Bladder Cancer Cell Line T24 Research Articles

Activation of a c-Jun N-terminal kinase-mediated autophagy pathway attenuates the anticancer activity of gemcitabine in human bladder cancer cells.

The role of autophagy in the anticancer activity of gemcitabine (GEM) in bladder cancer is unclear. The aim of this study is to determine whether GEM activates autophagy, the role of autophagy in the anticancer activity of GEM, and the underlying mechanism by which GEM induces autophagy. Human bladder cancer cell lines T24 and BIU87 were treated with GEM in vitro. Cell viability was measured using the Cell Counting Kit-8 assay. Apoptosis was detected by annexin V assay and western blot. Autophagy was measured by western blot and transmission electron microscopy. c-Jun N-terminal kinase (JNK) activation was detected by western blot. Chemical inhibitors were used for intervention of JNK and autophagy. GEM killed bladder cancer cells, which was associated with apoptosis induction. Autophagy was effectively activated by GEM. Suppressing autophagy in GEM-treated cells significantly decreased cell viability, which was associated with increased apoptosis. GEM-induced JNK activation and suppressed B-cell lymphoma 2 expression. The JNK inhibitor SP600125 inhibited GEM-induced autophagy activation and increased GEM's cytotoxicity. GEM kills bladder cancer cells through apoptosis. Meanwhile, JNK-mediated autophagy was activated, which protects the cells against apoptosis. Therefore, inhibition of autophagy could be exploited to enhance the anticancer efficacy of GEM for treating bladder cancer.

Curcumin inhibits bladder cancer progression via regulation of β-catenin expression.

Bladder cancer has a considerable morbidity and mortality impact with particularly poor prognosis. Curcumin has been recently noticed as a polyphenolic compound separated from turmeric to regulate tumor progression. However, the precise molecular mechanism by which curcumin inhibits the invasion and metastasis of bladder cancer cells is not fully elucidated. In this study, we investigate the effect of curcumin on the bladder cancer as well as possible mechanisms of curcumin. The expression of β-catenin was detected by quantitative real-time polymerase chain reaction and immunohistochemical analysis in a series of bladder cancer tissues. In addition, bladder cancer cell lines T24 and 5637 cells were treated with different concentrations of curcumin. The cytotoxic effect of curcumin on cell proliferation of T24 and 5637 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The migration and invasion capacity of T24 and 5637 cells were measured by transwell assay. The effects of curcumin on expression levels of β-catenin and epithelial-mesenchymal transition marker were determined by western blotting. The β-catenin expression was significantly upregulated in bladder cancer tissues when compared with corresponding peri-tumor tissues. Furthermore, curcumin inhibited the cell proliferation of T24 and 5637 cells, and curcumin reduced the migration and invasive ability of T24 and 5637 cells via regulating β-catenin expression and reversing epithelial-mesenchymal transition. Curcumin may be a new drug for bladder cancer.

Effects of programmed death-ligand 1 expression on OK-432 immunotherapy following transurethral resection in non-muscle invasive bladder cancer.

The present study aimed to investigate the effect of the negative costimulatory molecule programmed death-ligand 1 (PD-L1) on immunotherapy with OK-432, following transurethral resection of bladder tumors in non-muscle invasive bladder cancer (NMIBC), and to elucidate the underlying mechanism. PD-L1 was detected by immunohistochemical staining in tumor specimens from 55 cases of NMIBC following postoperative immunotherapy with OK-432. The PD-L1 mRNA and protein expression levels were measured in the bladder cancer T24 cell line and the human uroepithelial SV-HUC-1 cell line, following treatment with interleukin (IL)-2, interferon (IFN)-α and IFN-γ, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. PD-L1 was widely expressed in the NMIBC tumors, with 56.4% (31/55) of specimens exhibiting positive staining. When compared with PD-L1-negative patients, PD-L1-positive patients exhibited significantly increased recurrence [48.4% (15/31) vs. 16.7% (4/24)] and progression [16.1% (5/31) vs. 4.2% (1/24)] rates (P<0.05). RT-qPCR and western blotting demonstrated that cytokines IL-2, IFN-α and IFN-γ markedly upregulated PD-L1 mRNA expression rates and protein levels in bladder cancer T24 cells (P<0.05), but had no significant effect in non-tumor SV-HUC-1 cells. In conclusion, PD-L1 expression was negatively-associated with the efficacy of OK-432 intravesical immunotherapy in patients with NMIBC. The results indicated that the involved mechanism occurred via upregulation of PD-L1 by immune cytokines, which in turn suppressed the antitumor effectiveness of the immune system, thereby promoting tumor recurrence and progression.

