Human Urinary Bladder Carcinoma Research Articles

Indole‐3‐Acetic Acid/Horseradish Peroxidase Induces Apoptosis in TCCSUP Human Urinary Bladder Carcinoma Cells

In the present study, we determined the effects of IAA/HRP treatment on TCCSUP human urinary bladder carcinoma cells. IAA/HRP combination decreased cell viability of TCCSUP cells in a time‐ and dose‐dependent manner, whereas IAA or HRP alone showed no such effect. In addition, the decreased cell viability was restored by pretreatment with ascorbic acid. To clarify the mechanism of death of TCCSUP cells by IAA/HRP, we investigated the signal transduction pathways related to the apoptosis. IAA/HRP activates p38 mitogen‐activated protein (MAP) kinase and c‐Jun N‐terminal kinase (JNK). We further investigated the IAA/HRP‐mediated apoptotic pathways and showed that IAA/HRP induces caspase‐8 and caspase‐9 activation, which results in caspase‐3 activation and poly(ADP‐ribose) polymerase (PARP) cleavage. To further confirm whether IAA/HRP induces apoptotic cell death, we performed a DNA fragmentation assay after IAA/HRP treatment and found that IAA/HRP‐treated cells showed typical apoptotic DNA ladder formation. From these results, we suggest that IAA/HRP induces apoptosis of TCCSUP human urinary bladder carcinoma cells via both death receptor‐mediated and mitochondrial apoptotic pathways.

Synthesis of model morpholine derivatives with biological activities by laccase‐catalysed reactions

The efficient enzyme-catalysed reaction of morpholines as model structures for bioactive compounds with para-dihydroxylated aromatic systems was carried out using the oxidoreductase laccase and atmospheric oxygen to produce eight novel morpholine-substituted aromatics. The laccase of Myceliophthora thermophila was used for cross-linking morpholines containing primary or secondary amino groups with para-dihydroxylated laccase substrates. We demonstrate that not only primary amino groups, but also secondary amino groups, are able to couple with para-dihydroxylated aromatic systems in laccase-catalysed reactions. The resulting model products (yields up to 80%) were isolated, structurally characterized and tested for their antibacterial, antifungal and cytotoxic activities. Four of the eight products showed low to moderate growth inhibition against several Gram-positive and -negative bacterial strains and against the yeasts Candida maltosa and Candida albicans. The antibacterial and antifungal activities were determined by an agar disc diffusion test and a modified method according to the EUCAST discussion document E.Dis 7.1 [Rodríguez-Tudela et al. (2003) Clin. Microbiol. Infect. 9, i-viii] for the evaluation of MIC (minimal inhibitory concentration). Differences in cytotoxicity against the human urinary bladder carcinoma cell line 5637 are discussed.

Supraphysiological Thermal Injury in Different Human Bladder Carcinoma Cell Lines

Depending on the duration of exposure to supraphysiological temperatures, cellular proteins and organelles can suffer from structural alternations and irreversible denaturation, which may induce cell death. The thermotolerance of three human urinary bladder carcinoma cell lines, TSGH-8301, J82 and TCC-SUP (cytological grade 2, 3 and 4, respectively), was investigated in the present study. A home-made heating stage was used to provide a constant temperature for different cell lines of bladder carcinoma. The experimental data showed that the TCC-SUP and TSGH-8301 cells exhibited the lowest and highest thermotolerances, respectively, while J82 cells were intermediate. Moreover, the differences in the thermotolerances for the TSGH-8301 and J82 cells are significant when the supraphysiological temperature is higher than 43 degrees C. As for TSGH-8301 and TCC-SUP cells, the thermotolerances are significantly different for all of the thermal treatments tested. Furthermore, the thermotolerances of J82 and TCC-SUP are significantly different when the cells are exposed to a temperature less than 50 degrees C for longer than 2 min. Overall, the results suggest that the high cytological grade of the cell line of bladder cancer exhibits a low thermotolerance. The kinematic parameters of the activation energy and frequency factor for bladder cancer cell lines with different cytological grades were also quantitatively evaluated in this work.

