Human Bladder Tumor Cells Research Articles

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

Targeting urothelial neoplasia using an investigational virus-like drug conjugate.

514 Background: Human papillomavirus virus-like particles (HPV VLP) preferentially target tumor cells via cell surface modified heparan-sulfate proteoglycans (HSPG). The investigational virus-like drug conjugate, belzupacap sarotalocan (AU-011), is currently in phase 2 clinical trials for treatment of primary choroidal melanoma. We previously demonstrated AU-011’s in vivo tumor acute cytotoxicity upon activation with near-infrared light (nIR), resulting in tumor-free survival for at least 100 days and protection from tumor re-challenge in the MB49 murine flank model of bladder cancer. Here we provide additional data supporting further clinical development in urothelial neoplasia as an additional oncology indication. Methods: We examined the cytotoxicity of AU-011 in a panel of six human bladder cancer cell lines in vitro and binding and distribution in human tumor biopsy samples ex vivo. Tumor distribution of AU-011 was also assessed in vivo after intravesical instillation in the orthotopic MB49 murine model. In consideration of the glycocalyx layer often found coating the interior of the bladder wall and the surface of bladder tumors, pre-treatment with hyauronidase was tested both on human biopsy samples and in the murine orthotopic model. Lastly, tumor distribution of AU-011 was evaluated when formulated with the polyamide Syn3. Results: When tested in vitro on a panel of human bladder tumor cell lines representing grades I-IV carcinomas, we found AU-011’s potency to be in the picomolar range (EC50 range 16.6 pM – 62.75 pM). We assessed the VLPs ability to bind ex vivo to a panel of human urothelial tumor biopsies and observed binding in samples representing both invasive T2 and non-invasive Ta papillary tumors. Tumor biopsy samples were pre-treated with hyaluronidase to break down the glycocalyx layer surrounding the tumor, and an increase in signal was observed in the Ta biopsy sample. Similarly, using the orthotopic MB49 murine tumor model with intravesical delivery of AU-011, we observed an enhancement of AU-011 distribution into the tumor when the bladders were pre-treated with hyaluronidase. Additionally, when formulated with Syn3, AU-011 distribution throughout the tumors was augmented without the need for hyaluronidase pre-treatment. Conclusions: We have demonstrated AU-011 targeted cytotoxicity in vitro using a panel of human bladder cancer cell lines indicating that its binding to bladder cancer cells is tumor grade and genetic mutation agnostic. Additionally, tumor binding and distribution was observed ex vivo using human bladder cancer biopsy samples and in vivo in the murine MB49 orthotopic model. Human and murine tumor distribution was improved with a pre-treatment of hyaluronidase or when formulated with Syn3. Collectively, these data support the further investigation of the use of AU-011 for the indication of uroepithelial cancer.

Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by Caveolin-1-Dependent Adenosine Monophosphate-Activated Protein Kinase Activity.

PurposeAdenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells.MethodsIn cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay.ResultsThe level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated.ConclusionsFor the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.

Biomimetic TiO2-chitosan/sodium alginate blended nanocomposite scaffolds for tissue engineering applications.

The study is aimed to synthesize potent metal oxide based biomimetic nanocomposites to overcome the risk associated with artificial bone tissue engineering. High purity TiO2 nanoparticles are synthesized via hydrothermal route. A biomimetic nanocomposite scaffolds containing chitosan-sodium alginate (4: 4) blended with three different (0.5, 1, and 1.5wt%) concentrations of hydrothermally synthesized TiO2 nanoparticles are obtained by solvent casting technique. The physico-chemical and thermal degradation properties of as-synthesized TiO2 nanoparticles and their nanocomposite scaffolds are analyzed. In-vitro cytotoxicity and biocompatibility of the prepared TiO2 nanoparticles and nanocomposites are tested against human bladder tumor (UC6) and osteosarcoma (MG-63) cell lines. Antibacterial property is tested against Escherichia coli and Staphylococcus aureus. These studies reveal that TiO2 nanoparticles and polymeric nanocomposites contain good physico-chemical and mechanical properties for enhanced in-vitro biocompatibility suitable for biomedical applications. Biomimetically prepared chitosan-sodium alginate scaffold containing TiO2 nanoparticles (1wt%) is found to exhibit superior biocompatibility for bone tissue engineering applications.