Resistance to nanoparticle albumin-bound paclitaxel is mediated by ABCB1 in urothelial cancer cells.

Nanoparticle albumin-bound (nab)-paclitaxel appears to exhibit better response rates in patients with metastatic urothelial cancer of the bladder whom are pretreated with nab-paclitaxel compared with conventional paclitaxel. Paclitaxel may induce multidrug resistance in patients with cancer, while the mechanisms of resistance against paclitaxel are manifold. These include reduced function of pro-apoptotic proteins, mutations of tubulin and overexpression of the drug transporter adenosine 5′-triphosphate-binding cassette transporter subfamily B, member 1 (ABCB1). To evaluate the role of ABCB1 in nab-paclitaxel resistance in urothelial cancer cells, the bladder cancer cell lines T24 and TCC-SUP, as well as sub-lines with acquired resistance against gemcitabine (T24rGEMCI20 and TCC-SUPrGEMCI20) and vinblastine (T24rVBL20 and TCC-SUPrVBL20) were examined. For the functional inhibition of ABCB1, multi-tyrosine kinase inhibitors with ABCB1-inhibiting properties, including cabozantinib and crizotinib, were used. Additional functional assessment was performed with cell lines stably transduced with a lentiviral vector encoding for ABCB1, and protein expression was determined by western blotting. It was indicated that cell lines overexpressing ABCB1 exhibited similar resistance profiles to nab-paclitaxel and paclitaxel. Cabozantinib and crizotinib sensitized tumor cells to nab-paclitaxel and paclitaxel in the same dose-dependent manner in cell lines overexpressing ABCB1, without altering the downstream signaling of tyrosine kinases. These results suggest that the overexpression of ABCB1 confers resistance to nab-paclitaxel in urothelial cancer cells. Additionally, small molecules may overcome resistance to anticancer drugs that are substrates of ABCB1.

MicroRNA-294 Promotes Cellular Proliferation and Motility through the PI3K/AKT and JAK/STAT Pathways by Upregulation of NRAS in Bladder Cancer.

In our study we examined the role of microRNA-294 (miR-294) in bladder cancer and related mechanisms. Real-time polymerase chain reaction (RT-PCR) was performed to determine the expression level of miR-294. Western blot was used to determine the expression of NRAS, mainly factors in the PI3K/AKT and JAK/STAT pathways. Cell counting kit-8 assay, clonogenic assay, wound-healing assay, transwell and flow cytometry were used to explore, respectively, cell proliferation, survival, migration, invasion, and apoptosis of bladder cancer cell line T24. The expressions of miR-294 in bladder cancer cells including J82, HT1376, T24, and SW780 were significantly increased compared to those in human bladder epithelium cells (both HCV29 and SV-HUC-1). The proliferation rate, surviving fraction, migration, and invasion of T24 cells in miR-294 mimetic transfected group were significantly increased, while they were significantly decreased by miR-294 inhibitor transfection. Moreover, miR-294 suppression could increase the apoptotic rate of T24 cells. In addition, drug resistance of T24 cells to cisplatin was increased in miR-294 mimetic-treated group, while it was decreased by miR-294 inhibitor compared to empty control. Overexpression of miR-294 could upregulate NRAS expression in T24 cells and activate PI3K/AKT and JAK/STAT pathways. We found that miR-294 expression was positively related with proliferation and motility of T24 cells. Moreover, miR-294 suppression could promote the sensitivity of T24 cells to cisplatin. We also found miR-294 could upregulate NRAS and activate the PI3K/AKT and JAK/STAT pathways in T24 cells.

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.

Centromere protein U is a potential target for gene therapy of human bladder cancer.

