interleukin-8 and Urinary-Bladder-Neoplasms

In the majority of cases, death from bladder cancer results from metastatic disease. Understanding the closely linked mechanisms of invasion, metastasis and angiogenesis in bladder cancer has allowed us to develop new therapeutic strategies that harbor the promise of decisive improvements in patient survival. The essential link between cell based experiments and the translation of novel agents into human patients with bladder cancer is the animal model. With emphasis on the orthotopic xenograft model, this review outlines some key mechanisms relevant to angiogenesis and the development of metastasis in bladder cancer. We highlight especially pathways related to MMP-9, IL-8, VEGF and EGFR. Most commonly, expression patterns of these markers in patients have correlated to disease progression and patient survival, which has led to laboratory investigations of these markers and eventually novel targeted therapies that are translated back into the clinic by means of clinical trials. Although imperfect in their translatability into clinical efficacy, animal models remain a critical tool in bladder cancer research.

Topics: Animals; Carcinoma, Transitional Cell; Clinical Trials as Topic; Disease Models, Animal; ErbB Receptors; Humans; Interleukin-8; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

This study was designed to investigate whether there is a correlation between interleukin (IL)-8 secretion rate and recurrences in induction bacillus Calmette-Guérin (BCG) immunotherapy following transurethral resection (TUR) in cases of primary non-muscle-invasive bladder cancer (NMIBC).. A total of 41 patients with NMIBC were randomized to receive a 6-week course with a standard dose of 81 mg intravesical BCG. Voided urine samples were collected immediately before and after (at 2 and 4 hours) BCG instillation. IL-8 was measured using enzyme-linked immunosorbent assay. Patients were monitored according to European Association of Urology Guidelines.. Patients were monitored for a mean duration of 21.0 +/- 13.86 months. The mean time to recurrence for the 15 patients who had recurrences was 11.2 months. After adjusting for risk factors, the change in IL-8 levels at 2 hours after the first BCG compared with the levels before BCG instillation was found to be significantly predictive of recurrence (P = .047), and the best cutoff point was estimated as 112 pg/mL. The sensitivity of this measure for prediction of recurrences was 53.3%, specificity was 88.5%, positive predictive value was 72.7%, and negative predictive value was 76.7%. Comparison of patients who had values below and above this cutoff point revealed that the recurrence-free survival rate was 76.7% versus 27.3%, and the expected recurrence-free survival time was 34.9 months versus 18.8 months (P = .006).. Besides numerous other prognostic factors that have been suggested so far, a cutoff point of 112 pg/mL for IL-8 levels measured 2 hours after the first BCG instillation appears to be a good predictive factor for successful outcome in BCG treatment following TUR.

Topics: Adult; Aged; BCG Vaccine; Combined Modality Therapy; Female; Humans; Interleukin-8; Male; Middle Aged; Neoplasm Recurrence, Local; Predictive Value of Tests; Sensitivity and Specificity; Urinary Bladder Neoplasms; Urologic Surgical Procedures

We determined whether the proinflammatory cytokine interleukin-8 (IL-8) can serve as a predictor for the response to standard (120 mg.) and low (40 mg.) dose intravesical bacillus Calmette-Guerin (BCG) (modified Danish 1331 strain) for managing superficial bladder cancer in patients at risk for recurrence and progression.. We randomized 26 patients with superficial bladder cancer to receive a 6-week course of standard dose 120 mg. or low dose 40 mg. intravesical BCG. Voided urine samples were collected immediately before and after (2 and 4 hours) BCG instillation. Urine samples were centrifuged at 1,500 rpm for 8 minutes and stored at -80C. IL-8 was measured using a commercial enzyme-linked immunosorbent assay. Patients were monitored for recurrence, progression and side effects of BCG treatment at 3-month intervals.. At a median followup of 24 months (range 12 to 30), 5 and 6 patients who received a standard and low dose, respectively had disease recurrence and/or progression (nonresponders). At 4 hours after BCG mean Il-8 levels plus or minus SD were significantly higher in responders than in nonresponders (1,099.33 +/- 708.51 versus 261.82 +/- 182.66 pg./ml., p = 0.001). There was no difference at 4 hours in mean IL-8 levels in the standard and low dose groups (596.92 +/- 546 and 893 +/- 798.67 pg./ml., respectively, p = 0.28). In all patients who remained disease-free IL-8 levels were greater than 400 pg./ml. In 9 of the 11 patients with disease recurrence/progression IL-8 levels were less than 400 pg./ml.. IL-8 secretion after the initial intravesical BCG instillation strongly correlates with the possibility of future recurrence/progression. The quantitative IL-8 response to low and standard dose intravesical BCG (Danish 1331) is similar.