Phytochemicals in olive‐leaf extracts and their antiproliferative activity against cancer and endothelial cells

Olive oil compounds is a dynamic research area because Mediterranean diet has been shown to protect against cardiovascular disease and cancer. Olive leaves, an easily available natural material of low cost, share possibly a similar wealth of health benefiting bioactive phytochemicals. In this work, we investigated the antioxidant potency and antiproliferative activity against cancer and endothelial cells of water and methanol olive leaves extracts and analyzed their content in phytochemicals using LC-MS and LC-UV-SPE-NMR hyphenated techniques. Olive-leaf crude extracts were found to inhibit cell proliferation of human breast adenocarcinoma (MCF-7), human urinary bladder carcinoma (T-24) and bovine brain capillary endothelial (BBCE). The dominant compound of the extracts was oleuropein; phenols and flavonoids were also identified. These phytochemicals demonstrated strong antioxidant potency and inhibited cancer and endothelial cell proliferation at low micromolar concentrations, which is significant considering their high abundance in fruits and vegetables. The antiproliferative activity of crude extracts and phytochemicals against the cell lines used in this study is demonstrated for the first time.

Staphylococcus saprophyticusATCC 15305 is internalized into human urinary bladder carcinoma cell line 5637

Invasion of bacteria into nonphagocytic host cells is an important pathogenicity factor for escaping the host defence system. Gram-positive organisms, for example Staphylococcus aureus and Listeria monocytogenes, are invasive in nonphagocytic cells, and this mechanism is discussed as an important part of the infection process. Uropathogenic Escherichia coli and Staphylococcus saprophyticus can cause acute and recurrent urinary tract infections as well as bloodstream infections. Staphylococcus saprophyticus shows strong adhesion to human urinary bladder carcinoma and Hep2 cells and expresses the 'Microbial Surface Components Recognizing Adhesive Matrix molecule' (MSCRAMM)-protein SdrI with collagen-binding activity. MSCRAMMs are responsible for adhesion and collagen binding in S. aureus and are discussed as an important pathogenicity factor for invasion. To investigate internalization in S. aureus, several fluorescence activated cell sorting (FACS) assays have been described recently. We used a previously described FACS assay, with slight modifications, in addition to an antibiotic protection assay and transmission electron microscopy to show that S. saprophyticus ATCC 15305 and the wild-type strain 7108 were internalized into the human urinary bladder carcinoma cell line 5637. The discovery of the internalization of S. saprophyticus may be an important step for understanding the pathogenicity of recurrent infections caused by this organism.

Detection of urinary bladder cancer with flow cytometry and hexaminolevulinate in urine samples

Urinary bladder urothelial carcinoma is diagnosed by a combination of cystoscopy and biopsy, with cytology as a valuable additional technique. The accuracy of cytological diagnosis depends on the experience of the cytologist and can inevitably vary from one cytologist to another. There is a need for an easy, reliable and objective diagnostic method. In the present study a new method was designed for the detection of bladder cancer cells in urine. Flow cytometry was utilized to detect protoporphyrin IX in an artificial model consisting of normal urinary bladder transitional epithelial cells (NBECs) from healthy volunteers' urine and an established human urinary bladder carcinoma cell line, TCCSUP, after incubation with hexaminolevulinate (HAL). In addition, urine samples from 19 patients with histopathologically confirmed superficial bladder cancer were examined. Incubation of NBECs or TCCSUP cells with HAL for 1 hour resulted in production of protoporphyrin IX only in the TCCSUP cells. Incubation of a mixture of NBECs and TCCSUP cells with HAL gave rise to a separated subpopulation of cells with protoporphyrin IX fluorescence. After cell sorting by flow cytometry the protoporphyrin IX-containing subpopulation of cells was confirmed as TCCSUP cells on cytological examination. It was possible to detect 5% TCCSUP cells in the mixture of NBECs/TCCSUP cells. To test the feasibility of the method in clinica diagnosis, urine samples from patients with bladder cancer were also measured with comparable, although preliminary and limited, results to those of cytological examination. The preliminary results show that the technique may be feasible for the detection of bladder cancer cells in urine with possible advantages of simplicity, reliability and objectivity.

Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC50s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.

Tumor grade-dependent alterations in the protein kinase C isoform pattern in urinary bladder carcinomas.

