Human Bladder Cancer Cell Lines Research Articles

A Galactose Dendritic Silicon (IV) Phthalocyanine as a Photosensitizing Agent in Cancer Photodynamic Therapy.

Two protected galacto-dendritic units have been axially coordinated to the central ion of a silicon(IV) phthalocyanine to afford SiPcPGal4 containing four units of galactose per macrocycle. These biological moieties provided better solubility in aqueous medium and a sensitizer with higher absorption peaks at 680-690 nm. The photodynamic activity of SiPcPGal4 was evaluated against UM-UC-3 human bladder cancer cell line and the results were compared with the activity of the reported SiPcPGal2 and SiPc(OH)2 . SiPcPGal4 had a better uptake and it was a better toxicity inducer than SiPcPGal2 and SiPc(OH)2 owing to its four galactose units, protected by isopropylidene groups, which can act as targeted micelles.

Upregulation of lncRNA snoRNA host gene 6 regulates NUAK family SnF1-like kinase-1 expression by competitively binding microRNA-125b and interacting with Snail1/2 in bladder cancer.

Numerous studies have reported that long noncoding RNAs (lncRNAs) play critical roles in the development and progression of bladder cancer (BC). LncRNA snoRNA host gene 6 (SNHG6) is ectopically expressed in tumor tissues of patients with BC and BC cell lines. However, little is known about the molecular mechanism of SNHG6-mediated bladder urothelial carcinoma cell migration and invasion. We detected the SNHG6 levels in human BC specimens and cell lines byquantitativereal-time polymerase chain reaction and Western blot, and investigated its role in BC using in vitro assays. We showed that overexpression of SNHG6 induced epithelial-mesenchymal transition (EMT) and promoted the migration and invasion capabilities of BC cells. Mechanistically, SNHG6 induced EMT of BC cells by upregulating the expression levels of Snail1/2 and regulated BC cell migration and invasion by tumor suppressive hsa-miR-125b and its target gene NUAK Family Kinase 1 (NUAK1). Furthermore, we found that SNHG6 was positively correlated with Snail1/2 expression, and negatively correlated with hsa-miR-125b expression in BC specimens. Further study showed that SNHG6 repressed hsa-miR-125b expression to upregulate Snail1/2. Conversely, hsa-miR-125b knockdown augmented SNHG6 expression in BC cells. Overall, our study demonstrated that SNHG6 promotes BC cell migration and invasion partly via the hsa-miR-125b/Snail1/2/NUAK1 pathway. Therefore, SNHG6 may be a potential prognostic biomarker in BC, and targeting hsa-miR-125b/Snail1/2/NUAK1 axis may be a promising therapeutic approach for BC patients.

Additive antitumor effect of arsenic trioxide combined with intravesical bacillus Calmette–Guerin immunotherapy against bladder cancer through blockade of the IER3/Nrf2 pathway

BackgroundAs an inorganic compound used to treat various cancers and other diseases, arsenic trioxide (As2O3) has been reported to induce cellular apoptosis in certain kinds of cancers including bladder cancer. The aim of the present study was to elucidate the crucial cooperative role of As2O3 and intravesical bacillus Calmette-Guerin (BCG) immunotherapy and its ability to protect against bladder cancer by targeting the IER3/Nrf2 pathway. MethodInitially, an orthotopic bladder cancer model was established in mice by means of intravesical instillation of the human bladder cancer cell line 5637. The expression of IL-6/IL-8 in dendritic cells (DCs) and the proportion of CD4+ cells and ratio of CD4+/CD8+ T cells were subsequently determined. RT-qPCR and Western blot assay methods were employed to determine the expressions of IER3, Nrf2, NQO1, IL-6 and IL-8. Finally, tumor cell apoptosis and the volume and weight of the in vivo tumors were evaluated in an attempt to determine the contributory role of As2O3 in combination with BCG immunotherapy in treating bladder cancer. ResultsThe additive effect of As2O3 and BCG was demonstrated to promote the expressions of IL-6/IL-8 among DCs. Additionally, the proportion of CD4+ cells, ratio of CD4+/CD8+ T cells and rate of tumor cell apoptosis were all elevated, while decreased in vivo tumor volume and weight were detected. Of importance, we determined the role that ad-shNrf2 (adenoviral vectors expressing shRNA against Nrf2) played in inhibiting the effects of As2O3 on bladder cancer. ConclusionTaken together, the key findings of the present study provide evidence defining the effect of As2O3 on inducing the inhibitory effect of BCG on the development of bladder cancer via the IER3/Nrf2 pathway, highlighting the potential of As2O3 as a treatment option for bladder cancer through its enhancement of intravesical BCG.