Topics: BCG Vaccine; Carcinoma in Situ; Disease Progression; Dose-Response Relationship, Drug; Double-Blind Method; Female; Follow-Up Studies; Humans; India; Interleukin-8; Male; Neoplasm Recurrence, Local; Neoplasm Staging; Precancerous Conditions; Prospective Studies; Treatment Outcome; Urinary Bladder; Urinary Bladder Neoplasms

Our purpose was to determine whether urinary interleukin-8 (IL-8) levels could be used to predict a tumor-free response to intravesical bacillus Calmette-Guerin (BCG) therapy in bladder cancer patients.. A total of 46 patients with superficial bladder cancer underwent an initial 6-week course of intravesical BCG therapy after transurethral resection. Voided urine samples were collected immediately before BCG instillations 1 and 6. Urine samples were centrifuged at 1,500 rpm for 8 minutes, and the supernatant was stored at -20 C. An enzyme-linked immunosorbent assay technique was used to measure urinary IL-8 levels. The Jaffé method was used to measure urinary creatinine. Results were expressed as the IL-8/creatinine ratio. Patients were followed with cystoscopy and urinary cytology every 3 months to detect bladder tumor recurrence.. IL-8/creatinine ratios were measured in 31 patients before BCG therapy and were undetectable in 15. After 5 weeks of intravesical BCG therapy, IL-8/creatinine ratios fell in 27 patients (59%), were unchanged in 10 (22%) and rose in 9 (19%). Mean followup was 20.9 months (range 3 to 48). There was no association between the direction of change in IL-8/creatinine ratio and response to intravesical BCG therapy (p = 0.5).. Urinary IL-8 levels obtained before intravesical BCG therapy and after instillation 5 of BCG were not helpful in predicting tumor recurrences in bladder cancer patients.

Topics: Adjuvants, Immunologic; Administration, Intravesical; BCG Vaccine; Carcinoma, Transitional Cell; Creatinine; Humans; Interleukin-8; Predictive Value of Tests; Urinary Bladder Neoplasms

The aim of our study is to investigate the roles of IL-8 (+ 781 C/T) and MMP-2 (-735 C/T) gene variations in early diagnosis and progression of BCA.. Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) methods were used to determine the genotype distributions of IL-8 (+ 781 C/T) and MMP-2 (-735 C/T) gene variations.. In our study, the genotype distributions of IL-8 (+ 781 C/T) and MMP-2 (-735 C/T) gene variations were not found to be significantly different between the patient and control groups. In addition, C and T allele frequencies for these gene variations were not different from the Hardy-Weinberg distribution in patient and control groups. However, when the combined genotype analyzes for these gene variations were evaluated, CC-CC and CT-CC combined genotypes for + 781 C/T / -735 C/T gene variations were observed significantly more in the patient group compared to other genotypes.. Although IL-8 (+ 781 C/T) and MMP-2 (-735 C/T) gene variations were not found to be genetic risk factors in the Thrace population in our study, CC-CC and CT-CC combined genotypes were determined as genetic risk factors for BCA susceptibility. The combined genotypes obtained as a result of the combined genotype analysis of these genetic variations that are effective in tumor progression may be considered to be important biomarkers for the early diagnosis and progression of BCA.

Topics: Case-Control Studies; Early Detection of Cancer; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Interleukin-8; Matrix Metalloproteinase 2; Polymorphism, Single Nucleotide; Urinary Bladder Neoplasms