A sensitive refining of in vitro and in vivo toxicological behavior of green synthesized ZnO nanoparticles from the shells of Jatropha curcas for multifunctional biomaterials development

ZnO nanoparticles (NPs) possess a wide range of biological functions in pharmaceutical and cosmetic applications due to their excellent antimicrobial, optical and UV protective properties. This study first reports the toxicological assessment of ZnO NPs green synthesized from Jatropha curcas shells for multifunctional biomedical applications. The hot water extract of J.curcas shells is utilized as a chelating agent for the reduction of zinc acetate and then, the prepared ZnO NPs are broadly characterized using X-ray spectroscopic and electron microscopic observations. The prepared ZnO NPs acquire high purity (100%) wurtzite crystal with hexagonal structure with the average particle size of 53 nm. In vitro and in vivo toxicity evaluation against human tumor cell lines and zebrafish embryos have ascertained the purpose of ZnO NPs in clinical research. Toxic effects of ZnO NPs were observed by a dose-dependent reduction of bacterial growth at ≥1 μg ml−1, by teratogenicity and genotoxicity in zebrafish embryos (from 3 to 90 μg ml−1) and by a significant nanoparticle uptake (0.5 ng μl−1) by a fish serum. In contrast, ZnO NPs fail to reduce the proliferation of human bladder tumor cells (UC6) and cell viability of A549 cells in vitro up to 500 μg ml−1. All these observations limit the unobstructed application of ZnO NPs at higher concentrations. Thus, abundantly used metal oxide nanoparticles like ZnO NPs examined in our present study in different animal models under in vitro and in vivo conditions will be the significant screening strategy to determine the nanotoxicity.

Two telomerase-targeting Pt(ii) complexes of jatrorrhizine and berberine derivatives induce apoptosis in human bladder tumor cells.

Two novel Pt(ii) complexes, [Pt(B-TFA)Cl]Cl (Pt1) and [Pt(J-TFA)Cl]Cl (Pt2) with jatrorrhizine and berberine derivatives (B-TFA and J-TFA) were first prepared as desirable luminescent agents for cellular applications and potent telomerase inhibitors, which can induce bladder T-24 tumor cell apoptosis by targeting telomerase, together with induction of mitochondrial dysfunction, telomere DNA damage and cell-cycle arrest. Importantly, T-24 tumor inhibition rate (TIR) was 50.4% for Pt2, which was higher than that of Pt1 (26.4%) and cisplatin (37.1%). Taken together, all the results indicated that jatrorrhizine and berberine derivatives Pt1 and Pt2 show low toxicity and could be novel Pt-based anti-cancer drug candidates.

Therapeutic efficacy of the live-attenuated Mycobacterium tuberculosis vaccine, MTBVAC, in a preclinical model of bladder cancer

Intravesical instillation of bacillus Calmette-Guérin (BCG) has been a first-line therapy for non-muscle-invasive bladder cancer for the last 4 decades. However, this treatment causes serious adverse events in a significant number of patients and a substantial percentage of recurrence episodes. MTBVAC is a live-attenuated vaccine derived from a Mycobacterium tuberculosis clinical isolate and is currently under evaluation in clinical trials to replace BCG as a tuberculosis vaccine. Here, we describe for the first time the potential of MTBVAC as a bladder cancer therapy in vitro and in vivo in a preclinical model. MTBVAC colonized human bladder tumor cells to a much greater extent than BCG via a mechanism mediated by macropinocytosis and induced cell growth inhibition after internalization. In vivo testing in an orthotopic murine model of bladder cancer demonstrated a higher antitumor effect of MTBVAC in experimental conditions in which BCG did not work. Our data encourage further studies to support the possible application of MTBVAC as a new immunotherapeutic agent for bladder cancer.

Biological effects of eukaryotic recombinant plasmid pReceiver-M61-BAI-1 transfection on T24cells and HUVECs.

