RT4 Cells Research Articles

N-Formylated Peptide Induces Increased Expression of Both Formyl Peptide Receptor 2 (Fpr2) and Toll-Like Receptor 9 (TLR9) in Schwannoma Cells-An In Vitro Model for Early Inflammatory Profiling of Schwann Cells.

Following nerve injury, disintegrated axonal mitochondria distal to the injury site release mitochondrial formylated peptides and DNA that can induce activation and inflammatory profiling of Schwann cells via formyl peptide receptor 2 (Fpr2) and toll-like receptor 9 (TLR9), respectively. We studied RT4 schwannoma cells to investigate the regulation of Fpr2 and TLR9 after stimulation with fMLF as a prototypical formylated peptide. RT4 cells were treated with fMLF at various concentrations and times with and without pretreatment with inhibitors (chloroquine for activated TLR9, PBP10 for Fpr2). Western blots of Fpr2, TLR9, p-p38, p-NFκB, and IL-6 were compared in relation to inflammatory profiling of RT4 cells and chemokine receptors (CCR2, CXCR4) as potential co-receptors of Fpr2. fMLF stimulation upregulated Fpr2 in RT4 cells at low concentrations (10 nM and 100 nM) but higher concentrations were required (10 µM and 50 µM) when the cells were pretreated with an activated TLR9 inhibitor. Moreover, the higher concentrations of fMLF could modulate TLR9 and inflammatory markers. Upregulation of Fpr2 triggered by 10 nM and 100 nM fMLF coincided with higher levels of chemokine receptors (CCR2, CXCR4) and PKCβ. Treating RT4 cells with fMLF, as an in vitro model of Schwann cells, uncovered Schwann cells’ complex responses to molecular patterns of release from injured axonal mitochondria.

Profiling and Targeting of Energy and Redox Metabolism in Grade 2 Bladder Cancer Cells with Different Invasiveness Properties.

Bladder cancer is one of the most prevalent deadly diseases worldwide. Grade 2 tumors represent a good window of therapeutic intervention, whose optimization requires high resolution biomarker identification. Here we characterize energy metabolism and cellular properties associated with spreading and tumor progression of RT112 and 5637, two Grade 2 cancer cell lines derived from human bladder, representative of luminal-like and basal-like tumors, respectively. The two cell lines have similar proliferation rates, but only 5637 cells show efficient lateral migration. In contrast, RT112 cells are more prone to form spheroids. RT112 cells produce more ATP by glycolysis and OXPHOS, present overall higher metabolic plasticity and are less sensitive than 5637 to nutritional perturbation of cell proliferation and migration induced by treatment with 2-deoxyglucose and metformin. On the contrary, spheroid formation is less sensitive to metabolic perturbations in 5637 than RT112 cells. The ability of metformin to reduce, although with different efficiency, cell proliferation, sphere formation and migration in both cell lines, suggests that OXPHOS targeting could be an effective strategy to reduce the invasiveness of Grade 2 bladder cancer cells.

LncRNA HCP5 Promotes Cell Invasion and Migration by Sponging miR-29b-3p in Human Bladder Cancer.

BackgroundBladder cancer (BC) is one of the most common malignant tumors in the urinary system. In this study, the roles of lncRNA HCP5 (human major histocompatibility complex p5) and miR-29b-3p in human BC were investigated. Their regulations involved in cell invasion and migration were also evaluated.MethodsLuciferase reporter assay was performed to detect the binding between miR-29b-3p and HCP5 or high-mobility group box 1 (HMGB1). Cell viability, migration, invasion and apoptosis were assessed by CCK-8, colony formation, transwell assay and flow cytometry, respectively. Expression levels of HMGB1/toll‐like receptor 4 (TLR4) proteins were measured by Western blot. Xenograft model was built, and tumor volumes and weights were calculated.ResultsThe results revealed dysregulation of HCP5 and miR-29b-3p in BC samples and cells. HCP5 negatively regulated the expression of miR-29b-3p and enhanced cell viability, migration and invasion. MiR-29b-3p mediated the effect of HCP5 on cell viability, proliferation, migration and invasion in RT4 cells. In addition, miR-29b-3p could regulate the expression of HMGB1 through interaction with HMGB1.ConclusionThe findings in this study supported that lncRNA HCP5 could promote cell invasion and migration by sponging miR-29b-3p in human BC.