Bladder cancer (BC) is among the most common cancers diagnosed in men in the USA. The current gold standards for the diagnosis of BC are invasive or lack the sensitivity to correctly identify the disease.. An aptamer-based screen analyzed the expression of 1317 proteins in BC compared to urology clinic controls. The top hits were subjected to systems biology analyses. Next, 30 urine proteins were ELISA-validated in an independent cohort of 68 subjects. Three of these proteins were next validated in an independent BC cohort of differing ethnicity.. Systems biology analysis implicated molecular functions related to the extracellular matrix, collagen, integrin, heparin, and transmembrane tyrosine kinase signaling in BC susceptibility, with HNF4A and NFKB1 emerging as key molecular regulators. STEM analysis of the dysregulated pathways implicated a functional role for the immune system, complement, and interleukins in BC disease progression. Of 21 urine proteins that discriminated BC from urology clinic controls (UC), urine D-dimer displayed the highest accuracy (0.96) and sensitivity of 97%. Furthermore, 8 urine proteins significantly discriminated MIBC from NMIBC (AUC = 0.75-0.99), with IL-8 and IgA being the best performers. Urine IgA and fibronectin exhibited the highest specificity of 80% at fixed sensitivity for identifying advanced BC.. Given the high sensitivity (97%) of urine D-dimer for BC, it may have a role in the initial diagnosis or detection of cancer recurrence. On the other hand, urine IL-8 and IgA may have the potential in identifying disease progression during patient follow-up. The use of these biomarkers for initial triage could have a significant impact as the current cystoscopy-based diagnostic and surveillance approach is costly and invasive when compared to a simple urine test.

Topics: Biomarkers, Tumor; Disease Progression; Humans; Immunoglobulin A; Interleukin-8; Male; Neoplasm Recurrence, Local; Proteomics; Urinary Bladder Neoplasms

Bladder cancer (BC) is the 10th most common form of cancer globally, but its complete aetiology is still unknown. Nevertheless, there is evidence that chronic inflammation plays a role in the development and progression of BC. Therefore, the presented study aimed to detect a potential association between selected single nucleotide polymorphisms (SNPs)-rs1800797 and rs2069845 in

Topics: Case-Control Studies; Genetic Predisposition to Disease; Humans; Inflammation; Interleukin-6; Interleukin-8; Methylation; Polymorphism, Single Nucleotide; Urinary Bladder Neoplasms; Urologic Diseases

Bladder cancer (BLCA) is a common genitourinary cancer in patients, and tumour angiogenesis is indispensable for its occurrence and development. However, the indepth mechanism of tumour angiogenesis in BLCA remains elusive. According to recent studies, the tight junction protein family member occludin (OCLN) is expressed at high levels in BLCA tissues and correlates with a poor prognosis. Downregulation of OCLN inhibits tumour angiogenesis in BLCA cells and murine xenografts, whereas OCLN overexpression exerts the opposite effect. Mechanistically, the RT-qPCR analysis and Western blotting results showed that OCLN increased interleukin-8 (IL8) and p-signal transducer and activator of transcription 3 (STAT3) levels to promote BLCA angiogenesis. RNA sequencing analysis and dual-luciferase reporter assays indicated that OCLN regulated IL8 transcriptional activity via the transcription factor STAT4. In summary, our results provide new perspectives on OCLN, as this protein participates in the development of BLCA angiogenesis by activating the IL8/STAT3 pathway via STAT4 and may serve as a novel and unique therapeutic target.

Topics: Animals; Humans; Interleukin-8; Mice; Neovascularization, Pathologic; Occludin; STAT3 Transcription Factor; STAT4 Transcription Factor; Urinary Bladder Neoplasms

Tumors and neutrophils undergo an unexpected interaction, in which products released by tumor cells interact to support neutrophils that in turn support cancer growth, angiogenesis, and metastasis. A key protein that is highly expressed by cancer cells in tumors is the a2 isoform V-ATPase (a2V). A peptide from a2V (a2NTD) is secreted specifically by cancer cells, but not normal cells, into the tumor microenvironment. This peptide reprograms neutrophils to promote angiogenesis, cancer cell invasiveness, and neutrophil recruitment. Here, we provide evidence that cancer-associated a2V regulates the life span of protumorigenic neutrophils by influencing the intrinsic pathway of apoptosis. Immunohistochemical analysis of human cancer tissue sections collected from four different organs shows that levels of a2NTD and neutrophil counts are increased in cancer compared with normal tissues. Significant increases in neutrophil counts were present in both poorly and moderately differentiated tumors. In addition, there is a positive correlation between the number of neutrophils and a2NTD expression. Human neutrophils treated with recombinant a2NTD show significantly delayed apoptosis, and such prolonged survival was dependent on NF-κB activation and ROS generation. Induction of antiapoptotic protein expression (Bcl-xL and Bcl-2A1) and decreased expression of proapoptotic proteins (Bax, Apaf-1, caspase-3, caspase-6, and caspase-7) were a hallmark of these treated neutrophils. Autocrine secretion of prosurvival cytokines of TNF-α and IL-8 by treated neutrophils prolongs their survival. Our findings highlight the important role of cancer-associated a2V in regulating protumorigenic innate immunity, identifying a2V as a potential important target for cancer therapy.