Metastatic cells are preferentially vulnerable to lysosomal inhibition

Molecular alterations that confer phenotypic advantages to tumors can also expose specific therapeutic vulnerabilities. To search for potential treatments that would selectively affect metastatic cells, we examined the sensitivity of lineage-related human bladder cancer cell lines with different lung colonization abilities to chloroquine (CQ) or bafilomycin A1, which are inhibitors of lysosome function and autophagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro to highly metastatic cells compared with their less metastatic counterparts. Genetic inactivation of macroautophagy regulators and lysosomal proteins indicated that this was due to greater reliance on the lysosome but not upon macroautophagy. To identify the mechanism underlying these effects, we generated cells resistant to CQ in vitro. Surprisingly, selection for in vitro CQ resistance was sufficient to alter gene expression patterns such that unsupervised cluster analysis of whole-transcriptome data indicated that selection for CQ resistance alone created tumor cells that were more similar to the poorly metastatic parental cells from which the metastatic cells were derived; importantly, these tumor cells also had diminished metastatic ability in vivo. These effects were mediated in part by differential expression of the transcriptional regulator ID4 (inhibitor of DNA binding 4); depletion of ID4 both promoted in vitro CQ sensitivity and restored lung colonization and metastasis of CQ-resistant cells. These data demonstrate that selection for metastasis ability confers selective vulnerability to lysosomal inhibitors and identify ID4 as a potential biomarker for the use of lysosomal inhibitors to reduce metastasis in patients.

Predicting the tumorigenic phenotype of human bladder cancer cells by combining with fetal rat mesenchyme

BackgroundIn nonmuscle invasive bladder cancer patients, prediction of pTa and pT1 bladder cancer recurrence and progression must be established. Micropapillary structures have been defined as small clusters of invasive cancer cells having features of the epithelial-mesenchymal transition. Since the stromal microenvironment helps to induce the epithelial-mesenchymal transition, interactions between cancer cells and stroma should be closely examined to predict the tumorigenic phenotype of human bladder cancer cells. Materials and methodsTo investigate differences in the responsiveness of cancer cells to stroma, we combined 3 established human bladder cancer cell lines (high-grade T24 and UM-UC-3 cells, and low-grade papillary RT4 cells) with fetal rat mesenchyme. ResultsAmong 3 bladder cancer cell lines, the expression profiles of p63 isoforms were distinct, i.e., p63γ in T24 cells, p63β in UM-UC-3 cells, and p63α in RT4 cells. Tumors formed by T24 cells combined with fetal mesenchyme formed micropapillary-like structures, whereas those formed by T24 cells alone did not. T24 cells combined with fetal mesenchyme showed poor differentiation, e.g., innumerable chromatic atypia in the nuclei, higher levels of chromatic condensation, and increased nucleoli. In contrast, both UM-UC-3 and RT4 cells combined with fetal mesenchyme did not form micropapillary-like structures. Ki-67 and p63 labeling indices were significantly elevated by combining fetal mesenchyme with T24 cells but not with the others. ConclusionsBy mixing cancer cells with fetal mesenchyme, our data demonstrated that formation of micropapillary-like structures may predict the tumorigenic phenotype of invasive bladder cancer cells. Taken together, distinct expression profiles of p63 isoforms may predict poor outcomes in invasive bladder cancer.