The Interplay between Oxidative Phosphorylation and Glycolysis as a Potential Marker of Bladder Cancer Progression.

Urothelial bladder cancer (UBC) is the most common tumor of the urinary system. One of the biggest problems related to this disease is the lack of markers that can anticipate the progression of the cancer. Genomics and transcriptomics have greatly improved the prediction of risk of recurrence and progression. Further progress can be expected including information from other omics sciences such as metabolomics. In this study, we used 1H-NMR to characterize the intake of nutrients and the excretion of products in the extracellular medium of three UBC cell lines, which are representatives of low-grade tumors, RT4, high-grade, 5637, and a cell line that shares genotypic features with both, RT112. We have observed that RT4 cells show an activated oxidative phosphorylation, 5637 cells depend mostly on glycolysis to grow, while RT112 cells show a mixed metabolic state. Our results reveal the relative importance of glycolysis and oxidative phosphorylation in the growth and maintenance of different UBC cell lines, and the relationship with their genomic signatures. They suggest that cell lines associated with a low risk of progression present an activated oxidative metabolic state, while those associated with a high risk present a non-oxidative state and high glycolytic activity.

A Clinical Genomics-Guided Prioritizing Strategy Enables Selecting Proper Cancer Cell Lines for Biomedical Research.

SummarySelecting appropriate cell lines to represent a disease is crucial for the success of biomedical research, because the usage of less relevant cell lines could deliver misleading results. However, systematic guidance on cell line selection is unavailable. Here we developed a clinical Genomics-guided Prioritizing Strategy for Cancer Cell Lines (CCL-cGPS) and help to guide this process. Statistical analyses revealed CCL-cGPS selected cell lines were among the most appropriate models. Moreover, we observed a linear correlation between the drug response and CCL-cGPS score of cell lines for breast and thyroid cancers. Using RT4 cells selected by CCL-GPS, we identified mebendazole and digitoxin as candidate drugs against bladder cancer and validate their promising anticancer effect through in vitro and in vivo experiments. Additionally, a web tool was developed. In conclusion, CCL-cGPS bridges the gap between tumors and cell lines, presenting a helpful guide to select the most suitable cell line models.

1-Methyl-D-tryptophan activates aryl hydrocarbon receptor, a pathway associated with bladder cancer progression

BackgroundIndoleamine 2, 3-dioxygenase-1 (IDO1) is a promising target for immunotherapy in bladder cancer (BC). IDO1 breaks-down tryptophan to generate kynurenine derivatives, which may activate the aryl hydrocarbon receptor (AHR). AHR is an important target for carcinogens, but its association with BC progression was unknown. Two IDO1 inhibitors used in clinical trials are 1-methyl-D-tryptophan (MT) and INCB240360. Because MT is an aromatic hydrocarbon, it may be a ligand for AHR. We hypothesized that AHR could be associated with BC progression and that MT could activate AHR in BC.MethodsBC patients (n = 165) were selected from the Gene Expression Omnibus database. A cut-off point for relative expression of AHR and cytochrome 450 enzymes (CYP1A1, CYP1A2, and CYP1B1; markers of AHR activation) was determined to compare with the grade, stage, and tumor progression. For in vitro experiments, RT4 (grade 1) and T24 (grade 3) BC cells were incubated with MT and INCB240360 to evaluate the expression of AHR and CYP1A1.ResultsAHR activation was associated with grade, stage, and progression of BC. T24 cells express more CYP1A1 than RT4 cells. Although IDO1 expression and kynurenine production are elevated in T24 cells concomitantly to CYP1A1 expression, IDO1 inhibitors were not able to decrease CYP1A1 expression, in contrast, MT significantly increased it in both cell lines.ConclusionIn conclusion, it is rational to inhibit IDO1 in BC, among other factors because it contributes to AHR activation. However, MT needs to be carefully evaluated for BC because it is an AHR pathway agonist independently of its effects on IDO1.