To investigate the role of centromere protein U (CENPU) in human bladder cancer (BCa), CENPU gene expression was evaluated in human BCa tissues. We used real-time quantitative PCR (qPCR) and found that CENPU gene expression in human BCa tissues was higher compared to that observed in cancer-adjacent normal tissues. High CENPU expression was found to be strongly correlated with tumor size and TNM stage. Kaplan-Meier survival analysis indicated that high CENPU levels were associated with reduced survival. We used a lentivirus to silence endogenous CENPU gene expression in the BCa T24 cell line. CENPU knockdown was confirmed by qPCR. Cellomic imaging and BrdU assays showed that cell proliferation was significantly reduced in the CENPU-silenced cells compared to that noted in the control cells. Flow cytometry revealed that in the CENPU-silenced cells the cell cycle was arrested at the G1 phase relative to that in the control cells. In addition, apoptosis was significantly increased in the CENPU-silenced cells. Giemsa staining showed that CENPU-silenced cells, compared to control cells, displayed a significantly lower number of cell colonies. The genome-wide effect of CENPU knockdown showed that a total of 1,274 differentially expressed genes was found, including 809 downregulated genes and 465 upregulated genes. Network analysis by Ingenuity Pathway Analysis (IPA) resulted in 25 distinct signaling pathways, including the top-ranked network: ‘Cellular compromise, organismal injury and abnormalities, skeletal and muscular disorders’. In-depth IPA analysis revealed that CENPU was associated with the HMGB1 signaling pathway. qPCR and western blot analysis demonstrated that in the HMGB1 signaling pathway, CENPU knockdown downregulated expression levels of ILB, CXCL8, RAC1 and IL1A. In conclusion, our data may provide a potential pathway signature for therapeutic targets with which to treat BCa.

MicroRNA-218 Increases the Sensitivity of Bladder Cancer to Cisplatin by Targeting Glut1

Background/Aims: MicroRNA-218 (miR-218) is down-regulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. However, the involvement of miR-218 in chemo-sensitivity to cisplatin and the precise mechanism of this action remained unknown in bladder cancer. Methods: qRT-PCR was used to detect miR-218 and its target Glut1 expression in bladder cancer cell lines T24 and EJ. CCK-8 method was utilized to measure the cell viability. IC 50 was calculated via a probit regression model. Glut1 was detected by western blotting for analysis of potential mechanism. Luciferase reporter assay was utilized to validate Glut1 as a direct target gene of miR-218. The intracellular level of GSH and ROS were determined using a commercial colorimetric assay kit and 2’, 7’-dichlorodihydro-fluorescein diacetate, respectively. Results: Over-expression of miR-218 significantly reduced the rate of glucose uptake and total level of GSH and enhanced the chemo-sensitivity of bladder cancer to cisplatin. Mechanistically, Glut1 was found to be a direct and functional target of miR-218. Up-regulation of Glut1 could restore chemo-resistance in T24 and EJ cells. On the contrary, knockdown of Glut1 could generate a similar effect as up-regulating the expression of miR-218. Conclusions: MiR-218 increases the sensitivity of bladder cancer to cisplatin by targeting Glut1. Restoration of miR-218 and repression of glut1 may provide a potential strategy to restore chemo-sensitivity in bladder cancer.

Tea Polyphenol inhibits autophagy to sensitize Epirubicin-induced apoptosis in human bladder cancer cells.

Resistance to anticancer agents such as Epirubicin (EPI) becomes a great challenge for treating bladder cancer. However, the mechanism by which chemoresistance arised is still elusive. In the present study, we showed evidence that EPI induced cytoprotective autophagy in bladder cancer cell lines T24 and BIU87. In addition, EPI robustly activated JNK-mediated phosphorylation of Bcl-2 and disruption of Bcl-2/Beclin-1 complex. Furthermore, the green tea derivative tea polyphenol (TP) inhibited EPI-induced autophagy and promoted apoptosis induced by EPI in bladder cancer cells. These results revealed a pathway for EPI-induced autophagy that involved in JNK/Bcl-2/Beclin-1 in bladder cancer cells, and that TP synergistically promoted EPI-induced apoptosis at least partly through autophagy inhibition. Thus, TP could be utilized in combination with EPI to improve EPI-based bladder cancer therapy.

Overexpression of PTK6 predicts poor prognosis in bladder cancer patients.

Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase and works as an oncogene in various cancers. Recently, PTK6 has been used as a therapeutic target for breast cancer patients in a clinical study. However, the prognostic value of PTK6 in bladder cancer (BC) remains vague. Therefore, we retrieved 3 independent investigations of Oncomine database and found that PTK6 is highly expressed in BC tissues compared with corresponding normal controls. Similar results were also observed in clinical specimens at both mRNA and protein levels. Immunohistochemical analysis indicated that PTK6 overexpression was highly related to the T classification, N classification, grade, recurrence, and poor prognosis of BC patients. Furthermore, we demonstrated that when PTK6 expression was knocked down by siRNAs, cell proliferation and migration were considerably inhibited in BC cell lines T24 and EJ. By these approaches, we are intended to elucidate PTK6 may be a reliable therapeutic target in BC and might benefit from PTK6 inhibitors in the future.

Low Releasing Mitomycin C Molecule Encapsulated with Chitosan Nanoparticles for Intravesical Installation

The aim of this investigation is preparation of Mitomycin-C encapsulated with chitosan nanoparticles synthesis using ionic gelation technique for intravesical controlled drug delivery systems. This study was conducted in vitro. Cumulative amount of drug released from the nanoparticles was calculated. Mitomycin-C release studies were examined for different pH values. During the drug loading and release studies, initial amount of drug was changed (i.e., 0.5, 1.25 and 2.5 mg) to get different release profiles and the release studies were repeated (n = 6). The loading efficiencies of Mitomycin-C with three different initial concentrations 0.5mg/ml, 1.25 mg/ml and 2.5 mg/ml into chitosan nanoparticles were 54.5%, 47.1% and 36.4%, respectively. For different pH values, the cumulative releases of Mitomycin-C from chitosan nanoparticles were 47% and 53% for pH 6.0 and 7.4, respectively (p p san nanoparticles was measured in T24 bladder cancer cell line in vitro, and the results revealed that the 2.5 MMC coated Chitosan nanoparticles had better tumor cells decline activity. From this investigation, we conclude that the drug encapsulated synthesized chitosan nanoparticles possess a high ability to be used as pH and dose responsive drug delivery system. This systematic investigation demonstrates a promising future for the intravesical installation in treatment of the superficial bladder cancer.

Raman microscopy of bladder cancer cells expressing green fluorescent protein.

Gene engineering is a commonly used tool in cellular biology to determine changes in function or expression of downstream targets. However, the impact of genetic modulation on biochemical effects is less frequently evaluated. The aim of this study is to use Raman microscopy to assess the biochemical effects of gene silencing on T24 and UMUC-13 bladder cancer cell lines. Cellular biochemical information related to nucleic acid and lipogenic components was obtained from deconvolved Raman spectra. We show that the green fluorescence protein (GFP), the chromophore that served as a fluorescent reporter for gene silencing, could also be detected by Raman microscopy. Only the gene-silenced UMUC-13 cell lines exhibited low-to-moderate GFP fluorescence as determined by fluorescence imaging and Raman spectroscopic studies. Moreover, we show that gene silencing and cell phenotype had a greater effect on nucleic acid and lipogenic components with minimal interference from GFP expression. Gene silencing was also found to perturb cellular protein secondary structure in which the amount of disorderd protein increased at the expense of more ordered protein. Overall, our study identified the spectral signature for cellular GFP expression and elucidated the effects of gene silencing on cancer cell biochemistry and protein secondary structure.

Expression of PAK1 in bladder cancer and its influence on invasion of bladder cancer cells

Objective: To investigate the expression of p21-activated kinase 1 (PAK1) in bladder cancer and its biological influence on invasion ability of bladder cancer cells. Methods: A total of 54 paraffin-embedded bladder cancer tissue samples and 12 normal bladder tissue specimens were retrieved in Jinshan Hospital Affiliated to Fudan University between January 2009 and December 2012. The expression of PAK1 in these tissues was detected by immunohistochemical staining. The PAK1 mRNA and protein levels were measured in bladder cancer cell lines and human normal bladder epithelial cell line using real-time, fluorescence-based quantitative polymerase chain reaction (RT-PCR) and Western blot, respectively. A stable PAK1 gene silencing bladder cancer cell strain 5637 were successfully constructed. After treatment with PAK1 RNA interference(RNAi), the ability of migration and invasion of the 5637 cell was evaluated using a Transwell system. Results: The expression of PAK1 proteins was significantly higher in bladder cancer tissues than in normal bladder tissues (28/54 vs 1/12, P<0.05). The overexpression of PAK1 was positively correlated with high histological grade, lymph node metastasis, and tumor size (all P<0.05). The mRNA level and protein level of PAK1 was much higher in bladder cancer cell lines T24, 5637 than human normal urothelial cell line SV-HUC-1.PCR and Western blot showed satisfactory inhibitory effect of PAK1 short hairpin RNA (shRNA) on PAK1 expression in 5637 bladder cancer cells. The number of PAK1 RNAi-treated 5637 cells traversed the membrane was decreased compared with the control group in migration and invasion assays. Conclusions: Overexpression of PAK1 in bladder cancer tissues may be an important feature of bladder cancer and related with the metastasis and invasion of bladder cancer. The molecular mechanisms involved in regulation of PAK1 expression needs further research.

