Urinary Bladder Cancer Cell Lines Research Articles

MANNosylation of Mesoporous Silica Nanoparticles Modifies TLR4 Localization and NF-κB Translocation in T24 Bladder Cancer Cells.

D-mannose is widely used as non-antibiotic treatment for bacterial urinary tract infections. This application is based on a well-studied mechanism of binding to the type 1 bacterial pili and, therefore, blocking bacteria adhesion to the uroepithelial cells. To implement D-mannose into carrier systems, the mechanism of action of the sugar in the bladder environment is also relevant and requires investigation. Herein, two different MANNosylation strategies using mesoporous silica nanoparticles (MSNs) are described. The impact of different chemical linkers on bacterial adhesion and bladder cell response is studied via confocal microscopy imaging of the MSN interactions with the respective organisms. Cytotoxicity is assessed and the expression of Toll-like receptor 4 (TLR4) and caveolin-1 (CAV-1), in the presence or absence of simulated infection with bacterial lipopolysaccharide(LPS), is evaluated using the human urinary bladder cancer cell line T24. Further, localisation of the transcription factor NF-κB due to the MANNosylated materials is examined over time. The results show that MANNosylation modifies bacterial adhesion to the nanomaterials and significantly affects TLR4, caveolin-1, and NF-κB in bladder cells. These elements are essential components of the inflammatory cascade/pathogens response during urinary tract infections. These findings demonstrate that MANNosylation is a versatile tool to design hybrid nanocarriers for targeted biomedical applications.

Novel imino-thiazoloquinoxaline derivatives against renal cell carcinoma: less radiation-damaging approach

Renal cell carcinoma (RCC) is the most fatal tumor in the urinary system. Resistance development and unmet effective responses, request new anticancer agents with better therapeutic index. Ten new imino-thiazolo-quinoxaline derivatives (5a-j) were synthesized and preliminary evaluated for downregulation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) activity taking sorafenib as a reference drug. Compounds 5d & 5h showed potent inhibition to VEGFR-2 activity at IC50 89.35 nM & 60.64 nM, respectively, then they both were further evaluated in-vitro against urinary bladder cancer cell line T-24 taking sorafenib as a reference drug. Compound 5h displayed nearly anticancer activity to sorafenib against T-24 cell line in all concentrations tested except at concentration 10 µM where it highly suppressed cell viability to 6.71 % compared to 15.15% of sorafenib. Compound 5h was then evaluated for its ameliorative efect against radiation induced renal tissue injury. Assessment of pro-angiogenic (VEGFR-2), pro-fibrotic (transforming growth factor-beta 1 (TGF-β1)) and apoptotic (caspase-3) markers, as well as histopathological examinations were performed on kidney of irradiated mice. Results showed ability of compound 5h to downregulate VEGFR-2 activity and its cytotoxic effect against RCC, in addition to mitigation of radiation induced renal tissue injury. Ethyl imino-thiazoloquinoxaline carboxylate derivative 5h showed a potential cytotoxic activity against RCC and could be considered a promosing alleviative candidate when employed post radiotherapy regimen.Graphical

MiR-221-5p and miR-186-5p Are the Critical Bladder Cancer Derived Exosomal miRNAs in Natural Killer Cell Dysfunction

Bladder cancer (BC) is the tenth most commonly diagnosed cancer worldwide, and its carcinogenesis mechanism has not been fully elucidated. BC is able to induce natural killer (NK) cell dysfunction and escape immune surveillance. The present study found that exosomes derived from the urinary bladder cancer cell line (T24 cell) contribute in generating NK cell dysfunction by impairing viability, and inhibiting the cytotoxicity of the NK cell on target cells. Meanwhile, T24 cell-derived exosomes inhibited the expression of the important functional receptors NKG2D, NKp30, and CD226 on NK cells as well as the secretion of perforin and granzyme-B. The critical miRNAs with high expression in T24 cell-derived exosomes were identified using high-throughput sequencing. Furthermore, following dual-luciferase reporter assay and transfection experiments, miR-221-5p and miR-186-5p were confirmed as interfering with the stability of the mRNAs of DAP10, CD96, and the perforin gene in NK cells and may be potential targets used in the therapy for BC.