Sensitization of cisplatin resistant bladder tumor by combination of cisplatin treatment and co-culture of dendritic cells with apoptotic bladder cancer cells

Dendritic cells (DCs) are a population of professional antigen presenting cells (APCs), serving as central regulators of adaptive immunity by presenting antigens. In addition, DCs play essential roles in the connection of the innate and adaptive immune responses. Recently, therapeutic vaccines turn out to become a viable therapeutic approach for immunotherapy of cancers. Here we report a dendritic cell-based vaccine strategy which could suppress the bladder tumor growth in vivo and improve the efficiency of chemotherapy. In this study, we investigate the antitumor effects of mature DCs induced by antigen loading in bladder tumor in vivo. We demonstrate the co-culture of cisplatin induced apoptotic human bladder cancer cell line, T24 cells with immature DCs could promote the maturation of DCs. Cisplatin and these activated DCs were reintroduced into mice bearing the T24 cisplatin resistant cells-derived tumor growth to activate or boost the immune response. Mice with iDCs co-cultured with apoptotic T24 (iDCs T24 Apo) cells injection effectively initiate a cytotoxic effect against tumor growth and improve the survival rates of mice compared with controls. Moreover, we observed injection of iDCs T24 apoptosis cells combined with cisplatin into mice with T24 cisplatin resistant cancer cells-derived tumor resulted in a statistically significant suppression of tumor growth compared with mice injected with PBS alone, cisplatin alone, iDCs, iDCs T24 Apo cells, cisplatin plus iDCs. This study provides a dendritic cell-based vaccine strategy which might reduce the risk of tumor recurrence and improve the efficiency of anti-chemoresistance of bladder cancer.

MicroRNA-124 inhibits cell proliferation, invasion and migration by targeting CAV1 in bladder cancer.

MicroRNAs (miRs) may have promotive or suppressive roles in various human cancers types, but the molecular mechanisms underlying the role of miR-124 in bladder cancer (BC) progression have remained largely elusive. In the present study, it was observed that miR-124 was significantly downregulated in BC tissues compared with that in adjacent non-neoplastic tissues. Furthermore, its expression was also reduced in several human BC cell lines (T24, HT-1376 and 5637) compared with that in the normal bladder epithelial SV-HUC-1 cell line. A low expression of miR-124 in BC patients was significantly associated with advanced malignancy and a poor prognosis. Caveolin 1 (CAV1) was identified as a novel target gene of miR-124, and the expression of CAV1 was negatively regulated by miR-124 in T24 cells. Furthermore, CAV1 was identified to be significantly upregulated in BC tissues and cell lines, and a negative correlation was observed between the expression of miR-124 and CAV1 in BC tissues. Furthermore, restoration of miR-124 expression significantly inhibited the proliferation, migration and invasion of T24 cells, and these effects were impaired following overexpression of CAV1. Taken together, the present results demonstrate that miR-124 has a suppressive role in the proliferation, migration and invasion of BC cells by targeting CAV1, which suggests that miR-124 is a potential therapeutic candidate for BC.

Phenformin alone or combined with gefitinib inhibits bladder cancer via AMPK and EGFR pathways

BackgroundIn previous studies, we have shown that the combination of metformin and gefitinib inhibits the growth of bladder cancer cells. Here we examined whether the metformin analogue phenformin, either used alone or in combination with gefitinib, could inhibit growth of bladder cancer cells.MethodsThe growth-inhibitory effects of phenformin and gefitinib were tested in one murine and two human bladder cancer cell lines using MTT and clonogenic assays. Effects on cell migration were assessed in a wound healing assay. Synergistic action between the two drugs was assessed using CompuSyn software. The potential involvement of AMPK and EGFR pathways in the effects of phenformin and gefitinib was explored using Western blotting.ResultsIn MTT and clonogenic assays, phenformin was > 10-fold more potent than metformin in inhibiting bladder cancer cell growth. Phenformin also potently inhibited cell migration in wound healing assays, and promoted apoptosis. AMPK signaling was activated; EGFR signaling was inhibited. Phenformin was synergistic with gefitinib, with the combination of drugs showing much stronger anticancer activity and apoptotic activation than phenformin alone.ConclusionsPhenformin shows potential as an effective drug against bladder cancer, either alone or in combination with gefitinib.