Members of the protein kinase C (PKC) isoenzyme family play central role in the tumorigenesis of several tissues. In this study our goal was to determine the possible alterations in the protein kinase C (PKC) isoform pattern in relation with the different tumor grade in human urinary bladder carcinomas. Western blot analysis, followed by quantitative densitometry, was performed to define the expression of PKC isoforms in the epithelial tissue of human urinary bladder carcinomas with various tumor grades and in control samples. The human urinary bladder epithelium expressed five PKC isoforms (PKC alpha, beta, delta, zeta), the levels of which differentially altered as a function of tumor grade. Namely, whereas the expressions of PKC beta and delta decreased with increasing grade of the carcinomas, the levels of PKC alpha, and zeta showed opposite patterns of changes. These grade-dependent alterations in the PKC isoform pattern strongly argue for the central yet antagonistic roles of certain members of the PKC system in malignant transformation of human urinary bladder epithelium.

Expression of peroxisome proliferator-activated receptors (PPARs) in human urinary bladder carcinoma and growth inhibition by its agonists.

Recent studies have demonstrated that peroxisome proliferator activator-receptors(PPAR)-gamma is expressed in various cancer tissues and its ligand induces growth arrest of these cancer cells through apoptosis. In our study, we investigated the expression of PPAR-alpha, beta and gamma in human bladder tumor (BT) and normal bladder (NB) tissues as well as the effects of PPAR-gamma ligands. Specimens were obtained from 170 patients with BT and 20 with NB. The expressions were investigated using RT-PCR and immunohistochemical methods. We also investigated the inhibitory effect of PPAR-gamma ligands on BT-derived cell line. Immunoreactive PPAR-alpha and -beta were significantly apparent in both BT and NB tissues. Although no marked expression of PPAR-gamma was observed in NB tissue, significant expression was found in BT tissue. The extent and intensity of immunoreactive PPAR-gamma polypeptides in BT cells were statistically much greater than those of NB cells. Correlation between PPAR-gamma expression and tissue type or progression of bladder cancer was observed; PPAR-gamma expression was higher in G3 of bladder cancer than in G1 and was higher in advanced than in early cancer. PPAR-gamma agonists, troglitazone and 15-deoxy-Delta(12, 14)-prostaglandin J(2) inhibited the growth of the BT cells. PPAR-gamma is expressed in bladder tumor, and results suggest that PPAR-gamma ligands may mediate potent antiproliferative effects against BT cells. Thus, PPAR-gamma has the ability to become a new target in treatment of bladder tumor.

Gelsolin gene therapy using adenovirus vector for orthotopically transplanted human urinary bladder carcinoma in nude mice

Gelsolin gene therapy using adenovirus vector for orthotopically transplanted human urinary bladder carcinoma in nude mice

Human urinary bladder carcinomas express adenovirus attachment and internalization receptors.

The use of adenoviral vectors as potent gene delivery systems requires expression of the Coxsackievirus/adenovirus receptor (CVADR) on the target cell surface. This receptor is important for virus attachment to the cell surface. For effective internalization of the vector into the target cell the integrins alpha(v)beta(3) and/or alpha(v)beta(5) are needed. Since there have been reports of loss of CVADR in bladder cancer cell lines, we wanted to investigate the expression of this receptor in bladder carcinoma biopsies. Surgical biopsies, as well as five human bladder cancer cell lines, were analyzed for expression of CVADR, the integrins alpha(v)beta(3) and alpha(v)beta(5) and MHC class I. Further, we studied the ability to transduce these cell lines using adenoviral vectors. Immunohistochemistry revealed that all biopsies (27/27) were positive for CVADR. Some variation in expression was evident, and superficially growing tumors stained more strongly than invasive ones. Most human tumors expressed the integrin alpha(v)beta(5) (14/24), whereas integrin alpha(v)beta(3) was less frequently seen (3/20). The established cell lines were efficiently transduced with adenoviral vectors, and transduction could be reduced with anti-CVADR antibodies. The abundance of appropriate viral receptors on tumor biopsy cells is a further argument for using adenoviral vectors in gene therapy of bladder cancer.

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence

To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT-1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface-covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.

Bladder cancer cell implantation in reconstructed bladder in vitro: a model of tumour recurrence