Cytotoxic and chemosensitizing effects of glycoalkaloidic extract on 2D and 3D models using RT4 and patient derived xenografts bladder cancer cells

Glycoalkaloids have been widely demonstrated as potential anticancer agents. However, the chemosensitizing effect of these compounds with traditional chemotherapeutic agents has not been explored yet. In a quest for novel effective therapies to treat bladder cancer (BC), we evaluated the chemosensitizing potential of glycoalkaloidic extract (GE) with cisplatin (cDDP) in RT4 and PDX cells using 2D and 3D cell culture models. Additionally, we also investigated the underlying molecular mechanism behind this effect in RT4 cells. Herein, we observed that PDX cells were highly resistant to cisplatin when compared to RT4 cells. IC50 values showed at least 2.16-folds and 1.4-folds higher in 3D cultures when compared to 2D monolayers in RT4 cells and PDX cells, respectively. GE + cDDP inhibited colony formation (40%) and migration (28.38%) and induced apoptosis (57%) in RT4 cells. Combination therapy induced apoptosis by down-regulating the expression of Bcl-2 (p < 0.001), Bcl-xL (p < 0.001) and survivin (p < 0.01), and activating the caspase cascade in RT4 cells. Moreover, decreased expression of MMP-2 and 9 (p < 0.01) were observed with combination therapy, implying its effect on cell invasion/migration. Furthermore, we used 3D bioprinting to grow RT4 spheroids using sodium alginate-gelatin as a bioink and evaluated the effect of GE + cDDP on this system. Cell viability assay showed the chemosensitizing effect of GE with cDDP on bio-printed spheroids. In summary, we showed the cytotoxicity effect of GE on BC cells and also demonstrated that GE could sensitize BC cells to chemotherapy.

Bladder Cancer Metastasis Induced by Chronic Everolimus Application Can Be Counteracted by Sulforaphane In Vitro.

Chronic treatment with the mTOR inhibitor, everolimus, fails long-term in preventing tumor growth and dissemination in cancer patients. Thus, patients experiencing treatment resistance seek complementary measures, hoping to improve therapeutic efficacy. This study investigated metastatic characteristics of bladder carcinoma cells exposed to everolimus combined with the isothiocyanate sulforaphane (SFN), which has been shown to exert cancer inhibiting properties. RT112, UMUC3, or TCCSUP bladder carcinoma cells were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM), alone or in combination. Adhesion and chemotaxis along with profiling details of CD44 receptor variants (v) and integrin α and β subtypes were evaluated. The functional impact of CD44 and integrins was explored by blocking studies and siRNA knock-down. Long-term exposure to everolimus enhanced chemotactic activity, whereas long-term exposure to SFN or the SFN-everolimus combination diminished chemotaxis. CD44v4 and v7 increased on RT112 cells following exposure to SFN or SFN-everolimus. Up-regulation of the integrins α6, αV, and β1 and down-regulation of β4 that was present with everolimus alone could be prevented by combining SFN and everolimus. Down-regulation of αV, β1, and β4 reduced chemotactic activity, whereas knock-down of CD44 correlated with enhanced chemotaxis. SFN could, therefore, inhibit resistance-related tumor dissemination during everolimus-based bladder cancer treatment.

Tumor cell-intrinsic PD-L1 signals promote DNA damage responses that mediate resistance to Chk1 and PARP inhibitors in vivo