LINC00312 inhibits the migration and invasion of bladder cancer cells by targeting microRNA-197-3p

To investigate the influence of the long non-coding RNA LINC00312 on bladder cancer (BC) cell invasion and metastasis by targeting miR-197-3p. BC and corresponding adjacent tissues were collected. LINC00312 and miR-197-3p were measured, and their correlation was detected through quantitative real-time PCR (qRT-PCR). BC cell line T24 was transfected and grouped (five groups) according to different transfection conditions. A scratch test was applied to analyze cell migration, and a Transwell assay was used to test cell invasion ability. Western blotting was to measure matrix metalloproteinase (MMP)-2, MMP-9, and the tissue inhibitor of metalloproteinase 2 (TIMP2) protein levels. qRT-PCR indicated that LINC00312 expression was lower but miR-197-3p expression was higher in BC tissues compared with adjacent tissues; LINC00312 was negatively correlated with miR-197-3p. The migration test revealed that the downregulation of miR-197-3p and overexpression of LINC00312 inhibited cell migration and invasion abilities, while the overexpression of miR-197-3p and the upregulation of LINC00312 promoted cell migration and invasion. BC cells with downregulated miR-197-3p or upregulated LINC00312 had low MMP-2 and MMP-9 levels but high TIMP2. LINC00312 inhibited BC cell invasion and metastasis through mediating miR-197-3p.

MiR-489 Suppresses Proliferation and Invasion of Human Bladder Cancer Cells.

MicroRNAs (miRNAs) have been shown to be involved in bladder cancer progression. miR-489 (also known as miR-489-3p) was recently reported to be a tumor suppressor in several cancers. However, its exact role and mechanism in the progression of bladder cancer are largely unknown. In this study, we explore the role of miR-489 in the proliferation and invasion of human bladder cancer cells. The miR-489 expression levels were detected in bladder cancer and normal adjacent tissues, as well as in human normal bladder epithelial cells and bladder cancer cell lines. The results showed that miR-489 was sharply reduced in bladder cancer tissues and cell lines. Then the miR-489 mimic or oligo anta-miR-489 was transfected into T24 and UMUC3 bladder cancer cell lines. The results showed that the miR-489 mimic greatly increased the miR-489 level and significantly decreased the proliferation and invasion of T24 and UMUC3 cells. In contrast, the anta-miR-489 had a completely opposite effect on miR-489 expression, cell proliferation, and cell invasion. Moreover, bioinformatics and luciferase reporter gene assays confirmed that miR-489 targeted the mRNA 3′-untranslated region (3′-UTR) region of Jagged1 (JAG1), a Notch ligand. In conclusion, miR-489 suppressed proliferation and invasion of human bladder cancer cells.

Dynamin2 GTPase contributes to invadopodia formation in invasive bladder cancer cells

Cancer cell invasion is mediated by actin-based membrane protrusions termed invadopodia. Invadopodia consist of “core” F-actin bundles associated with adhesive and proteolytic machineries promoting cell invasion by degrading extracellular matrix (ECM). Formation of the F-actin core in invadopodia is regulated by various actin-binding proteins including Arp2/3 complex and cortactin. Dynamin GTPase localizes to the invadopodia and is implicated in cancer cell invasion, but its precise role at the invadopodia remained elusive.In this study, we examined the roles of dynamin at the invadopodia of bladder cancer cells. Although all three dynamin isoforms (dynamin1, 2 and 3) are expressed in human bladder cancer cell line T24, only dynamin2 localizes to the invadopodia. Inhibition of dynamin2 function, using either RNA interference (RNAi) or the dynamin specific inhibitor Dynasore, caused defects in invadopodia formation and suppressed invasive activity of T24 bladder cancer cells. Structure-function analysis using dynamin2 deletion fragments identified the proline/arginine-rich domain (PRD) of dynamin2 as indispensable for invadopodia formation and invasiveness of T24 cells. Thus, dynamin2 contributes to bladder cancer invasion by controlling invadopodia formation in bladder cancer cells and may prove a valuable therapeutic target.