Objective To evaluate the involvement of umbrella cells in tumour adhesion and growth, by examining whether human urinary bladder carcinoma cells (HUBCC) can grow on reconstructed urinary bladder mucosa in vitro, as the implantation of tumour cells after resection is thought to be a cause of bladder tumour recurrence. Materials and methods Normal transitional epithelial cells isolated from porcine bladder were cultured on reconstructed lamina propria using fibroblasts in type I collagen gel. The urothelium thus reconstructed was artificially injured either by a scalpel or by dilute acid, after which transitional epithelial cells began to grow in a stratified fashion within a few days of culture. A HUBCC line (HT‐1197) was seeded onto this impaired mucosa to determine whether the cells could become implanted. Cultured cells on the reconstructed mucosa were evaluated by histological observation of vertical paraffin sections. Results The inoculated transitional epithelial cells grew in a stratified fashion and closely resembled urothelium in vivo. The superficial cells that were in contact with the medium solution differentiated into umbrella cells. HUBCC were unable to adhere to reconstructed mucosa which had not been injured, but these cells could adhere to and become implanted on the reconstructed mucosa after it had been injured either by a scalpel or by dilute acid. After acid injury, only the surface‐covering cells were removed sporadically, while the lower epithelial cell layer remained intact. The bladder cancer cells adhered to and proliferated within these stripped regions. Conclusion These results suggest that the urothelium, especially umbrella cells, seems to be important in preventing the adhesion and growth of urinary bladder tumour cells.

Expression of collagenase‐3 (matrix metalloproteinase‐13) in transitional‐cell carcinoma of the urinary bladder

Expression of collagenase-3 [matrix metalloproteinase-13 (MMP-13)] has been previously demonstrated in squamous-cell carcinomas of both the head and neck and the vulva, cutaneous basal-cell carcinomas, chondrosarcomas and melanomas. Using in situ hybridization, MMP-13 mRNA expression was detected in 13 of 23 (52%) urinary bladder transitional-cell carcinomas (TCCs). Expression was restricted to cells in the invading edges of tumors. No expression of MMP-13 mRNA could be detected in normal urothelium. As detected by immunohistochemistry, MMP-13 protein showed an expression pattern similar to that of MMP-13 mRNA. Expression of MMP-13 mRNA and protein was also detected in 2 bladder carcinoma cell lines (RT4 and T24). In these cell lines, TNF-α potently induced MMP-13 mRNA expression. Retinoids and a selective p38 inhibitor, SB203580, potently inhibited MMP-13 mRNA expression. Our results demonstrate MMP-13 expression in human urinary bladder carcinoma cells in vivo and in vitro and suggest that MMP-13 may serve as a marker for transformation and invasion in urinary bladder TCCs. Int. J. Cancer 88:417–423, 2000. © 2000 Wiley-Liss, Inc.

Expression of collagenase-3 (matrix metalloproteinase-13) in transitional-cell carcinoma of the urinary bladder

Expression of collagenase-3 [matrix metalloproteinase-13 (MMP-13)] has been previously demonstrated in squamous-cell carcinomas of both the head and neck and the vulva, cutaneous basal-cell carcinomas, chondrosarcomas and melanomas. Using in situ hybridization, MMP-13 mRNA expression was detected in 13 of 23 (52%) urinary bladder transitional-cell carcinomas (TCCs). Expression was restricted to cells in the invading edges of tumors. No expression of MMP-13 mRNA could be detected in normal urothelium. As detected by immunohistochemistry, MMP-13 protein showed an expression pattern similar to that of MMP-13 mRNA. Expression of MMP-13 mRNA and protein was also detected in 2 bladder carcinoma cell lines (RT4 and T24). In these cell lines, TNF-alpha potently induced MMP-13 mRNA expression. Retinoids and a selective p38 inhibitor, SB203580, potently inhibited MMP-13 mRNA expression. Our results demonstrate MMP-13 expression in human urinary bladder carcinoma cells in vivo and in vitro and suggest that MMP-13 may serve as a marker for transformation and invasion in urinary bladder TCCs.

Immunohistochemical expression of retinoblastoma gene product (Rb), p53 protein, MDM2, c-erbB-2, HLA-DR and proliferation indices in human urinary bladder carcinoma.

Archival biopsy specimens from transitional cell bladder cancers (n=88) were analysed immunohistochemically for the expression of the retinoblastoma (Rb) gene protein, p53, mdm2, c-erbB-2, HLA-DR antigen and proliferation indices. An altered nuclear expression of Rb, p53 and mdm2 was observed in 55.2%, 33.3% and 18.2% of tumors respectively. Cytoplasmic membrane immunoreactivity (>25% tumor cells) for c-erbB-2 was detected in 14.1% of tumors and aberrant HLA-DR antigen cytoplasmic staining (>5% of tumor cells) in 22.2% of the cases. P53 overexpression was associated with higher tumor grade and stage. Aberrant HLA-DR antigen expression and PCNA were also correlated with the grade of differentiation and tumor stage. MIB1 was statistically correlated with stage. pRb scores and HLA-DR antigen expression were correlated with proliferation activity as determined by PCNA and MIB1 immunostaining. p53 protein was also strongly correlated with the proliferation index PCNA. A strong correlation between PCNA and MIB1 (p<0.0001) was also found. In addition a statistically positive correlation between p53 and HLA-DR antigen expression was observed. Our data show that, although pRb and p53 protein expressions are not associated between them, they may contribute to the growth fraction of the bladder cancer. In addition, p53 and HLA-DR antigen expression could be indicators of aggressive behavior of bladder cancer.