Abstract Tumor PD-L1 mediates diverse cell-intrinsic signals that increase cancer pathogenesis including the DNA damage response (DDR). Although immune consequences of tumor-extrinsic PD-L1 blockade are known, novel therapeutic vulnerabilities from inhibiting tumor-intrinsic PD-L1 signals are little studied. To assess tumor-intrinsic PD-L1 DDR control, we depleted PD-L1 by CRISPR/Cas9 or shRNA (PD-L1KO) in mouse B16 (melanoma), 4T1 (breast), ID8agg (ovarian), and human bladder RT4 cancer lines. We identified specific DDR signaling defects in PD-L1KO versus control (con) cells after exposure to distinct DNA damaging agents. 24h gemcitabine (or 2 Gy X-ray) in vitro in PD-L1KO vs. con RT4 cells elevated γH2AX (DNA damage marker) and reduced Chk2 DDR gene expression but not ATM, ATR, Chk1, or BRCA1 (immunoblots), and decreased BRCA1/p-RPA32 nuclear foci formation (confocal) altogether suggesting tumor-intrinsic PD-L1 promotes specific DDR/DNA repair pathways. PD-L1 loss could thus predict synthetic lethality to DDR inhibitors (DDRi) Chk1i or PARPi. PD-L1KORT4, ID8, and 4T1 cells were more sensitive to small molecule Chk1i in vitro (&amp;gt;15 fold vs. con, MTT) and in vivo in NSG mice. Chk1i and PARPi led to γH2AX increase in PD-L1KO B16, RT4, and 4T1 vs. sham treated tumor cells as predicted by DDR defects. Genetic cellular PD-L1 depletion, but not anti-PD-L1 (extrinsic), strongly sensitized B16 and 4T1 tumors to PARPi in vivo. Strikingly, PARPi had no effect on PD-L1KO B16 in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi treatment efficacy. Thus, DDRi plus inhibiting tumor intrinsic PD-L1 signals could improve immunotherapy in immunotherapy-sensitive or resistant tumors.

MP01-08 GPX2 PROMOTES THE DEVELOPMENT OF BLADDER CANCER WITH SQUAMOUS DIFFERENTIATION THROUGH APOPTOSIS CONTROL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-08 GPX2 PROMOTES THE DEVELOPMENT OF BLADDER CANCER WITH SQUAMOUS DIFFERENTIATION THROUGH APOPTOSIS CONTROL Taku Naiki*, Aya Naiki-Ito, Toshiki Etani, Keitaro Iida, Ryosuke Ando, Takashi Nagai, Noriyasu Kawai, Satoru Takahashi, and Takahiro Yasui Taku Naiki*Taku Naiki* More articles by this author , Aya Naiki-ItoAya Naiki-Ito More articles by this author , Toshiki EtaniToshiki Etani More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru Takahashi Satoru Takahashi More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Reactive oxygen species (ROS) have been identified as important chemical mediators in cell growth and differentiation. The glutathione redox system is the main mechanism protecting against the damage caused by ROS. We investigated the role and therapeutic potential of the glutathione redox system in bladder cancer. METHODS: The expression levels of glutathione peroxidase 2 (GPX2) and Ki-67 proteins were analyzed in human TUR specimens obtained from urethral carcinoma (UC) patients using immunohistochemistry; correlations between the GPX2 expression and prognosis were also analyzed. Further, male F344 rats were given 0.05% BBN in drinking water and 0.1% phenyl isothiocyanate in their diet for 36 weeks. Bladder tissue samples were collected from each animal for analyses. Furthermore, the rat cell line BC31 and human cell lines, were transfected with GPX2 siRNA and negative control siRNA (NC). Subsequently, the cell proliferation rates and ROS levels were investigated by cell counting, DCFH assay, western blotting, and flow cytometry. siRNA- or NC- transfected BC31 cells were subcutaneously implanted into nude mice. RESULTS: GPX2 was strongly expressed in low-grade and low-MIB-1 index cancers. PFS rates were significantly better in patients with higher GPX2 than in those with lower GPX2. Furthermore, GPX2 expression was significantly lower in the normal epithelium of the control group animals with bladder cancer than in the treated group. GPX2 expression was significantly higher in UC than in the normal epithelium with the strongest expression observed in cells with squamous differentiation. BC31 and RT4 cells strongly expressed GPX2 compared to the other cell lines. Silencing the GPX2 caused significant growth inhibition of BC31 and RT4 cells; the DCFH assay revealed significant reductions in the ROS levels in the siRNA-treated cells. Caspase-dependent apoptosis was found to be the cause of the decreased proliferation rates in the siRNA group. Notably, tumor growth was significantly inhibited in the BC31-implanted nude mice using the siRNA strategy for Gpx2. CONCLUSIONS: Our findings demonstrated that GPX2 plays several important roles in carcinogenesis through apoptosis regulation against intracellular ROS and may be a novel marker or therapeutic target in bladder cancer. Source of Funding: Grant-in-Aid for Scientific Research C (17K11153) The Ministry of Education, Culture, Sports, Science and Technology © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e4-e4 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Taku Naiki* More articles by this author Aya Naiki-Ito More articles by this author Toshiki Etani More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Takashi Nagai More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement PDF downloadLoading ...