MKAD-21 Suppresses the Oncogenic Activity of the miR-21/PPP2R2A/ERK Molecular Network in Bladder Cancer.

Bladder cancer represents a disease associated with significant morbidity and mortality. MiR-21 has been found to have oncogenic activity in multiple cancers, including bladder cancer, whereas inhibition of its expression suppresses tumor growth. Here, we examine the molecular network regulated by miR-21 in bladder cancer and evaluate the effects of i.v. and i.p. administration of a novel miR-21 chemical inhibitor in vivo LNA miR-21 reduced the oncogenic potential of bladder cancer cells, whereas the MKAD-21 chemically modified antisense oligo against miR-21 dose-dependently blocked xenograft growth. I.v. administration of LNA miR-21 was more effective in suppressing tumor growth than was i.p. administration. Integration of computational and transcriptomic analyses in a panel of 28 bladder cancer lines revealed a 15-gene signature that correlates with miR-21 levels. Protein Phosphatase 2 Regulatory Subunit Balpha (PPP2R2A) was one of these 15 genes and was experimentally validated as a novel miR-21 direct target gene. Gene network and molecular analyses showed that PPP2R2A is a potent negative regulator of the ERK pathway activation and bladder cancer cell proliferation. Importantly, we show that PPP2R2A acts as a mediator of miR-21-induced oncogenic effects in bladder cancer. Integrative analysis of human bladder cancer tumors and a large panel of human bladder cancer cell lines revealed a novel 15-gene signature that correlates with miR-21 levels. Importantly, we provide evidence that PPP2R2A represents a new miR-21 direct target and regulator of the ERK pathway and bladder cancer cell growth. Furthermore, i.v. administration of the MKAD-21 inhibitor effectively suppressed tumor growth through regulation of the PPP2R2A-ERK network in mice. Mol Cancer Ther; 17(7); 1430-40. ©2018 AACR.

Combination chemotherapy with Zyflamend reduced the acquired resistance of bladder cancer cells to cisplatin through inhibiting NFκB signaling pathway.

BackgroundCisplatin-based chemotherapy is mainstay treatment in urinary bladder cancer (UBC). However, tumor recurrence frequently occurs with the acquisition of cisplatin resistance. We explored the potential effect of a polyherbal preparation, Zyflamend, on UBC cells resistant to cisplatin treatment.MethodsTo establish a cisplatin-resistant human bladder cancer cell line, T24 cells were cultured in increasing concentrations of cisplatin for more than 10 months. These cells (T24R) were then treated with different concentrations of Zyflamend, and both proliferation and activity of nuclear factor kappaB (NFκB) signaling pathway were examined. To test the synergistic effect between Zyflamend and cisplatin, we treated T24R cells either with Zyflamend or cisplatin alone, or in combination. Apoptotic effect was evaluated by Annexin V/propidium iodide double staining, and the levels of the proteins involved in cell cycle and anti-apoptosis were examined by Western blotting. Finally, mice with palpable xenograft were treated either with cisplatin and Zyflamend alone or in combination for 28 days before they were sacrificed for measuring the sizes and weights of the tumor tissues. In addition, proliferation and apoptosis markers were examined by immunohistochemistry.ResultsComparing to that in the parental T24 cells, NFκB is constitutively active in cisplatin-resistant T24R cells. Zyflamend is capable of inhibiting the growth of T24, T24R, as well as another UBC cell line J82 in a concentration-dependent manner. Mechanistically, Zyflamend suppresses NFκB-mediated cell proliferation, survival, and invasion/angiogenesis and induces apoptosis. In addition, Zyflamend significantly increased the sensitivity of T24R and J82 cells to cisplatin treatment and these findings were confirmed in T24R xenograft model with reduced proliferation index and decreased expression of RelA and its downstream target MMP9.ConclusionZyflamend is capable of counteracting bladder cancer resistance to cisplatin by repressing proliferation and inducing apoptosis through targeting NFκB signaling pathway.