Antitumour Activity of Different Bioactive Compounds from Hemolymph and Mucus of Mollusca against Human Urinary Bladder Cancer Cell Lines

In this study, we isolated different bioactive compounds and fractions from Rapana venosa (RvH), Helix aspersa (HaH) and Helix lucorum (HlH) and then we evaluated their in vitro antitumour activity on CAL-29 and T- 24 cell lines (transitional cell carcinoma). Also, we performed fluorescence staining with propidium iodide to assess nuclear changes caused by molluscan extracts. Briefly, our results have demonstrated antineoplastic activity of: subunit βc-HaH of H. aspersa hemocyanins to CAL-29 cell line, subunit βc-HaH and subunit RvH II of R. venosa hemocyanin against T-24 cells. Also, fractions from R. venosa hemolymph exerted significant cytotoxic activity against CAL- 29 cells. Furthermore, fluorescence microscopy results demonstrated chromatin condensation and nuclear morphological changes which are typical for apoptosis induction. We assume that the observed death of T-24 and CAL-29 cells is due to the interaction between the oligosaccharide structures of the proteins on the tumour cell membranes and the hemolymph from R. venosa, active components isolated from hemolymph of R. venosa, H. aspersa and H. lucorum, as well as H. aspersa mucus.

Evaluation of the anti-proliferative activity of violacein, a natural pigment of bacterial origin, in urinary bladder cancer cell lines.

Violacein is a natural pigment, a pyrrolidone, and a bisindole derived from the condensation of two tryptophan molecules, which gives a blue violet color to several gram-negative violacein-producing bacteria. Violacein production provides a competitive advantage against antagonistic species or predators. In addition, the compound has antibacterial, antifungal, antiviral, and antioxidant activities. Several studies on colon, breast, and head and neck cancer lines have already demonstrated the anti-proliferative potential of violacein. Bladder cancer is one of the most common types of cancer in urology. The therapeutic approach is mainly based on surgery, chemotherapy, and immunotherapy. The aim of the present study was to evaluate the anti-proliferative activity of violacein against the human bladder cancer cell lines, HTB4 (T24) and HTB9 (5637), using low-grade and high-grade transitional cell carcinoma models, respectively, which has never been assayed before. For this purpose, the potential violacein anti-proliferative effect on T24 and 5637 cells was evaluated by studying the cell viability, proliferation, cell cycle, and caspase-3 activation. The results showed that violacein had anti-proliferative activity in the two cell lines, which was greater for the second-stage bladder cancer cell line (5637), and a different mode of action against the two cell lines.

Supplementation of Probiotic Butyricicoccus pullicaecorum Mediates Anticancer Effect on Bladder Urothelial Cells by Regulating Butyrate-Responsive Molecular Signatures.

In bladder cancer, urothelial carcinoma is the most common histologic subtype, accounting for more than 90% of cases. Pathogenic effects due to the dysbiosis of gut microbiota are localized not only in the colon, but also in regulating bladder cancer distally. Butyrate, a short-chain fatty acid produced by gut microbial metabolism, is mainly studied in colon diseases. Therefore, the resolution of the anti-cancer effects of butyrate-producing microbes on bladder urothelial cells and knowledge of the butyrate-responsive molecules must have clinical significance. Here, we demonstrate a correlation between urothelial cancer of the bladder and Butyricicoccus pullicaecorum. This butyrate-producing microbe or their metabolite, butyrate, mediated anti-cancer effects on bladder urothelial cells by regulating cell cycle, cell growth, apoptosis, and gene expression. For example, a tumor suppressor against urothelial cancer of the bladder, bladder cancer-associated protein, was induced in butyrate-treated HT1376 cells, a human urinary bladder cancer cell line. In conclusion, urothelial cancer of the bladder is a significant health problem. To improve the health of bladder urothelial cells, supplementation of B. pullicaecorum may be necessary and can further regulate butyrate-responsive molecular signatures.