Mitomycin C Induces Immunogenic Cell Death Biomarkers In Human Bladder Tumors

IntroductionStandard‐of‐care for non‐muscle invasive bladder cancer is complete transurethral tumor resection followed by intravesical therapy with BCG, mitomycin C (MMC), gemcitabine, or other agents. Tumor recurrence is common and occurs in 40% to 80% of patients, with 10–20% progressing to muscle‐invasive disease. Earlier studies have shown selected cytotoxic agents induce immunogenic cell death (ICD) that promotes tumor response to immunotherapy and thereby offers the opportunity of developing synergistic combinations of ICD‐inducing cytotoxics with immune checkpoint blockers. The current qualifications for an ICD‐inducing cytotoxic include induction of DAMPs (danger‐associated molecular patterns) in vitro and vaccination with cytotoxic‐treated tumor cells in immunocompetent mice. Under these criteria, MMC is considered a poor ICD inducer. The present study tested the hypothesis that MMC can induce ICD in a more clinically relevant model, i.e., 3D histocultures of bladder tumors obtained from patients.MethodsWe evaluated the ability of MMC to induce two ICD biomarkers (i.e., ecto‐CRT (calreticulin) and release of HMGB1 from nucleus) and/or dendritic cell (DC) maturation in 3 bladder cancer cell lines with different differentiation status (RT4, T24, and TCCSUP), and 21 MMC‐treated human bladder tumor histocultures including 11 archived paraffin‐embedded tumors and 10 freshly obtained surgical tumor specimens. The methods included immunocytochemical staining flow/immune‐cytometry, immunoblotting, and immunohistochemical staining and quantitative imaging. Co‐cultures of human peripheral blood DC with MMC‐treated tumor cells (48 h) were used to detect DC maturation markers (CD83 and HLA‐DR).ResultsOver 85% of human bladder tumors (7/10 fresh tumors and 11/11 archived tumors) showed ecto‐CRT and/or HMGB1 release upon treatments with MMC at clinically achievable concentrations (1 and 3 μM). Induction of ICD markers by MMC was time‐dependent (peaked at 18 h) and increased with tumor grade (TCCSUP (grade 4) &gt;T24 (grade 3)&gt;RT4 (grade 1) (3 experiments, P&lt;0.01, 2‐way ANOVA). HMGB1 release was increased in three cell lines with a dose‐dependent manner after 48 h MMC treatment (2 experiments, p value&lt;0.05, 2‐way ANOVA). MMC further induced DC maturation in TCCSUP cells but not RT4 cells.ConclusionOur results indicate MMC induced ICD biomarkers and/or DC maturation in cultured human tumor cells and histocultures of human bladder tumors; the MMC effects were dependent on time, tumor grade and culture conditions.Support or Funding InformationSupported in part by Mosier Endowed Chair in Pharmaceutical Sciences at University of Oklahoma, Albert Charitable Trust, 1R01CA163015 from National Cancer Institute, and University of Kansas Cancer Center P30‐CA168524‐03.

Oncogenic roles of the cholesterol metabolite 25-hydroxycholesterol in bladder cancer.

Oxysterols, such as 24S-hydroxycholesterol and 25-hydroxycholesterol are oxidation products of cholesterol generated by enzymatic reactions. The pathological effects of oxysterols have been described in multiple types of cancer, including cancers of the skin, lung, colon, breast and bile ducts. The molecular mechanisms underlying oxysterol-induced cancer initiation and progression have yet to be completely elucidated, and to the best of our knowledge, no prior data on the role of 24S-hydroxycholesterol and 25-hydroxycholesterol in bladder cancer exists. The results of the present study demonstrated that 25-hydroxycholesterol is increased in bladder cancer tissues, and that it promotes proliferation and the epithelial-to-mesenchymal transition in human T24 and RT4 bladder cancer cells. It was also observed that 25-hydroxycholesterol promotes Adriamycin resistance in T24 and RT4 cells, and that high levels of 25-hydroxycholesterol in bladder cancer are associated with a poor outcome. Therefore, 25-hydroxycholesterol, a primary metabolite of cholesterol, may serve an important role in the progression of bladder cancer.