Abstract 499: MicroRNA-411 downregulation promotes tumorigenesis and inhibits chemotherapy sensitivity by upregulating MLLT11 expression in human bladder cancer

Abstract Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in BC carcinogenesis and anti-cancer therapy are not explored yet at present. Here, we screened 1900 known miRNAs and first discovered that miR-411 was one of major miRNAs that was downregulated in a n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BC mouse model. This miR-411 downregulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA database also indicated that miR-411 was positively correlated with survival (>3 years). Our studies also showed that miR-411 inhibited tumorigenesis and increased chemotherapy sensitivity in human BC. Mechanistic studies revealed that overexpression of miR-411 repressed expression of ALL1-fused gene from the chromosome 1q (MLLT11) by binding to the 3′-untranslated region of MLLT11 mRNA, induced p21 expression, and caused cell cycle arrest at the G2/M phase, in turn inhibiting BC tumorigenic growth. Ectopic expression of miR-411 also increased the sensitivity of BC cells to the chemotherapeutic agents, whereas MLLT11 restoration in miR-411-overexpressing cells increased resistance to the chemotherapeutic agents. Citation Format: Honglei Jin, Haishan Huang, Chuanshu Huang, Xiaohui Hua, Junlan Zhu. MicroRNA-411 downregulation promotes tumorigenesis and inhibits chemotherapy sensitivity by upregulating MLLT11 expression in human bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 499.

Synthesis, Characterization and In Vitro Evaluation of Carbohydrate‐Containing Ruthenium Phthalocyanines as Third Generation Photosensitizers for Photodynamic Therapy

AbstractPyridyl ligands with one ketal‐protected carbohydrate unit (glucofuranose, galactopyranose or mannofuranose) were coordinated to the axial positions of ruthenium phthalocyanines (RuPc) in order to target the macrocycles to tumor cells. The biocompatibility of the resulting photosensitizers (PS) was enhanced by increasing their solubility in water through their peripheral and/or axial functionalization with polyether chains (PEG). All compounds generated singlet oxygen, with peripherally unsubstituted phthalocyanines displaying higher 1O2 yields. In vitro assays using the human bladder cancer cell line HT‐1376 revealed that peripherally unsubstituted phthalocyanines were also more efficient in terms of cellular uptake and therefore, phototoxicity. The protected carbohydrate functional groups at the axial positions did not improve significantly the cellular uptake of PSs, although they increased their photodynamic efficiency. Overall, mixed RuPcs functionalized with one carbohydrate unit at one of the axial positions and two PEG units at the other axial site showed promising properties as photosensitizers for photodynamic therapy applications and low toxicity in the dark.

LSINCT5 activates Wnt/β-catenin signaling by interacting with NCYM to promote bladder cancer progression

Accumulating evidence has indicated that long non-coding RNAs (lncRNAs) are critically involved in tumor progression. In current study, we reported a novel lncRNA signature correlated with bladder cancer development. Particularly, the lncRNA long stress-induced noncoding transcript 5 (LSINCT5) is significantly upregulated in human bladder cancer cell lines and tumor specimens. Meanwhile, high LSINCT5 expression correlates with poor prognosis, enhances tumor sphere formation and invasion in vitro. In vivo xenograft tumor growth is also elevated by LSINCT5 overexpression. Mechanistic investigations showed that LSINCT5 could physically interact with NCYM, a de novo gene product from the MYCN cis-antisense RNA and inhibit GSK3β activity leading to enhanced Wnt/β-catenin signaling activation and epithelial mesenchymal transition (EMT). Taken together, our findings have created a novel LSINCT5 mediated process which facilitates bladder cancer progression and may provide a potential target for therapeutic intervention.