LINC00312 inhibits the migration and invasion of bladder cancer cells by targeting miR-197-3p.

To investigate the influence of the long non-coding RNA LINC00312 on bladder cancer (BC) cell invasion and metastasis by targeting miR-197-3p. BC and corresponding adjacent tissues were collected. LINC00312 and miR-197-3p were measured, and their correlation was detected through quantitative real-time PCR (qRT-PCR). BC cell line T24 was transfected and grouped (five groups) according to different transfection conditions. A scratch test was applied to analyze cell migration, and a Transwell assay was used to test cell invasion ability. Western blotting was to measure matrix metalloproteinase (MMP)-2, MMP-9, and the tissue inhibitor of metalloproteinase 2 (TIMP2) protein levels. qRT-PCR indicated that LINC00312 expression was lower but miR-197-3p expression was higher in BC tissues compared with adjacent tissues; LINC00312 was negatively correlated with miR-197-3p. The migration test revealed that the downregulation of miR-197-3p and overexpression of LINC00312 inhibited cell migration and invasion abilities, while the overexpression of miR-197-3p and the upregulation of LINC00312 promoted cell migration and invasion. BC cells with downregulated miR-197-3p or upregulated LINC00312 had low MMP-2 and MMP-9 levels but high TIMP2. LINC00312 inhibited BC cell invasion and metastasis through mediating miR-197-3p.

Reprogramming bladder cancer cells for studying cancer initiation and progression.

The induced pluripotent stem cell (iPSC) technology is the forced expression of specific transcription factors in somatic cells resulting in transformation into self-renewing, pluripotent cells which possess the ability to differentiate into any type of cells in the human body. While malignant cells could also be reprogrammed into iPSC-like cells with lower efficiency due to the genetic and epigenetic barriers in cancer cells, only a limited number of cancer cell types could be successfully reprogrammed until today. In the present study, we aimed at reprogramming two bladder cancer cell lines HTB-9 and T24 using a non-integrating Sendai virus (SeV) system. We have generated six sub-clones using distinct combinations of four factors-OCT4, SOX2, KLF4 and c-MYC-in two bladder cancer cell lines. Only a single sub-clone, T24 transduced with 4Fs, gave rise to iPSC-like cells. Bladder cancer cell-derived T24 4F cells represent unique features of pluripotent cells such as epithelial-like morphology, colony-forming ability, expression of pluripotency-associated markers and bearing the ability to differentiate in vitro. This is the first study focusing on the reprogramming susceptibility of two different bladder cancer cell lines to nuclear reprogramming. Further molecular characterisation of T24 4F cells could provide a better insight for biomarker research in bladder carcinogenesis and could offer a valuable tool for the development of novel therapeutic approaches in bladder carcinoma.