Ethanolic extract of Tunisian propolis: chemical composition, antioxidant, antimicrobial and antiproliferative properties

Propolis is a byproduct widely used in folk medicine due to its valuable biological and pharmacological properties. In this study, in vitro antioxidant, antimicrobial and antiproliferative capacities of propolis ethanolic extracts (EEPs) collected from four Tunisian regions were evaluated. EEPs showed high total antioxidant and antiradical capacities and were active against all tested microorganisms. These biological capacities were correlated to the amounts of phenolics in the samples. Cytotoxicity of EEPs on human bladder cancer cells RT112 was assessed using both clonogenic and the tetrazolium salt colorimetric (MTT) assays. Clonogenic assay of RT112 cells showed that all EEPs (10 µg/ml) reduce cell survival, as compared to the control. In addition, MTT assay showed a significant cytotoxic capacity of all samples on RT112 cells with an estimated IC50 ranging between 8 and 11 µg/ml. Annexin V–FITC staining test was then used to study and quantify the induction of apoptosis. Data showed that propolis induces apoptosis of RT112 cells. These findings show that propolis extracted from different Tunisian regions is an attractive antioxidant/antimicrobial/antiproliferative agent suggesting its potential application in pharmaceutical and nutraceutical products.

In vitro evaluation of cytotoxic properties of 5-Aminolevulinic acid (5-ALA) on bladder cancer cells.

5-Aminolevulinic (5-ALA) may be used as a photo diagnostic agent in bladder cancer. The aim of this study was to investigate the cytotoxic effect of 5-ALA on bladder cancer cells. T24 cells treated with various concentrations of mitomycin (MC), 5-ALA and an MC/5-ALA mixture were evaluated to determine the role of 5-ALA on MC cytotoxicity. Cell cycle analysis was conducted, and apoptosis was analyzed by flow cytometry. Caspase 3 enzyme and reactive oxygen species were measured. Our initial studies exploring the impact of combination therapy on cell viability demonstrated improved cytotoxic effects on T24 and RT cells with relatively low doses of 5-ALA/MC in conjunction with MC alone. Indicated no significant difference between the IC50 of MC and MC/5-ALA in T24 cells, while IC50 value was decreased by 25 % in RT4 cells in 5-ALA/MC in comparing with MC alone. However, examination of cell cycle phase arrests by flow cytometry revealed significant PreG1 apoptosis and cell growth arrest in G2/M in T24 cells treated with the MC/5-ALA mixture compared with MC treatment. In addition, caspase 3 enzyme was increased by 1.15-fold in T24 cells treated with MC/5-ALA in comparison with MC alone. These results suggest that 5-ALA might possess anti-cancer properties and is not only a photo diagnostic element.

Cordycepin Resensitizes T24R2 Cisplatin-Resistant Human Bladder Cancer Cells to Cisplatin by Inactivating Ets-1 Dependent MDR1 Transcription.

Tumor cell resistance to anti-cancer drugs is a major obstacle in tumor therapy. In this study, we investigated the mechanism of cordycepin-mediated resensitization to cisplatin in T24R2 cells, a T24-derived cell line. Treatment with cordycepin or cisplatin (2 μg/mL) alone failed to induce cell death in T24R2 cells, but combination treatment with these drugs significantly induced apoptosis through mitochondrial pathways, including depolarization of mitochondrial membranes, decrease in anti-apoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, and increase in pro-apoptotic proteins Bak and Bax. High expression levels of MDR1 were the cause of cisplatin resistance in T24R2 cells, and cordycepin significantly reduced MDR1 expression through inhibition of MDR1 promoter activity. MDR1 promoter activity was dependent on transcription factor Ets-1 in T24R2 cells. Although correlation exists between MDR1 and Ets-1 expression in bladder cancer patients, active Ets-1, Thr38 phosphorylated form (pThr38), was critical to induce MDR1 expression. Cordycepin decreased pThr-38 Ets-1 levels and reduced MDR1 transcription, probably through its effects on PI3K signaling, inducing the resensitization of T24R2 cells to cisplatin. The results suggest that cordycepin effectively resensitizes cisplatin-resistant bladder cancer cells to cisplatin, thus serving as a potential strategy for treatment of cancer in patients with resistance to anti-cancer drugs.