Topics: Adenosine Triphosphatases; Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Endometrial Neoplasms; Female; Gene Expression; Humans; Immunohistochemistry; Interleukin-8; Kidney Neoplasms; Lung Neoplasms; Mitochondria; Neoplasms; Neutrophils; NF-kappa B; Reactive Oxygen Species; Recombinant Proteins; Signal Transduction; Toll-Like Receptor 2; Tumor Microenvironment; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms

Urothelial bladder cancers (UBCs) are distinct in two main molecular subtypes, namely basal and luminal type. Subtypes are also diverse in term of immune contexture, providing a rationale for patient selection to immunotherapy.. By digital microscopy analysis of a muscle-invasive BC (MIBC) cohort, we explored the density and clinical significance of CD66b. Basal type BC contained a significantly higher density of CD66b. TAN are recruited in basal type MIBC by pro-inflammatory CKs. This finding establishes a groundwork for a better understanding of the UBC immunity and its relevance.

Topics: Aged; Aged, 80 and over; Carcinoembryonic Antigen; CD3 Complex; Cell Line, Tumor; Cell Movement; Cell Survival; Chemokine CXCL1; Chemokine CXCL2; Databases, Genetic; Female; Gene Silencing; Humans; Immunohistochemistry; Interleukin-8; Kaplan-Meier Estimate; Male; Middle Aged; Muscle Neoplasms; Neutrophils; Retrospective Studies; STAT3 Transcription Factor; T-Lymphocytes; Urinary Bladder Neoplasms

Interactions between stromal and tumor cells in tumor microenvironment contribute to tumor progression. In bladder cancer (BCa), infiltration of macrophages in tumors correlates with cancer progression. Herein, the aim was to study the paracrine effects of tumor-associated macrophages (TAM) on BCa cells.. The correlation between TAMs and tumor grade and stages was examined in tumor tissue microarrays. In addition, a conditioned media (CM) model was employed to investigate the paracrine effects of macrophages on BCa cell growth, migration, and invasion, as well as on the cytokine profile of each cell line.. The correlation of tumor-infiltrating macrophages with high-grade and muscle-invasive BCa was demonstrated in human bladder tumor tissue microarrays. CM from co-cultures of macrophages and BCa cells increased BCa cell growth, migration and invasion. Moreover, higher mRNA and protein expression levels of CCL5 and IL-8 were found in cells and CM from co-cultures, respectively.. The paracrine interaction between BCa cells and TAMs led to enhanced BCa cell growth, migration, and invasiveness, and moreover, increased IL-8 and CCL5 cytokine production in tumor microenvironment.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL5; Culture Media, Conditioned; Cytokines; Disease Progression; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Macrophages; Neoplasm Invasiveness; Neoplasm Staging; Urinary Bladder Neoplasms

Tumor endothelial cells (TEC) lining tumor blood vessels actively contribute to tumor progression and metastasis. In addition to tumor cells, TEC may develop drug resistance during cancer treatment, allowing the tumor cells to survive chemotherapy and metastasize. We previously reported that TECs resist paclitaxel treatment via upregulation of ABCB1. However, whether TEC phenotypes are altered by anticancer drugs remains to be clarified. Here, we show that ABCB1 expression increases after chemotherapy in urothelial carcinoma cases. The ratio of ABCB1-positive TEC before and after first-line chemotherapy in urothelial carcinoma tissues (

Topics: Adult; Aged; Aged, 80 and over; Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Biomarkers, Tumor; Cell Proliferation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Induction Chemotherapy; Interleukin-8; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neovascularization, Pathologic; Paclitaxel; Prognosis; Survival Rate; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Bacillus Calmette-Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca

Topics: Calcium; Calcium Signaling; Cell Line, Tumor; Cytokines; Cytosol; Humans; Interleukin-8; Mycobacterium bovis; NF-kappa B; Toll-Like Receptor 4; Urinary Bladder Neoplasms

Differentiation of noninvasive from invasive papillary urothelial carcinoma can be challenging due to inability of proper orientation and thermal damage of transurethrally obtained material. The aim of this study was to analyze the presence and extent of peritumoral retractions in pT1 compared to pTa papillary urothelial carcinoma. Since peritumoral retractions may result from altered expression profiles of extracellular matrix proteins, we additionally analyzed the expression of matrix metalloproteinase 2 (MMP-2) and interleukin 8 (IL-8) in these tumors. The study comprised 50 noninvasive (pTa) and 50 invasive (pT1) cases of transurethrally obtained primary papillary urothelial carcinomas. The invasive nature of nests showing peritumoral retractions was confirmed immunohistochemically using antibody against collagen IV. Staining for MMP-2 and IL-8 was evaluated semiquantitatively using immunohistochemical staining index, calculated by multiplying the percentage of positive cells and staining intensity. Peritumoral retractions were found in 32% of pT1 carcinomas but in none of the pTa carcinomas. All tumors showing peritumoral retraction were high grade tumors. There was no statistically significant correlation between the expression of MMP-2 or IL-8 and the presence of peritumoral retractions or stage of the tumor (pTa vs. pT1). A statistically significant but weak correlation was found between MMP-2 and IL-8 expression (χ2-test, p=0,015). There was no statistically significant correlation between the presence of peritumoral retractions or MMP-2 expression and tumor recurrence and progression. Our study shows that, in doubtful cases, when differentiating between pTa and pT1 stages of papillary urothelial carcinoma, the presence of peritumoral retractions could favor the diagnosis of invasive neoplasm.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Papillary; Female; Follow-Up Studies; Humans; Interleukin-8; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Survival Rate; Urinary Bladder Neoplasms

A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors' offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin-carbon, (V) streptavidin (strp)-gold, and (VI) strp-horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5-9%).

Topics: Biomarkers, Tumor; Biosensing Techniques; Colorimetry; Decorin; Gold; Horseradish Peroxidase; Humans; Interleukin-8; Limit of Detection; Metal Nanoparticles; Neoplasm Recurrence, Local; Protein Array Analysis; Urinalysis; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Characterization of disease-associated proteins improves our understanding of disease pathophysiology. Obtaining a comprehensive coverage of the proteome is challenging, mainly due to limited statistical power and an inability to verify hundreds of putative biomarkers. In an effort to address these issues, we investigated the value of parallel analysis of compartment-specific proteomes with an assessment of findings by cross-strategy and cross-omics (proteomics-transcriptomics) agreement. The validity of the individual datasets and of a "verified" dataset based on cross-strategy/omics agreement was defined following their comparison with published literature. The proteomic analysis of the cell extract, Endoplasmic Reticulum/Golgi apparatus and conditioned medium of T24 vs. its metastatic subclone T24M bladder cancer cells allowed the identification of 253, 217 and 256 significant changes, respectively. Integration of these findings with transcriptomics resulted in 253 "verified" proteins based on the agreement of at least 2 strategies. This approach revealed findings of higher validity, as supported by a higher level of agreement in the literature data than those of individual datasets. As an example, the coverage and shortlisting of targets in the IL-8 signalling pathway are discussed. Collectively, an integrative analysis appears a safer way to evaluate -omics datasets and ultimately generate models from valid observations.

Topics: Cell Line, Tumor; Extracellular Space; Humans; Interleukin-8; Intracellular Space; Neoplasm Proteins; Peptides; Proteome; Proteomics; Reproducibility of Results; RNA, Messenger; Sequence Analysis, RNA; Signal Transduction; Transcriptome; Urinary Bladder Neoplasms