TRAIL attenuates sulforaphane-mediated Nrf2 and sustains ROS generation, leading to apoptosis of TRAIL-resistant human bladder cancer cells

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can preferentially initiate apoptosis in malignant cells with minimal toxicity to normal cells. Unfortunately, many human cancer cells are refractory to TRAIL-induced apoptosis through many unknown mechanisms. Here, we report that TRAIL resistance can be reversed in human bladder cancer cell lines by treatment with sulforaphane (SFN), a well-known chemopreventive isothiocyanate in various cruciferous vegetables. Combined treatment with SFN and TRAIL (SFN/TRAIL) significantly induced apoptosis concomitant with activation of caspases, loss of mitochondrial membrane potential (MMP), Bid truncation, and induction of death receptor 5. Transient knockdown of Bid prevented collapse of MMP induced by SFN/TRAIL, consequently reducing apoptotic effects. Furthermore, SFN increased both the generation of reactive oxygen species (ROS) and the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is an anti-oxidant enzyme. Interestingly, TRAIL effectively suppressed SFN-mediated nuclear translocation of Nrf2, and the period of ROS generation was more extended compared to that of treatment with SFN alone. In addition, silencing of Nrf2 increased apoptosis in cells treated with SFN/TRAIL; however, blockade of ROS generation inhibited apoptotic activity. These data suggest that SFN-induced ROS generation promotes TRAIL sensitivity and SFN can be used for the management of TRAIL-resistant cancer.

Paeoniflorin inhibits the growth of bladder carcinoma via deactivation of STAT3.

Bladder cancer (BCa) is one of the most common urinary cancers. The present study aims to investigate whether Paeoniflorin (Pae) can exert inhibitory effects on BCa. The results showed that Pae inhibited proliferation of human BCa cell lines in a concentration- and time-dependent manner. Pae and cisplatin (Cis) synergistically inhibited the growth of tumours in RT4-bearing mice. Pae treatment neutralized the body loss induced by Cis. Moreover, Pae induced apoptosis in RT4 cells and increased the activities of caspase3, caspase8 and caspase9. Western blotting and immunohistochemical analysis revealed that the phosphorylated signal transducer and activator of transcription-3 (p-STAT3) level were decreased in Pae-treated RT4 cells and Pae-treated tumour-bearing mice. Furthermore, STAT3 transcriptional target B-cell lymphoma-2 was decreased in Pae-treated RT4 cells. Interestingly, Pae prevented translocation of STAT3 to the nucleus in RT4 cells. Collectively, Pae inhibits the growth of BCa, at least in part, via a STAT3 pathway.

Gamma-Klotho exhibits multiple roles in tumor growth of human bladder cancer

Alpha-Klotho (KLα) and beta-Klotho (KLβ) have recently been reported to correlate with cancer prognosis in some malignancies and we previously reported the association between KLα, KLβ, and urothelial carcinoma of the bladder (UCB), indicating that KLβ acts as a tumor promoter. However, the association between gamma-Klotho (KLγ) and cancer prognosis remains unclear. In the present study, we evaluated the association between KLγ and UCB. To evaluate the effect of KLγ on human bladder cancer cell lines in vitro assays were performed. Exogenous KLγ increased the ability of human bladder cancer cells to proliferate, migrate, invade, form colonies, and provide anchorage-independent growth potential. In in vivo assays, eighteen mice bearing xenografts inoculated using UM-UC-3, were randomly divided into three groups and treated with a small interfering RNA (siRNA) by intratumoral administration once a week for four weeks. Knockdown of KLγ with siRNA led to a dramatic change in tumor growth and suggested that KLγ had effects on tumor growth, including promotion of cell proliferation, inhibition of apoptosis, and enhancement of the epithelial-mesenchymal transition. To confirm the study, human tissue samples were used and patients were divided into two groups according to KLγ expression level. High expression of KLγ was significantly associated with higher stage and grade cancer and the presence of lymphovascular invasion compared to patients with lower expression of KLγ. Our results suggest that KLγ plays an important role in tumor invasion and progression and these results may lead to the development of new therapies and diagnostic methods for UCB.

Bacillus Calmette-Guérin induces rapid gene expression changes in human bladder cancer cell lines that may modulate its survival.