MP61-10 DNA METHYLATION INHIBITORS MAY REVERSE DRUG-RESISTANCE IN HUMAN BLADDER CANCER CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2016MP61-10 DNA METHYLATION INHIBITORS MAY REVERSE DRUG-RESISTANCE IN HUMAN BLADDER CANCER CELLS Naotaka Nishiyama, Stephanie Yi, Christopher E. Duymich, Siamak Daneshmand, Peter A. Jones, Hooman Djaladat, Naoya Masumori, and Gangning Liang Naotaka NishiyamaNaotaka Nishiyama More articles by this author , Stephanie YiStephanie Yi More articles by this author , Christopher E. DuymichChristopher E. Duymich More articles by this author , Siamak DaneshmandSiamak Daneshmand More articles by this author , Peter A. JonesPeter A. Jones More articles by this author , Hooman DjaladatHooman Djaladat More articles by this author , Naoya MasumoriNaoya Masumori More articles by this author , and Gangning LiangGangning Liang More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.884AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Aberrant DNA methylation is among common events during tumorigenesis. Although heritable, the dynamic nature and its potential reversibility with pharmacological interventions make it an interesting target for drugs. The aim of this study was to evaluate the chemotherapeutic re-sensitivity of cisplatin (CDDP) and gemcitabine (GEM) resistance in bladder cancer cell lines induced by the hypomethylating agent 5-aza-2-deoxycytidine (5-Aza-CdR). METHODS Six bladder cancer cell lines, T24, UMUC3, T24-RCDDP, UMUC3-RCDDP, T24-RGEM and UMUC3-RGEM cells were used. The drug resistance cell lines, T24-RCDDP/UMUC3-RCDDP and T24-RGEM/UMUC3-RGEM, were newly established at our laboratory. Cell lines were treated with 1µM 5-Aza-CdR for 24 hours on day 1, with CDDP (5µg/ml) and GEM (30nmol/ml) on day 2, or combinatorial treatment of 5-Aza-CdR (day 1) and either CDDP or GEM (day 2). The cell phonotypical alterations were monitored by cell double time, DNA methylation changes and gene expression changes. RESULTS We used the Illumina Infinium Human Methylation450 BeadArray to measure the DNA methylation changes at 482,421 CpGs, and found dramatic DNA methylation alterations between T24 and UMUC3 bladder cancer cell lines, as well as their drug-resistance counterparts (T24-RCDDP/T24-RGEM and UMUC3-RCDDP/UMUC3-RGEM). We identified DNA hypermethylation at 28,271 and 45,927 CpG sites in T24 and UMUC3 drug resistant cells, respectively. Furthermore, we identified DNA hypomethylation at 41,576 and 18,711 CpG sites in T24 and UMUC3 drug resistant cells, respectively. In addition, 5-Aza-CdR may re-sensitize CDDP-resistant cell lines, since the cell double time significantly increased from 70 (T24-RCDDP) or 86 (UMUC3-RCDDP) hours by CDDP alone to 495 (T24-RCDDP) or 721(UMUC3-RCDDP) hours after the combination treatments. However, it should be noted that the combination treatment of 5-Aza-CdR with GEM did not re-sensitize the chemotherapeutic effect on GEM-resistant cells. CONCLUSIONS Overall, our results indicate that alteration of DNA methylation may play an important role in drug-resistance, while DNA methylation re-programming may also sensitize the chemotherapeutic efficacy for drug resistance cells especially in metastatic urothelial carcinoma. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e806-e807 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Naotaka Nishiyama More articles by this author Stephanie Yi More articles by this author Christopher E. Duymich More articles by this author Siamak Daneshmand More articles by this author Peter A. Jones More articles by this author Hooman Djaladat More articles by this author Naoya Masumori More articles by this author Gangning Liang More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Targeted p53 activation by saRNA suppresses human bladder cancer cells growth and metastasis.

BackgroundPrevious study showed that dsP53-285 has the capacity to induce tumor suppressor gene p53 expression by targeting promoter in non-human primates’ cells. And it is well known that TP53 gene is frequently mutant or inactivated in human bladder cancer. Hereby, whether this small RNA can activate the expression of wild-type p53 and inhibit human bladder cancer cells remains to be elucidated.MethodsOligonucleotide and lentivirus were used to overexpress dsP53-285 and dsControl. Real-time PCR and western blot were used to detect genes’ mRNA and protein expression, respectively. Cell proliferation assay, colony formation, flow cytometry, transwell assay and wound healing assay were performed to determine the effects on bladder cancer cells proliferation and migration/invasion in vitro. Animal models were carried out to analyze the effects on cells growth and metastasis in vivo.ResultsTransfection of dsP53-285 into human bladder cancer cell lines T24 and EJ readily activate wild-type p53 expression by targeting promoter. Moreover, dsP53-285 exhibited robust capacity to inhibit cells proliferation and colony formation, induce cells G0/G1 arrest, suppress migration and invasion. Besides, the Cyclin-CDK genes (Cyclin D1 and CDK4/6) were down-regulated and the EMT-associated genes (E-cadherin, β-catenin, ZEB1 and Vimentin) were also expressed inversely after dsP53-285 treatment. In addition, dsP53-285 could also significantly suppress the growth of bladder cancer xenografts and metastasis in nude mice. Most importantly, the anti-tumor effects mediated by dsP53-285 were mainly achieved by manipulating wild-type p53 expression.ConclusionOur findings indicate that the dsP53-285 can upregulate wild-type p53 expression in human bladder cancer cells through RNA activation, and suppresses cells proliferation and metastasis in vitro and in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0329-8) contains supplementary material, which is available to authorized users.