Bladder cancer is among the five most common malignancies worldwide. Altered expression of suppressor of cytokine signaling -3 (SOCS-3) has been implicated in various types of human cancers; however, its role in bladder cancer is not well established.. The present study was undertaken to investigate the mRNA expression of SOCS-3 in normal and cancerous bladder tissue and to explore its correlation with urinary levels of some proinflammatory cytokines, cytokeratin-18 (CK -18) and with tumor histopathological grading, in order to evaluate their role as potential diagnostic markers.. SOCS3 mRNA expression levels were evaluated using quantitative real time PCR. Urinary levels of interleukins 6 and 8 were estimated by enzyme linked immunosorbent assay (ELISA). Cytokeratin-18 expression was analyzed by immuunohistochemistry then validated by ELISA.. SOC3 m RNA expression levels were significantly lower in high grade urothelial carcinoma (0.36±0.12) compared to low grade carcinoma (1.22±0.38) and controls (4.08±0.88), (p<0.001). However, in high grade urothelial carcinoma the urinary levels of IL-6, IL-8, total CK-18(221.33±22.84 pg/ml, 325.2±53.6 pg/ ml, 466.7±57.40 U/L respectively) were significantly higher than their levels in low grade carcinoma (58.6±18.6 pg/ ml, 58.3±50.2 pg/ml, 185.5±60.3 U/L respectively) and controls (50.9±23.0 pg/ml, 7.12±2.74 pg/ml, 106.7±47.3U/L respectively), (p<0.001).. Advanced grade of urothelial bladder carcinoma is significantly associated with lowered mRNA expression of SOC3 as well as elevated urinary levels of proinflammatory cytokines and CK-18. Furthermore, our results suggested that urinary IL-8, IL-6 and CK-18 may benefit as noninvasive biomarkers for early detection as well as histopathological subtyping of urothelial carcinoma.

Topics: Biomarkers, Tumor; Carcinoma, Transitional Cell; Female; Humans; Interleukin-6; Interleukin-8; Keratin-18; Male; Middle Aged; Neoplasm Grading; ROC Curve; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

The microenvironment of tumor cells is critically involved in tumor development and progression. Tumor-associated fibroblasts (TAFs) represent a major constituent of the tumor stroma. Tumor cells are operative in the activation of TAFs, whereas TAFs in turn contribute to tumor cell malignancy. This report describes mechanisms of communication between fibroblasts and urinary bladder cancer (UBC) cells. Migration of bladder cancer cell lines RT112 and Cal-29, representing two different grades of dedifferentiation, was enhanced by cocultivation with TAFs. Conditioned medium from tumor cells induced the release of interleukin (IL)-8, hepatocyte growth factor (HGF), matrix metalloproteinase-2, granulocyte macrophage colony-stimulating factor, and monocyte chemotactic protein (MCP)-1 by TAFs. Tumor cell-derived IL-1α was identified as a major mediator of these stimulatory effects. Fibroblasts, on the other hand, exerted a migration and invasion stimulating influence on UBC cells. MCP-1 and HGF were shown to promote cell migration of both bladder cancer cell lines.

Topics: Cell Communication; Cell Line, Tumor; Cell Movement; Chemokine CCL2; Chemokines; Coculture Techniques; Culture Media, Conditioned; Epithelial-Mesenchymal Transition; Fibroblasts; Granulocyte-Macrophage Colony-Stimulating Factor; Hepatocyte Growth Factor; Humans; Interleukin-1alpha; Interleukin-8; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Stromal Cells; Urinary Bladder; Urinary Bladder Neoplasms

While B cells in the tumor microenvironment may play important roles in cancer progression, their impacts on the bladder cancer (BCa) metastasis remain unclear. Here we found from human clinical BCa samples that BCa tissues could recruit more B cells than the surrounding normal bladder tissues and the in vitro co-culture assay also demonstrated that B cells could be recruited more easily towards BCa cells compared to normal bladder cells. Chamber invasion and 3D invasion assays showed the recruited B cells could then significantly increase the BCa cell invasion. Mechanism dissection found that recruited B cells could increase IL-8/androgen receptor (AR) signals in BCa cells that could then promote the expression of metastasis genes including MMP1 and MMP13. Blocking the IL-8/AR/MMPs signals either by anti-IL-8 neutralizing antibody, AR-siRNA, or MMPs inhibitors all partially reversed the infiltrating B cells capacity to increase the BCa cell invasion. The in vivo data from orthotopically xenografted BCa mouse model also confirmed that infiltrating B cells could increase BCa cell invasion via increasing AR signals. Together, these results demonstrate the key roles of B cells within the bladder tumor microenvironment that increase the BCa metastasis and may help us to develop the potential therapies via targeting these newly identified IL-8/AR/MMPs signals to better battle the BCa progression.