Royal Jelly Decreases MMP-9 Expression and Induces Apoptosis in Human Bladder Cancer 5637 Cells

Royal jelly (RJ) from queen honeybee larva as a traditional medicine agent has a variety of pharmacological benefits. In the present study, the effect of Royal jelly was investigated on the urinary bladder cancer cell line (HTB-9 5637). To determine the cell viability in different concentrations of Royal jelly, MTT assay was performed. An in vitro wound healing assay was applied to investigate the effect of RJ on cell migration. The activity and gene expression level of matrix metalloproteinase 2 and 9 was assessed by zymography and Real time PCR respectively. R.J.S at the concentration of 0.7 mg/ml had a significant effect on reducing the proliferation rate of 5637 cells after 72h (p < 0.009). R.J.S significantly decreased cell migration and induced a significant decrease in the transcriptional level of MMP9 after 72h (0.5x; P < 0.049). However R.J.S did not impose any effect on the expression level and activity of matrix metalloproteinase 2. These results indicate the potential of RJ as a promised natural anti-proliferative and anti-metastatic drug in combination with advanced therapy methods for cancer treatment. Royal jelly has the potential to be more focused as an anti-metastatic drug to control tumor growth and can be considered as a more effective alternative to the current chemotherapy drugs.

CircCA12 Promotes Malignant Process via Sponging miR-1184 and Upregulating RAS Family in Bladder Cancer.

Circular RNAs (circRNAs) are a panel of non-coding RNAs that mediate the regulation of gene expression, as well as pathological responses. Nonetheless, the function and expression pattern of circRNAs in urinary bladder cancer (UBC) remain unclear. Herein, we examined the function of circCA12 in UBC development. qRT-PCR results demonstrated remarkable circCA12 upregulation in UBC cell lines, as well as tissues. CCK-8, colony formation, and xenograft assays were employed to determine the effect of circCA12 on UBC. Our data illustrated silencing circCA12 repressed the proliferation along with the colony-formation capability of UBC cells. The migration and metastasis potential of UBC cells were remarkably abated in vivo, as well as in vitro after transfection with si-cirCA12 or sh-circCA12. Moreover, luciferase reporter and RIP assays indicated that circCA12 binds to miRNA-1184 through sponging miRNA, thereby up-regulating the expression of RAS family genes (NRAS, KRAS, and HRAS). In conclusion, the circCA12/miRNA-1184/RAS family was identified as a regulatory axis in UBC progression.

Centromere protein U enhances the progression of bladder cancer by promoting mitochondrial ribosomal protein s28 expression.

Bladder cancer is one of the most common types of cancer. Most gene mutations related to bladder cancer are dominantly acquired gene mutations and are not inherited. Previous comparative transcriptome analysis of urinary bladder cancer and control samples has revealed a set of genes that may play a role in tumor progression. Here we set out to investigate further the expression of two candidate genes, centromere protein U (CENPU) and mitochondrial ribosomal protein s28 (MRPS28) to better understand their role in bladder cancer pathogenesis. Our results confirmed that CENPU is up-regulated in human bladder cancer tissues at mRNA and protein levels. Gain-of-function and loss-of-function studies in T24 human urinary bladder cancer cell line revealed a hierarchical relationship between CENPU and MRPS28 in the regulation of cell viability, migration and invasion activity. CENPU expression was also up-regulated in in vivo nude mice xenograft model of bladder cancer and mice overexpressing CENPU had significantly higher tumor volume. In summary, our findings identify CENPU and MRPS28 in the molecular pathogenesis of bladder cancer and suggest that CENPU enhances the progression of bladder cancer by promoting MRPS28 expression.

Antiproliferative and apoptosis inducing effect of delphinidin against human bladder cancer cell line

Background: Urinary bladder cancer is one of the leading causes of death worldwide. This study was conducted to evaluate the effect of delphinidin on proliferation, apoptosis, and cell cycle arrest in the urinary bladder cancer cell line (T24). Materials and Methods: We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to analyze the cytotoxic effect of delphinidin on T24 cell proliferation. We tested different concentrations of delphinidin. Furthermore, the effect of delphinidin on cell cycle arrest was studied using flow cytometric assays and the percentage of apoptotic cell death was estimated via annexin V/propidium iodide. Results: According to our results, delphinidin (10–60 μg/mL) demonstrated a significant cytotoxic effect against T24 cells. Morphological analysis and analysis of the oxidative stress revealed a significant number of dead cells in comparison to the control group. The results of flow cytometry revealed that delphinidin caused dose-dependent apoptosis of T24 cells. Conclusion: In summary, delphinidin can be a promising option for the treatment of urinary bladder cancer in the future.