Hyperthermic therapy using warm sterile water enhances cytocidal effects on bladder cancer cells

Objectives: To investigate whether warm sterile water enhances the cytocidal effect of hypotonic shock on bladder cancer cells that show resistance to sterile water.Methods: Four bladder cancer cell lines of varying grades (T24, RT4, J82, and RT112) were exposed to sterile water, and morphological changes were closely observed under microscopy. Changes in cell membrane integrity and cell viability after water exposure were measured to determine the effects of water-induced hypotonic shock. Additionally, the effects of warm sterile water were analyzed.Results: T24, RT4, and J82 cells started swelling immediately upon exposure to water, followed by rupture within five minutes. RT112 cells demonstrated limited hypotonic swelling with significantly less cell rupture after 10 min. The percentages of viable cells at 10 min were 1.6 ± 0.8%, 3.5 ± 3.5%, 5.0 ± 3.2%, and 22.0 ± 10.3% for T24, RT4, J82, and RT112, respectively. The percentage of viable cells with 48 °C sterile water at one minute was 0% for RT112 cells.Conclusions: These findings support the efficacy of sterile water against bladder cancer cells and reveal that warm sterile water enhances the cytocidal effects of hypotonic shock, potentially avoiding the need for radical surgery.

Overexpression of DDR1 Promotes Migration, Invasion, Though EMT-Related Molecule Expression and COL4A1/DDR1/MMP-2 Signaling Axis.

Purpose:Discoidin domain receptor 1 (DDR1) belongs to a novel class of receptor tyrosine kinases. Previous evidence indicates that DDR1 overexpression promotes the aggressive growth of bladder cancer (BC) cells. This study aimed to investigate the molecular mechanisms by which DDR1 influences BC.Methods:DDR1 was transfected into human BC RT4 cells. DDR1, COL4A1, and MMP-2 expression in 30 BC tissues and paired adjacent tissues were examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Transwell assays were conducted to determine cell migration and invasion. RT-PCR and western blot (WB) were also used to measure the DDR1, COL4A1, MMP-2, and EMT-related gene (ZEB1 and SLUG) expression in RT4 cells after DDR1 overexpression.Results:COL4A1 and MMP-2 interacted with DDR1 in the PPI network. RT-PCR and immunohistochemistry results showed that both mRNA and protein levels of DDR1 and COL4A1 were significantly increased in BC tissue, while the expression of MMP-2 was increased only at the mRNA level (P < 0.05). Overexpression of DDR1 in RT4 cells significantly promoted their migratory and invasive capabilities in vitro (P < 0.05). Moreover, overexpression of DDR1 in RT4 cells increased the mRNA and protein expression of ZEB1, SLUG, COL4A1, and MMP-2 (P < 0.01). DDR1-mediated migration and invasion of RT4 cells were reversed after COL4A1-siRNA treatment.Conclusion:DDR1 may be a potential therapeutic target in BC patients.

Increased expression of Psoriasin is correlated with poor prognosis of bladder transitional cell carcinoma by promoting invasion and proliferation.

Psoriasin, otherwise known as S100A7, is a member of the S100 protein family. With the key function of binding calcium, it is able to regulate a range of cellular functions. Altered Psoriasin expression is associated with poor clinical outcomes in several solid cancers. The present study aimed to examine the implication of Psoriasin in bladder cancer (BC). Expression of Psoriasin was examined in BC cell lines using PCR. Immunohistochemical (IHC) staining of Psoriasin was performed on a bladder disease spectrum tissue array. Plasmids were constructed to effectively knockdown and overexpress Psoriasin in BC cells and further utilized for in vitro BC cellular function assays. Association between Psoriasin expression and survival of patients with BC was evaluated using Kaplan-Meier survival analysis. Psoriasin was revealed to be expressed by both bladder epithelia and cancer cells as determined by IHC. Increased expression of Psoriasin was significantly correlated with a poor overall BC patient survival. Overexpression of Psoriasin in the EJ138 cell line increased cellular proliferation, adhesion and invasion, whereas knockdown exhibited the opposite effect on cellular functions in RT112 cells. Matrix metalloprotease (MMP)9 appeared to be the most affected of the three MMPs examined in these two BC cell lines. The analysis revealed a positive correlation in BC tumours between Psoriasin and MMP9. Overall, high Psoriasin expression was correlated with poor overall survival in BC patients and promoted invasiveness of BC cells via upregulation of MMPs. Psoriasin possesses certain prognostic and therapeutic potential in BC which requires further exploration.