The aim of the current study was to investigate the biological effect on T24 cells and human umbilical vein endothelial cells (HUVECs) of transfection with brain-specific angiogenesis inhibitor-1 (BAI-1). The recombinant plasmid pReceiver-M61-BAI-1 was transfected into human superficial bladder tumor cells (T24) and HUVECs, in parallel with the vector control. mRNA and protein expression levels of BAI-1 were then detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Cell apoptosis of T24 cells and HUVECs prior and subsequent to transfection with BAI-1 was analyzed by flow cytometric analysis. Proliferation of T24 cells and HUVECs prior and subsequent to transfection of BAI-1 was assessed by the MTT method. T24 cells and HUVECs transfected with pReceiver-M61-BA1-1 were classed as the experimental group; T24 cells and HUVECs transfected with p-Receiver-M61 were the control group. qPCR and western blotting methods confirmed that there was positive expression of BAI-1 in T24 cells and HUVECs transfected with pReceiver-M61-BAI-1, however BAI-1 was not expressed in T24 cells and HUVECs transfected with pReceiver-M61. The results of the MTT assay demonstrated that absorbance was markedly reduced in HUVECs at 12, 48 and 72 h subsequent to transfection with pReceiver-M61-BAI-1 when compared with that of the control group and in T24 cells transfected with p-Receiver-M61-BAI-1. Furthermore, flow cytometry results also indicated that the apoptotic rate of HUVECs transfected with p-Receiver-M61-BAI-1 was significantly increased compared with that of the control group and T24 cells transfected with p-Receiver-M61-BAI-1. BAI-1 was observed to markedly inhibit the proliferation of vascular endothelial cells in vitro, however, no direct inhibition by BAI-1 was observed in T24 cells. In conclusion, BAI-1 is suggested to be a potential novel therapautic target for the inhibition of tumor neovascularization.

Metabolism and growth inhibitory activity of cranberry derived flavonoids in bladder cancer cells.

In the present study, anti-proliferative activities of cranberry derived flavonoids and some of their in vivo metabolites were evaluated using a panel of human bladder tumor cell lines (RT4, SCABER, and SW-780) and non-tumorigenic immortalized human uroepithelial cells (SV-HUC). Among the compounds tested, quercetin 3-O-glucoside, isorhamnetin (3'-O-methylquercetin), myricetin and quercetin showed strong concentration-dependent cell growth inhibitory activities in bladder cancer cells with IC50 values in a range of 8-92 μM. Furthermore, isorhamnetin and myricetin had very low inhibitory activity against SV-HUC even at very high concentrations (>200 μM) compared to bladder cancer cells, indicating that their cytotoxicity is selective for cancer cells. To determine whether the differential cell growth inhibitory effects of isomeric flavonoids quercetin 3-O-glucoside (active) and hyperoside (quercetin 3-O-galactoside) (inactive) are related to their metabolism by the cancer cells, SW-780 cells were incubated with these compounds and their metabolism was examined by LC-MS/MS. Compared to quercetin 3-O-glucoside, hyperoside undergoes relatively less metabolic biotransformation (methylation, glucuronidation and quinone formation). These data suggest that isorhamnetin and quercetin 3-O-glucoside may be the active forms of quercetin in prevention of bladder cancer in vivo and emphasize the importance of metabolism for the prevention of bladder cancer by diets rich in cranberries.

Simultaneous Targeting of Bladder Tumor Growth, Survival, and Epithelial-to-Mesenchymal Transition with a Novel Therapeutic Combination of Acetazolamide (AZ) and Sulforaphane (SFN).

Current chemotherapies for advanced stage metastatic bladder cancer often result in severe side effects, and most patients become drug resistant over time. Thus, there is a need for more effective therapies with minimal side effects. The acid/base balance in tumor cells is essential for tumor cell functioning. We reasoned that simultaneous targeting of pH homeostasis and survival pathways would improve therapeutic efficacy. We evaluated the effectiveness of targeting pH homeostasis with the carbonic anhydrase inhibitor acetazolamide (AZ) in combination with the survival pathway targeting isothiocyanate sulforaphane (SFN) on the HTB-9 and RT112(H) human bladder tumor cell lines. We assessed viability, proliferation, and survival in vitro and effect on xenografts in vivo. Combination AZ + SFN treatment induced dose-dependent suppression of growth, produced a potent anti-proliferative and anti-clonogenic effect, and induced apoptosis through caspase-3 and PARP activation. The anti-proliferative effect was corroborated by significant reductions in Ki-67, pHH3, cyclin D1, and sustained induction of the cell cycle inhibitors, p21 and p27. Both active p-Akt (Ser473) and p-S6 were significantly downregulated in the AZ + SFN combination treated cells with a concomitant inhibition of Akt kinase activity. The inhibitory effects of the AZ + SFN combination treatment showed similar efficacy as the dual PI3K/mTOR pathway inhibitor NVP-BEZ235, albeit at an expected higher dose. In terms of the effect on the metastatic potential of these bladder cancers, we found downregulated expression of carbonic anhydrase 9 (CA9) concomitant with reductions in both E-cadherin, N-cadherin, and vimentin proteins mitigating the epithelial-to-mesenchymal transition (EMT), suggesting negation of this program. We suggest that reductions in these components could be linked with downregulation of the survival mediated Akt pathway and suggested an active role of the Akt pathway in bladder cancer. Altogether, our in vitro and pre-clinical model data support the potential use of an AZ + SFN combination for the treatment of bladder cancer.