New role for tRNA and its fragment purified from human urinary bladder carcinoma conditioned medium: inhibition of endothelial cell growth.

The growth of endothelial cells is necessary for angiogenesis, which in turn is required for later steps of tumor progression. In an attempt to purify new modulators of endothelial cell growth from the conditioned medium of human urinary bladder carcinoma cells, we isolated a small and stable oligonucleotide containing 10 to 16 bases. This oligonucleotide inhibited the growth of endothelial cells in vitro and was identified as a fragment of transfer RNA (tRNA). When unfractionated bovine tRNA was added to the cell culture, it specifically inhibited growth of endothelial cells, but not smooth muscle cells, bovine kidney cells, 3T3 fibroblasts, and several cancer cell lines. In contrast, ribosomal RNA, total yeast RNA, and single nucleosides from tRNA hydrolysate had no effect. These results demonstrate a new role for tRNA and its fragment as a selective endothelial cell inhibitor in vitro.

Human urinary bladder carcinoma cell lines respond to treatment with alkylphosphocholines

Alkylphosphocholines (APC) constitute a new group of antineoplastic agents without haematological toxicity. Their first clinically available derivative hexadecylphosphocholine (miltefosine) is locally used to control skin metastases of breast cancer. Since intravesical chemotherapy represents a form of topical treatment we investigated whether a new APC with a long alkyl chain would be active against 5637 and EJ bladder cancer cell lines. Their antineoplastic activity was inversely related to the alkyl chain length of the respective APC. Erucylphosphocholine and its congener with modified phosphocholine head erucylphospho- N, N, N-trimethylpropanolamine were the most effective derivatives. APC with alkyl chains over 16 carbons in length induced programmed cell death in both cell lines, as determined by oligonucleosomal DNA fragmentation and morphology. The distinct antineoplastic effects lead us to predict that urinary bladder instillation of APC will be of therapeutic benefit for patients with urinary bladder neoplasia.

Gelsolin gene therapy by retrovirus producer cells for human bladder cancer in nude mice.

Gelsolin, a regulator of the actin cytoskeleton, has been shown previously to act as a tumor suppressor in vitro and in vivo when introduced into certain cancer cell lines. To investigate the in vivo efficacy of gene therapy with the gelsolin gene, we inoculated nude mice with human urinary bladder cancer cells (UMUC-2 or DAB-1) and tested the effects of adding either retroviral DNA constructs containing gelsolin cDNA or retrovirus producer cells that produce the same retroviral constructs at high levels. The addition of retroviral gelsolin cDNA constructs did not inhibit tumor growth; however, this form of gene therapy, in which retrovirus producer cells were introduced, resulted in marked and reproducible tumor growth inhibition and prolonged survival time in the majority of animals tested. These findings demonstrate the potential for treating human urinary bladder carcinomas with the gelsolin gene.

Retinoid signaling in immortalized and carcinoma-derived human uroepithelial cells

This paper investigates the presence and functionality of retinoid signaling pathways in human urinary bladder carcinoma and SV40-immortalized uroepithelial cell lines. Only two of eight cell lines were proliferation-inhibited by 10 μM of either all- trans or 13- cis-retinoic acid. Transactivation of the CAT gene under control of a retinoid-responsive element demonstrated functionality of the signaling pathway in both sensitive cell lines and four of six resistant cell lines. Relative RT-PCR analysis of a panel of retinoid-responsive and inducible genes demonstrated changes in expression levels of all the genes in response to-retinoic acid treatment together with numerous aberrations dysregulations. We conclude that retinoid signaling may be a target for inactivation during tumorigenesis by uncoupling gene expression, proliferation and differentiation. Therefore retinoids are more likely to be effective for chemoprevention than for treatment of bladder carcinomas.