Quantitative Proteomics of Urinary Bladder Cancer Cell Lines Identify UAP1 as a Potential Therapeutic Target.

Bladder carcinoma (BC) incidence and mortality rates are increasing worldwide. The development of novel therapeutic strategies is required to improve clinical management of this cancer. Aberrant protein expression may lead to cancer initiation and progression. Therefore, the identification of these potential protein targets and limiting their expression levels would provide alternative treatment options. In this study, we utilized a liquid-chromatography tandem mass spectrometry-based global proteomics approach to identify differentially expressed proteins in bladder cancer cell lines. A total of 3913 proteins were identified in this study, of which 479 proteins were overexpressed and 141 proteins were downregulated in 4 out of 6 BC cell lines when compared with normal human urothelial cell line (TERT-NHUC). We evaluated the role of UDP-N-acetylhexosamine pyrophosphorylase (UAP1) in bladder cancer pathogenesis. The silencing of UAP1 led to reduction in proliferation, invasion, colony formation and migration capability of bladder cancer cell lines. Thus, our study reveals UAP1 as a promising therapeutic target for bladder cancer.

A new method for purifying Brazilin from lignum sappan – Cytotoxic and anti-proliferative effects in cell lines and improved survival in mice bearing urinary bladder carcinoma

Background: Urinary bladder carcinoma is characterized by high post-operative recurrence rates. Sufu'ning lotion, prepared with Lignum Sappan, can reduce the recurrence of urinary bladder carcinoma. Objectives: The present study describes a methodology for extracting and obtaining high purity brazilin from Lignum sappan and evaluates its effects on urinary bladder carcinoma, in vitro and in vivo . Materials and Methods: The brazilin was extracted from Lignum sappan, purified and identified. The purified brazilin was assessed in vitro for its effects on tumor cell viability and growth using human (T24) and mouse urinary bladder (BTT) cancer cell lines. The role of brazilin in the inhibition of the cell cycle was determined by flow cytometry. The in vivo activity of brazilin was assessed based on the survival of tumor-bearing mice. Results: Brazilin was purified of 99.10% purity and was bioactive as it significantly (P

Structure-activity relationship studies and in vitro and in vivo anticancer activity of novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies

Novel 3-aroyl-1,4-diarylpyrrole derivatives were synthesized to explore structure-activity relationships at the phenyls at positions 1 and 4 of the pyrrole. The presence of amino phenyl rings at positions 1 and 4 of the pyrrole ring were found to be a crucial requirement for potent antitumor activity. Several compounds strongly inhibited tubulin assembly through binding to the colchicine site. Compounds 42, 44, 48, 62 and 69 showed antitumor activity with low nanomolar IC50 values in several cancer cell lines. Compound 48 was generally more effective as an inhibitor of glioblastoma, colorectal and urinary bladder cancer cell lines; 69 consistently inhibited CML cell lines and demonstrated superiority in nilotinib and imatinib resistant LAMA84-R and KBM5-T315I cells. In animal models, compound 48 exhibited significant inhibition of the growth of T24 bladder carcinoma and ES-2 ovarian clear cell carcinoma tumors. Compounds 48 and 69 represent robust lead compounds for the design of new broad-spectrum anticancer agents active in different types of solid and hematological tumors.

Abstract 1421: Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells

Abstract INTRODUCTION AND OBJECTIVES: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human γδT cell immunotherapy. We herein provide new strategies for the enhancement of γδT cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of γδT cells. METHODS: The surface expression of NKG2D, TCRVγ9, TCRVδ2 and the intracellular levels of perforin and Granzyme B in γδT cells were examined. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of Carboxyfluorescein diacetate succinimidyl ester (CFSE) /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human γδT cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded γδT cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS). RESULTS: Ex vivo-expanded γδT cell expressed NKG2D receptor on the cell surface and both TCRVγ9, TCRVδ2 positive lineage were well expanded. Activation of ex vivo-expanded γδT cell was confirmed by intracellular staining of granules, containing perforin and Granzyme B. We examined the synergistic effects of anticancer agents and γδT cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which γδT cells use to recognize cancer cells and resulted in the increased cytotoxicity of γδT cells. The cytotoxicity was abrogated by induction of siRNA against MICA/B. These findings were also confirmed in MICA/B or NKG2D blocking experiments. We also found that intravesical administration of γδT cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of γδT cells in vivo. CONCLUSIONS: These results indicated that γδT cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC. Citation Format: Teruki Shimizu, Mako Tomogane, Yusuke Sano, Daiki Shimizu, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara. Low dose gemcitabine increases the sensitivity of human Vgamma9Vdelta2T cell mediated cytotoxicity through NKG2D-NKG2D ligands axis in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1421.