Topics: Animals; B-Lymphocytes; Blotting, Western; Cell Line, Tumor; Cell Movement; Cells, Cultured; Coculture Techniques; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 13; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice, Nude; Neoplasm Metastasis; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Transplantation, Heterologous; Tumor Microenvironment; Urinary Bladder Neoplasms

Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guérin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG.. Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCG's direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)-like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition.. Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG's direct biologic effect on UC cells for all of the end points evaluated.. iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.

Topics: Amidines; BCG Vaccine; Benzylamines; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Proliferation; Chemokine CCL20; Chemokine CXCL1; Chemokines, CXC; Cyclin-Dependent Kinase Inhibitor p21; Gene Expression Regulation, Neoplastic; Humans; Intercellular Adhesion Molecule-1; Interleukin-6; Interleukin-8; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Urinary Bladder Neoplasms

The ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of a myriad of clinical assays. Here, we tested the performance of a new, multiplex protein array platform to quantitate three bladder cancer-associated proteins in urine samples. The following analytes, interleukin 8 (IL8), matrix metallopeptidase 9 (MMP9), and vascular endothelial growth factor A (VEGFA) were monitored using Q-plex, a customized multiplex ELISA system from Quansys Biosciences, and individual target commercial ELISA kits. The performance of the two approaches was compared by evaluating the diagnostic accuracy of the biomarker assays in samples from a cohort of 73 subjects of known bladder cancer status.. The combination biomarker panel analyses revealed an AUROC value of 0.9476 for the Q-plex assay, and 0.9119 for the combination of the single-target ELISA assays. The Q-plex assay achieved an overall diagnostic sensitivity of 0.93 and specificity of 0.81, and the individual target ELISA assays achieved an overall sensitivity of 0.77 and specificity of 0.91.. Based on these encouraging preliminary data, we believe that the Q-Plex technology is a viable platform that can be exploited as an efficient, highly accurate tool to quantitate multiplex panels of diagnostic proteins in biologic specimens.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Matrix Metalloproteinase 9; Middle Aged; Protein Array Analysis; Sensitivity and Specificity; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Bacillus Calmette-Guérin (BCG), a vaccine against tuberculosis(TB), has been used and proven to be one of the most effective treatments for non-muscle invasive bladder cancer (BCa). However, the mechanisms of BCG action have not been completely understood, thereby limiting the improvement of BCG therapy. Vitamin D deficiency has been associated with a high risk of TB infection, and the beneficial effect of UV exposure in TB patients was proven to be mediated via activation of vitamin D signals of innate immune cells. Thus, vitamin D signals might be involved in mediating BCG immunotherapy. To test this hypothesis, we examined the impact of 1 alpha, 25-dihydroxyvitamin D3 (1,25-VD) on BCG-induced response in BCa cells and macrophage cells. Our data revealed that 1,25-VD promotes BCG-induced interleukin 8 (IL-8) secretion by BCa cells, consequently inducing the migration of macrophage, THP-1. This THP-1 cell migration promoted by 1,25-VD can be blocked by IL-8 neutralized antibody. Furthermore, 1,25-VD increased BCG-induced expression of macrophage markers in THP-1 cell, and enhanced the BCG-induced THP-1 cytotoxicity against low-grade BCa cells. Importantly, a pre-clinical trial using the N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCa mouse model revealed that intravesical co-treatment of 1,25-VD with BCG can prolong mice survival. These data demonstrate a novel mechanism by which 1,25-VD promotes BCG-mediated anti-BCa pathways and provides a platform for improving BCG efficacy with combination of 1,25-VD.