Bacillus Calmette-Guérin (BCG) immunotherapy is the standard therapy for non-muscle invasive bladder cancer. The aim of the present study was to identify genes that are induced in response to BCG immunotherapy, as these may be potential biomarkers for the response to clinical therapy. To model clinical therapy, human bladder cancer cell lines were incubated with BCG (live or lyophilized BCG Connaught) for 2 h. RNA was extracted and evaluated by Representational Differential Analysis (RDA) and oligo arrays. Gene expression was confirmed by reverse transcription polymerase chain reaction on fresh cell lines with differential abilities to internalize BCG. The effect of 2 major BCG soluble proteins, antigen 85B (Ag85B) and Mycobacterium protein tyrosine phosphatase A (MptpA) and BCG Tice® on gene expression was also determined. GAPDH and β-actin, which are normally used as control genes, were upregulated by BCG. Therefore, the ribosomal RNA gene ribosomal protein S27a was used to normalize gene expression. The genes likely to be induced by BCG internalization and soluble factors were: GSTT2, MGST2, CCL20, TNFα, CCNE1 and IL10RB. Those induced by BCG membrane interactions and/or soluble factors were: MGST1, CXCL6, IL12A, CSF2, IL1β and TOLLIP. MptpA decreased GSTT2 expression, and Ag85B increased TNFα expression. The two BCG strains significantly increased GSTT2, TNFα and TOLLIP levels in MGH cells. However, in J82 cells there was a BCG strain-dependent difference in TNFα expression. An important outcome of the present study was the determination that neither GAPDH nor β-actin were suitable control genes for the analysis of BCG-induced gene expression. BCG Connaught and Tice® induced similar expression levels of genes in bladder cancer cell lines. BCG soluble proteins modulated gene expression and therefore may affect therapeutic outcomes. The genes identified may be novel biomarkers of the response to BCG therapy.

Effects of HDAC inhibitor Vorinostat and radiation on HTB4 and MDA‐MB‐435 cancer cells