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.

Hsa-microRNA-101-3p expression in human urinary bladder cancer: Correlation with tumor pathology

IntroductionMicroRNAs (miRs) are a class of small, conserved, non-protein-coding RNAs that regulate gene expression by controlling translation of target messenger RNA. In cancer, miRs can act either as tumor suppressors or oncogenes. Reduced expression of hsa-miR-101-3p has been observed in different types of cancers, and was thus considered to be a tumor suppressor. In urinary bladder cancer (BC) cell lines, hsa-miR-101-3p was found to suppress cancer cells motility, however studies in human BC patients, correlating hsa-miR-101-3p expression to pathological findings are very limited. This study aims at assessing hsa-miR-101-3p expression in human BC in correlation to tumor pathology in transurethral resection biopsies. Material and methodsTransuretheral bladder tissue specimens were collected from 100 bladder cancer patients. Only bladder transitional cell carcinoma (BTCC) patients (n = 80) were considered. Total RNA was extracted and following reverse transcription, expression of hsa-miR-101-3p was assessed by quantitative real time PCR using Taqman assay. The fold change for hsa-miR-101-3p expression between malignant and surrounding non-malignant tissues was calculated using relative quantification method. Expression of hsa-miR-101-3p was correlated to tumor stage, grade, and lymphatic/ vascular involvement. ROC curve was done to calculate the discriminative ability of hsa-miR-101-3p expression between malignant and non-malignant tissues. ResultsHsa-miR-101-3p was significantly under-expressed in all malignant versus non-malignant tissue samples (median 13.6 (9.8–19.4) vs. median 10.9 (7.4–16.2), p < 0.001) respectively. Mann-Whitney test showed statistically significant hsa-miR-101-3p underexpression in high vs. low tumor grades (p < 0.001), and T2 vs. T1 tumor stages (p < 0.001). There was no significant association between hsa-miR-101-3p underexpression and lymphatic/vascular invasion. ROC curve demonstrated hsa-miR-101-3p to highly discriminate between malignant and non-malignant tissue (p < 0.001).At cut-off value >10.85, hsa-miR-101-3p showed sensitivity of 83% and specificity of 56%. ConclusionsHsa-miR-101-3p can highly discriminate between malignant and non-malignant samples in BTCC, and seems to associate with tumor progression and aggressiveness. It might be a promising diagnostic and perhaps prognostic marker for BTCC.

An analysis of suppressing migratory effect on human urinary bladder cancer cell line by silencing of snail-1

BackgroundSnail-1 actively participates in tumor progression, invasion, and migration. Targeting snail-1 expression can suppress the EMT process in cancer. The aim of this study was to investigate the effect of snail1 silencing on urinary bladder cancer. MethodsQuantitative RT-PCR was used to detect snail-1 and other related metastatic genes expression following siRNA knockdown in urinary bladder cancer EJ-138 cells. The protein level of snail1 was assessed by Western blot. MTT and TUNEL assays were assessed to understand if snail-1 had survival effects on EJ-138 cells. Scratch wound healing assay measured cell motility effects after snail1 suppression. ResultsThe significant silencing of snail-1 reached 60pmol siRNA in a 48-h post-transfection. The result of scratch assay showed that snail-1 silencing significantly decreased Vimentin, MMPs, and CXCR4 expression; however, expression of E-cadherin was induced. The cell death assay indicated that snail-1 played the crucial role in bladder cancer survival rate. ConclusionThese results propose that snail-1 plays a major role in the progression and migration of urinary bladder cancer, and can be a potential therapeutic target for target therapy of invasive urinary bladder cancer.

