that repair of the TP53 gene region is most rapid within the first 4 h following MMC treatment. We conclude that the novel experimental protocol presented here has considerable potential in evaluating DNA damage and sequence-related repair responses to cross-linking agents.