IGFBP-2 acts as a tumour suppressor and plays a role in determining chemosensitivity in bladder cancer cells.

There are mixed reports on the role that IGFBP-2 plays in cancer progression, with some indicating a tumour suppressive role and others showing that IGFBP-2 may act as an oncogene. These apparent contradictions may be context and tissue specific. In this study we determined the role that IGFBP-2 played on the phenotype and chemosensitivity of a selection of bladder cancer cell lines and investigated how the abundance of IGFBP-2 was regulated. We found that IGFBP-2 was more abundant in the epithelial bladder cancer cells, RT4 and UMUC3 and absent in the more mesenchymal T24 and TCCSUP cells. Silencing IGFBP-2 using siRNA in epithelial RT4 cells promoted cell proliferation, invasion, colony formation, resulted in a reduction in epithelial (E-cadherin) and an increase in mesenchymal (N-cadherin) markers and increased sensitivity to cisplatin-induced cell death. Conversely, we observed the opposite effects when adding exogenous IGFBP-2 to the mesenchymal T24 cells. We determined that IGFBP-2 was epigenetically silenced via DNA methylation as the cells adopted a mesenchymal phenotype. Collectively these data suggest that IGFBP-2 acts as a tumour suppressor and marker of chemosensitivity in epithelial bladder cancer cells and that IGFBP-2 is epigenetically silenced by methylation to promote bladder cancer progression.

Tunneling Nanotubes Mediated microRNA-155 Intercellular Transportation Promotes Bladder Cancer Cells' Invasive and Proliferative Capacity.

ObjectiveTo investigate differential microRNAs' expression in heterogeneous bladder cancer cells, as well as to investigate the mechanism of changes in invasive and proliferative capacity induced by tunneling nanotubes (TNTs) mediated transport of microRNA between bladder cancer cells of varying histological grade.Materials and methodsDifferences in microRNA expression between bladder cancer cells of different grade were identified from a literature review. The identified heterogeneous microRNAs were analyzed by qPCR in T24 (high grade) and RT4 (low grade) bladder cancer cells. Scanning electron microscopy (SEM) and laser confocal fluorescence microscopy (LCM) were used to observe tunneling nanotubes (TNTs) between RT4 and T24 cells. Differentially expressed microRNA was labeled and traced by Fluorescent In Situ Hybridization (FISH) following co-culture of T24 and RT4 cells. MicroRNA mimic and inhibition technologies were applied to investigate how TNTs-mediated intercellular transport of microRNA affects the invasive and proliferative behavior of bladder cancer cells.ResultsMicroRNA-155 (miR-155) levels were highly expressed in T24 cells, whereas the same was not true in RT4 cells. MiR-155 was confirmed to be a crucial factor sustaining T24 bladder cancer cell proliferation, migration and cell cycle progression by CCK8, Matrigel test and cell cycle analysis, respectively. After T24 and RT4 co-culture, TNTs were assessed by SEM and LCM between T24 and RT4 cells. In addition, we observed TNTs mediated transport of miR-155 from T24 cells to RT4 cells, which thereby acquired a higher proliferative rate, an increased frequency of cells in the S phase, and increased invasive ability in Matrigel test. At the same time, Deptor, the target protein of miR-155 in RT4 cells, was downregulated, followed by mTOR/4EBP1/p70S6K- eIF4e/S6RP signaling activation.ConclusionMiR-155 was differentially expressed between RT4 and T24 bladder cancer cells. Intercellular transport of miR-155 via TNTs can promote bladder cancer cell reprogramming by Deptor-mTOR signal pathway activation.