Synthesis of a new 18F labeled porphyrin for potential application in positron emission tomography. In vivo imaging and cellular uptake

Synthesis, labeling and initial biodistribution studies of a new [18F] radiolabeled meso-tetraphenylporphyrin (radiochemical purity &gt;95%). Includes human bladder tumor cell uptake and biodistribution data.

Cytotoxic compounds isolated from Murraya tetramera Huang.

A new compound and seven known compounds were isolated from Murraya tetramera Huang for the first time, and they were identified with NMR and MS spectral analysis. It was confirmed that the new compound was 10-methoxy-7-methyl-2H-benzo[g]chromen-2-one (3) and the others were β-eudesmol (1), trans-3β-(1-hydroxy-1-methylethyl)-8aβ-methyl-5-methylenedecalin-2-one (2), 5,7-dimethoxy-8-[(Z)-3'-methyl-butan-1',3'-dienyl]coumarin (4), 7-geranyloxy-6-methoxycoumarin (5), 5,7-dimethoxy-8-(3-methyl-2-oxo-butyl)coumarin (6), murrangatin acetate (7) and toddalenone (8). Furthermore, the cytotoxic activity against human lung adenocarcinoma (A549), human hepatocellular carcinoma cells (SMMC-7721), human bladder tumor cells (EJ), human cervical carcinoma cells (HeLa), and human B-lineage acute lymphoblastic leukemia 1 cells (BALL-1) was evaluated for all compounds. It was found that five of them displayed various degrees of cytotoxicity against different testing targets. Compound 1 showed significant cytotoxic activity against the five cell lines (A549, SMMC-7721, EJ, Hela and BALL-1). Compounds 2 and 5 showed significant cytotoxicity against three cell lines (A549, SMMC-7721 and BALL-1). Compound 4 showed significant cytotoxicity against three cell lines (A549, EJ and BALL-1). However, compound 3 only showed fair cytotoxicity against the BALL-1 cell line. The structure-active relationships were investigated as well. These active compounds might be potential lead compounds for the treatment of cancer.

Bladder Cancer: The Hormonal Dependence Enigma and a New Hormonal Player.

Dear Editor, Bladder cancer is considered to be a hormonal-independent malignancy. However, an increasing number of studies show that sex hormonesmight have an important role in the etiology or progression of specific urothelial carcinoma subtypes. The most important indication that bladder cancer might be influenced by sex hormone is that the incidence and risk of developing bladder cancer in men is notably greater comparing to women, even when established risk factors such as smoking, occupational exposure, and urinary tract infection are counterbalanced between the sexes. The effect of sex hormones in bladder tissues is documented during pregnancy and menopause where the bladder undergoes dramatic changes in structure, function, histology, and gene expression. Bladder tissues are shown to express androgen, estrogen, and progesterone receptors. Additionally, in animal models, estrogens are shown to inhibit and androgens to promote bladder cancer development even when carcinogenetic substances are used such as N-butyl-N-(4hydroylbutyl)nitrosamine [1]. Another indication of the hormonal contribution in bladder cancer development is the relatively lower risk of parous women to develop this malignancy compared to the nulliparous females. A human bladder tumor cell line, R198, was shown to develop under the effect of androgens while it regressed under the presence of estrogen [2]. Finally, there is also the report of a man who developed endometriosis of urinary bladder, after 5 years treatment with estrogen therapy for prostate cancer [3]. The lesion regressed after estrogen withdrawal, indicating a metaplasia potential of the urothelium under extreme changes of hormonal environment. Under these circumstances, we would like to report our experience of a 65-year-old woman treated for breast cancer who developed urothelial carcinoma at the trigone of the urinary bladder, 1 year after the onset of adjuvant hormonal therapy with third-generation non-steroidal aromatase inhibitor. The patient underwent modified radical mastectomy with ipsilateral axillary lymphadenectomy 4 years ago and subsequently chemotherapy (FEC and docetaxel) and radiotherapy. Three months after the onset of aromatase inhibitor treatment, the patient claimed to have aggravated hirsutism of both arms and no hair growth on the parietal skull following a pattern of androgenetic alopecia. A year since the onset of adjuvant hormonal therapy, a papillary protrusion was detected at the trigone of the urinary bladder. The histological report showed a non-muscle invasive low-grade papillary urothelial carcinoma, grade 1 by WHO-ISUP. One and a half year later, the patient was found to have bladder cancer relapse with the same histological urothelial tumor and the same androgenetic effects of the adjuvant hormonal therapy. The current hypothesis we propose is that the excessive androgen environment produced by the aromatase inhibitor in a postmenopausal woman with lack of the counterbalancing hormones (estrogen and progesterone) could contribute to the development of bladder cancer. There are some hypotheses which try to explain the hormonal dependence of bladder cancer and especially of the trigone of the urinary bladder. The trigone and the prostate have the same embryologic endodermic origin. This hypothesis is supported by the finding that the tumor cells of R198 cell line have some similar characteristics with human prostatic carcinomas [2]. Additionally, one of the ways that K. Ntzeros (*) :G. Louka Department of Urology, Hospital of Korinthos, Korinthos, Greece e-mail: konstantinos.ntzeros@gmail.com

CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.

Discrimination of bladder cancer cells from normal urothelial cells with high specificity and sensitivity: Combined application of atomic force microscopy and modulated Raman spectroscopy

Atomic force microscopy (AFM) and modulated Raman spectroscopy (MRS) were used to discriminate between living normal human urothelial cells (SV-HUC-1) and bladder tumour cells (MGH-U1) with high specificity and sensitivity. MGH-U1 cells were 1.5-fold smaller, 1.7-fold thicker and 1.4-fold rougher than normal SV-HUC-1 cells. The adhesion energy was 2.6-fold higher in the MGH-U1 cells compared to normal SV-HUC-1 cells, which possibly indicates that bladder tumour cells are more deformable than normal cells. The elastic modulus of MGH-U1 cells was 12-fold lower than SV-HUC-1 cells, suggesting a higher elasticity of the bladder cancer cell membranes. The biochemical fingerprints of cancer cells displayed a higher DNA and lipid content, probably due to an increase in the nuclear to cytoplasm ratio. Normal cells were characterized by higher protein contents. AFM studies revealed a decrease in the lateral dimensions and an increase in thickness of cancer cells compared to normal cells; these studies authenticate the observations from MRS. Nanostructural, nanomechanical and biochemical profiles of bladder cells provide qualitative and quantitative markers to differentiate between normal and cancerous cells at the single cellular level. AFM and MRS allow discrimination between adhesion energy, elasticity and Raman spectra of SV-HUC-1 and MGH-U1 cells with high specificity (83, 98 and 95%) and sensitivity (97, 93 and 98%). Such single-cell-level studies could have a pivotal impact on the development of AFM–Raman combined methodologies for cancer profiling and screening with translational significance.

Effect of surface charge of polymeric micelles on in vitro cellular uptake

This work focuses on the interaction between polymeric micelles with different charged surfaces and cancer cells in order to study the influence of surface charge on the in vitro cellular uptake efficiency. The amphiphilic diblock copolymers poly(ɛ-caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) with different functional groups at the end of hydrophilic block were synthesized. The functional groups endue the micelles with different charges on the surfaces. The cellular uptake of micelles to T-24 cells (human bladder tumor cells), HepG2 cells (human liver hepatocellular carcinoma cell line) and Hela cells (human epithelial cervical cancer cells) was studied by means of flow cytometer and confocal laser scanning microscopy. The results indicate that the surface charges showed great influence on zeta potential of micelles at different pH values. The in vitro cellular uptake efficiency of micelles with different charged surfaces demonstrated different cellular uptake patterns to three kinds of cancer cells.

Synergistic Antitumor Activity of Vitamins C and K3 on Human Bladder Cancer Cell Lines

Exponentially growing cultures of human bladder tumor cells (RT4 and T24) were treated with Vitamin C (VC) alone, Vitamin K3 (VK3) alone, or with a VC:VK3 combination continuously for 5 days or treated with vitamins for 1 h, washed with PBS and then incubated in culture medium for 5 days. Co-administration of the vitamins enhanced the antitumor activity 12- to 24-fold for the RT-4 cells and 6- to 41-fold for the T24 cells. Flow cytometry of RT4 cells exposed to the vitamins revealed a growth arrested population and a population undergoing cell death. Growth arrested cells were blocked near the G0/G1-S-phase interface, while cell death was due to autoschizis. Catalase treatment abrogated both cell cycle arrest and cell death which implicated hydrogen peroxide (H2O2) in these processes. The H2O2 production resulted in a moderate increase in lipid peroxidation and depletion of cell thiol levels. Analysis of cellular ATP levels revealed a transient increase in ATP production for VC and the VC:VK3 combination, but decreased ATP levels following VK3 treatment. Lipid peroxidation, thiol depletion and ATP modulation occurred at a 17-fold lower concentration in the vitamin combination than with either vitamin alone. These results suggested that the increased cytotoxicity of the vitamin combination was due to redox cycling and increased oxidative stress.