Characterization of CNL like protein fragment (CNL-LPF) from mature Lageneria siceraria seeds

Coiled coil domain-nucleotide binding site-leucine rich repeat (CC-NBS-LRR; CNL) proteins are highly conserved family of plant disease resistance proteins, remarkably comprise of coiled-coil domain, which plays significant role in plant innate immunity. The present study reports that moderately elicited oligomerization of plant CNL like protein fragment (CNL-LPF) in presence of ATP/Mg using various biophysical methods Circular dichroism (CD) results depicted a substantial increase in β-sheet structure content of CNL-LPF. ATP/Mg induced conformational change in protein was observed by increase in blue shift with extrinsic fluorescence measurement, which indicates the exposure of hydrophobic regions of CNL-LPF and leads to self-association i.e. oligomerization. Likewise, cluster of protein oligomer and alteration in protein surface morphology were observed in presence of ATP/Mg by Transmission electron microscopy (TEM) and Atomic force microscopy (AFM), respectively. Also, augmented antiproliferation of HT1376 cells (urinary bladder cancer cell lines) was observed by CNL-LPF in presence of ATP/Mg. In conclusion, the current study illustrates that extent of CNL-LPF oligomerization was enhanced in presence of ATP/Mg (as compared to its absence). Utilization of enhanced oligomerization property of CNL-LPF as an anti-proliferative agent needs more assessment.

Abstract 2014: Genitourinary cancer-derived cells produce microenvironment proteinases that regulate proteinase activated receptors (PARs) to drive oncogenic signaling

Abstract Background: Thrombin-triggered activation of proteinase-activated receptor-1 (PAR1) is recognized as a key stimulus for driving epithelial malignancies (IUBMB life.63:397, 2011; PMID:21557443). However, the enzymes for regulating PAR1 in the tumour microenvironment are not known. Hypothesis: We hypothesize that prostate (PC) and urinary bladder cancer (UBC) cells can regulate microenvironment signaling to drive cancer progression via the secretion of proteinases that cleave/regulate proteinase-activated receptors (PARs). Aims: 1.) Visualize the cleavage status of N- and C-terminally-dual-labelled PARs in receptor-transfected UBC- and PC-derived cells. 2.) Measure PAR cleavage by UBC and PC-secreted proteinases. 3.) Identify the UBC &amp; PC-derived proteinases that cleave/regulate PAR function. Methods: The cleavage status of dual-labeled PARs (N-terminus-mCherry/C-terminus-YFP) expressed in PC &amp; UBC cells was determined by confocal image analysis as already described (JBC 288:32979, 2013; PMID: 26957205). Intact receptors look 'yellow' and cleaved receptors look 'green'. The cleavage of PARs in UBC and PC cells was studied for dual-tagged-PAR-expressing UBC and PC cells exposed or not to cell-derived culture supernatants, enzyme agonists for PAR1 and 2 (thrombin and trypsin, respectively), and proteinase inhibitors (e.g. for MMPs and other enzymes). The impact of CRISPR-mediated elimination of MMPs from PC3 cells on PAR1 cleavage status was also monitored. Results: UBC- and PC-produced enzymes can cleave PAR1 as efficiently as thrombin, an established PAR1 agonist. None of the UBC cell lines produce proteinases that cleave PAR2. Inhibition of MMP activity partially prevents PAR1 cleavage by UBC cells and completely prevents PAR1 cleavage in PC3-PC cells. Conclusions: We conclude that UBC and PC cells secrete PAR-regulating proteinases that can directly regulate PAR1 in the tumour microenvironment so as to drive cancer progression. Funding: Alberta Innovates CRIO Grant, Prostate Cancer Canada Discovery Grant, Motorcycle Ride for Dad, Johnson &amp; Johnson Alberta Health Partnership and CIHR Note: This abstract was not presented at the meeting. Citation Format: Stacy G. Gibson, Koichiro Mihara, Andries Zijlstra, Matthew E. Hyndman, Morley D. Hollenberg. Genitourinary cancer-derived cells produce microenvironment proteinases that regulate proteinase activated receptors (PARs) to drive oncogenic signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2014. doi:10.1158/1538-7445.AM2017-2014

2200 - siRNA-mediated silencing of Snail-1 induces apoptosis and alters microRNA expression in human urinary bladder cancer cell line

2200 - siRNA-mediated silencing of Snail-1 induces apoptosis and alters microRNA expression in human urinary bladder cancer cell line