HDAC inhibitors have shown promise in the treatment of various types of cancer. The HDAC inhibitor vorinostat was first introduced during treatments for cutaneous T‐cell lymphoma and has been shown to cause an increase in the effectiveness of radiation on multiple cancer cell lines. For the purpose of these experiments, the effects of vorinostat and radiation on HTB4 cells (a human bladder cancer cell line) and MDA‐MB‐435 cells (a human melanoma cell line) are being investigated in order to determine the efficacy of this combined treatment. Using cell cycle analysis and MTT assays, we were able to analyze the effects of radiation and vorinostat alone compared to the combination of both. For cell cycle analysis, we plated the cells in four flasks and treated three of them with either vorinostat (2.5 μM) alone, radiation (~20Gy) alone or the combination of the two. The cells were harvested, stained and analyzed five hours after radiation and 24 hours after vorinostat treatment by flow cytometry. Preliminary results for the HTB4 cells treated with both radiation and vorinostat showed an increase in the percent of cells in G2 and a reduction in the percent of those in both G1 and S phase compared to either treatment alone or the control population. Results for the 435's did not show a similar trend with distribution of cells between the control and three treatments. The MTT assay was performed using two 96‐well microtiter plates with 16μM vorinostat serially diluted 1:2 across 10 columns, and only cells in the 11th column and medium alone in the 12th column. One plate was irradiated using a lower dose of 5 Gy based on a literature survey. MTT was added to both plates directly after the one plate was irradiated. Results of the MTT assay for HTB4's and 435's showed that increasing concentrations of vorinostat alone and when coupled with radiation resulted in less MTT product produced compared to the radiation treatment alone. Based on the MTT data, the HTB4 cell line appears to be more sensitive than the 435's to the effects of vorinostat. Future experiments will focus on the effects of radiation on multiple tumor cell lines and investigate the molecular mechanisms by which HDAC inhibition influences radiation‐based cancer treatment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2018MP65-09 NEAR INFRARED PHOTOIMMUNOTHERAPY USING ANTI-CD47 ANTIBODY FOR HUMAN BLADDER CANCER Bernhard Kiss, Kelly McKenna, Robert Ertsey, Nynke Sjoerdtje Van den Berg, Kathleen Mach, Eben Rosenthal, and Joseph Liao Bernhard KissBernhard Kiss More articles by this author , Kelly McKennaKelly McKenna More articles by this author , Robert ErtseyRobert Ertsey More articles by this author , Nynke Sjoerdtje Van den BergNynke Sjoerdtje Van den Berg More articles by this author , Kathleen MachKathleen Mach More articles by this author , Eben RosenthalEben Rosenthal More articles by this author , and Joseph LiaoJoseph Liao More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2075AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Near infrared photoimmunotherapy (PIT) is a new molecular-targeted cancer therapy that uses a specific photosensitizer based on a near-infrared (NIR) phthalocyanine dye, IRDye700DX, conjugated to a monoclonal antibody. CD47 is a cell surface protein that elicits a ″don′t eat me signal″ to prevent macrophage engulfment and is expressed on over 80% of bladder cancer tumors but absent on the luminal cell layer of normal bladder urothelium. Thus, targeting CD47 for PIT has the potential to selectively induce cell death in CD47 expressing tumor cells as well as increase phagocytosis of tumor cells. METHODS The anti-CD47 antibody (B6H12) was conjugated to IRDye700DX NHS ester and purified using a dialysis spin column. Anti-CD47-IRDye700 concentration was determined with a Coomassie Plus protein assay. The molecular weight of the conjugated antibody was verified by SDS-page and IRDye700 conjugation verified using the 700nm fluorescence channel of a PEARL imager. Binding of anti-CD47-IRDye700DX to CD47 was verified by ELISA. Three different human urothelial cell lines (UMUC3, HT-1376 and 639V) were used for in vitro NIR-PIT experiments. 639V cells were stably transfected with GFP using a lentiviral vector. After plating in a 96well plate and incubation with 10 µg/ml of anti-CD47-IRDye700 or unconjugated anti-CD47 antibody (1h on ice), cells were irradiated with a red light-emitting diode (LED) at 670-710nm wavelength at different energy levels. The cytotoxic effects of NIR-PIT with anti-CD47-IRDye700DX were determined by propidium iodide staining and flow cytometry. To evaluate macrophage activity, a phagocytosis assay using human macrophages and analyzed by flow cytometry was performed. RESULTS Cancer-cell death significantly increased with anti-CD47-IRDye700DX in a light-dose dependent manner in all 3 human bladder cancer cell lines. Significant cancer-cell death was first observed with 2J exposure and when exposed to 40J of NIR light over 90% of 639V and UMUC3 cells and over 50% of HT1376 died. Phagocytosis of cancer-cells incubated with anti-CD47-IRDye700DX and irradiated with 8J was significantly increased compared to anti-CD47-antibody alone (p=0.0002). There was no cytotoxicity associated with anti-CD47 antibody alone with NIR light, with NIR light alone or with anti-CD47-IRDye700DX without NIR light. CONCLUSIONS NIR-PIT with anti-CD47-IRDye700 shows significantly increased cancer-cell cytotoxicity and significantly increased cancer-cell phagocytosis making this combination a highly promising novel approach for molecular targeted bladder cancer therapy. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e862 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Bernhard Kiss More articles by this author Kelly McKenna More articles by this author Robert Ertsey More articles by this author Nynke Sjoerdtje Van den Berg More articles by this author Kathleen Mach More articles by this author Eben Rosenthal More articles by this author Joseph Liao More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lentiviral Vector-mediated Gene Transfer in Human Bladder Cancer Cell Lines.

Currently, treatment of non-muscle invasive bladder cancer causes significant deterioration in a patient's quality of life (QOL). Therefore, development of novel therapeutic options without the deterioration of QOL is very important. In this study, we assessed the anti-tumor effect of lentivirus-mediated gene transfection of tumor-suppressor genes in human bladder cancer cells. Lentiviral vectors that contained the tumor suppressor genes, p53, p16, and PTEN, were transfected into human bladder cancer cell lines, 5637, T24, 253J, and UMUC3, and the normal human uroepithelial cell line, SV-HUC-1. Significant growth inhibition was observed in bladder cancer cells on transfection with the p16 and PTEN vectors. However, the effect of the p53 vector was limited. In normal cells, the lentiviral vectors did not exhibit a significant growth inhibitory effect. Lentiviral vector-mediated gene transfection is useful for the application of gene therapy in bladder cancers.