886 ADENOVIRAL VECTOR EXPRESSING SHORT HAIRPIN RNA TARGETING G-PROTEIN COUPLED RECEPTOR (GPR87) HAS A STRONG ANTITUMOR ACTIVITY AGAINST GPR87-OVEREXPRESSING BLADDER CARCINOMAS

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2012886 ADENOVIRAL VECTOR EXPRESSING SHORT HAIRPIN RNA TARGETING G-PROTEIN COUPLED RECEPTOR (GPR87) HAS A STRONG ANTITUMOR ACTIVITY AGAINST GPR87-OVEREXPRESSING BLADDER CARCINOMAS Xia Zhang, dage liu, Shinsuke shibuya, Mikio Sugimoto, and Yoshiyuki kakehi Xia ZhangXia Zhang kagawa, Japan More articles by this author , dage liudage liu kagawa, Japan More articles by this author , Shinsuke shibuyaShinsuke shibuya kagawa, Japan More articles by this author , Mikio SugimotoMikio Sugimoto kagawa, Japan More articles by this author , and Yoshiyuki kakehiYoshiyuki kakehi kagawa, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.981AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES GPR87 belongs to G-protein coupled receptor family and had long been an orphan receptor. Recently, it was demonstrated that lysophosphatidic acid (LPA) was its ligand. LPA is a lipid mediator that is associated with several function including cellular proliferation and angiogenesis. Overexpression of GPR87 was found in many cancers including bladder cancer. Recent our studies revealed that the GPR87-overexpression affect the tumor progression through the induction of tumor-associated GPR87-targets in bladder cancer cell lines. Therefore, we performed experimental studies using a GPR87-suppressing adenoviral vector in order to establish the gene therapy against GPR87-overexpressing tumors. METHODS Three human bladder tumor cells with an overexpression of GPR87, including HT1197, RT112, and Tcssup cell lines, were used. We constructed adenoviral vector expressing shRNA targeting GPR87 (Ad-shGPR87). RESULTS The infection of Ad-shGPR87 effectively downregulated the GPR87 expression in all three GPR87-overexpressing tumor cell lines. Regarding the in vitro experiment, MTT assays demonstrated that the infection of Ad-shGPR87 significantly inhibited the growth of all three GPR87-overexpressing tumor cells (p<0.001, respectively). A flow cytometric analysis revealed the infection with Ad-shGPR87 into RT112 tumor cells to increase the percentage of apoptotic cells. Regarding in vivo experiments, a human tumor xenograft model in Scid mice was prepared by the implantation of GPR87-overexpressing tumors derived from RT112 cells. The antitumor effects were compared between the Ad-shGPR87 treatment group and the saline control group. Ad-shGPR87 or saline was administered by intratumoral injection every 4 days. Consequently, the Ad-shGPR87 treatment was found to have the strongest antitumor effect against the GPR87-overexpressing tumor xenografts (Fig.1A and 1B p<0.001). CONCLUSIONS Conclusions: The cancer gene therapy using the adenoviral vector expressing shRNA against GPR87 has a strong antitumor effect against GPR87-overexpressing tumors. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e360-e361 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xia Zhang kagawa, Japan More articles by this author dage liu kagawa, Japan More articles by this author Shinsuke shibuya kagawa, Japan More articles by this author Mikio Sugimoto kagawa, Japan More articles by this author Yoshiyuki kakehi kagawa, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Cytotoxic Triterpenoid Saponins fromArdisia gigantifolia

Three new triterpenoid saponins, 1- 3, together with two known saponins, 4 and 5, were isolated from the rhizome of Ardisia gigantifolia. Their structures were elucidated by 1D and 2D NMR spectroscopic studies. Saponins 1, 2, 4, and 5 exhibited significant cytotoxicity against four human cancer cell lines, namely, Hela human cervical carcinoma cells, EJ human bladder tumor cells, HepG-2 human hepatoma cells, and BCG human gastric carcinoma cells with IC (50) values in the range of 1.9-4.8 µM.