Topics: Adjuvants, Immunologic; Animals; BCG Vaccine; Calcitriol; Cell Line; Drug Synergism; Female; HL-60 Cells; Humans; Immunotherapy; Interleukin-8; Macrophages; Mice; Urinary Bladder Neoplasms

Recent studies support cell-based therapies for cancer treatment. An advantageous cell type for such therapeutic schemes are the mesenchymal stem cells (MSCs) that can be easily propagated in culture, genetically modified to express therapeutic proteins, and exhibit an innate tropism to solid tumors in vivo. Recently, we successfully isolated and expanded MSCs from second-trimester amniotic fluid (AF-MSCs). The main characteristic of AF-MSCs is their efficient and rapid expansion in vitro. Herein, we investigated the AF-MSCs tropism and capability to transport interferon beta (IFNβ) to the region of neoplasia in a bladder tumor model. To this end, we used the T24M bladder cancer cell line, previously generated from our studies, and developed a disease progression model in immunosuppressed mice, that can recapitulate the molecular events of bladder carcinogenesis. Our results documented that AF-MSCs exhibited high motility, when migrated either to T24M cells or to T24M-conditioned medium, and we further identified and studied the secreted factors which may trigger these enhanced migratory properties. Further, lentivirus-transduced AF-MSCs, expressing green fluorescent protein (GFP) or IFNβ, were intravenously administered to T24M tumor-bearing animals at multiple doses to examine their therapeutic effect. GFP- and IFNβ-AF-MSCs successfully migrated and colonized at the tumor site. Notably, significant inhibition of tumor growth as well as prolonged survival of mice were observed in the presence of IFNβ-AF-MSCs. Collectively, these results document the great potential of AF-MSCs as anti-cancer vehicles, implemented by the targeting of the tumor site and further facilitated by their high proliferation rate and expansion efficiency in culture.

Topics: Amniotic Fluid; Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Drug Delivery Systems; Green Fluorescent Proteins; Humans; Interferon-beta; Interleukin-8; Kaplan-Meier Estimate; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; Proteome; Recombinant Proteins; Tumor Burden; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

The association between chronic human exposure to arsenicals and bladder cancer development is well recognized; however, the underlying molecular mechanisms have not been fully determined. We propose that inflammatory responses can play a pathogenic role in arsenic-related bladder carcinogenesis. In previous studies, it was demonstrated that chronic exposure to 50 nM monomethylarsenous acid [MMA(III)] leads to malignant transformation of an immortalized model of urothelial cells (UROtsa), with only 3 mo of exposure necessary to trigger the transformation-related changes. In the three-month window of exposure, the cells over-expressed pro-inflammatory cytokines (IL-1β, IL-6 and IL-8), consistent with the sustained activation of NFKβ and AP1/c-jun, ERK2, and STAT3. IL-8 was over-expressed within hours after exposure to MMA(III), and sustained over-expression was observed during chronic exposure. In this study, we profiled IL-8 expression in UROtsa cells exposed to 50 nM MMA(III) for 1 to 5 mo. IL-8 expression was increased mainly in cells after 3 mo MMA(III) exposure, and its production was also found increased in tumors derived from these cells after heterotransplantation in SCID mice. UROtsa cells do express both receptors, CXCR1 and CXCR2, suggesting that autocrine cell activation could be important in cell transformation. Supporting this observation and consistent with IL-8 over-expression, CXCR1 internalization was significantly increased after three months of exposure to MMA(III). The expression of MMP-9, cyclin D1, bcl-2, and VGEF was significantly increased in cells exposed to MMA(III) for 3 mo, but these mitogen-activated kinases were significantly decreased after IL-8 gene silencing, together with a decrease in cell proliferation rate and in anchorage-independent colony formation. These results suggest a relevant role of IL-8 in MMA(III)-induced UROtsa cell transformation.

Topics: Animals; Cell Proliferation; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Interleukin-8; Male; Mice; Mice, SCID; Neoplasm Invasiveness; Organometallic Compounds; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Urinary Bladder Neoplasms; Urothelium

BLCA-4 is a specific nuclear matrix protein found in bladder cancer and there is a dearth of study on functional analysis upon this factor. We aimed to discover whether BLCA-4 is related to angiogenesis in bladder cancer.. Fifty-three bladder cancer samples were included for immunohistochemical staining of BLCA-4, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), interleukin-1α (IL-1α), IL-8, pigment epithelium-derived factor (PEDF), tumour necrosis factor-α (TNF-α) and von Willebrand factor (vWF) for microvessel density (MVD). Expressional levels were scored and grouped by clinicopathological parametres for statistical analysis for correlations.. Positive correlations were identified between expression of BLCA-4 and IL-1α (p=0.038), IL-8 (p=0.001), VEGF (p=0.002), and MMP-9 (p=0.013). No correlation was found for PEDF (p=0.182), TNF-α (p=0.531) or MVD (p=0.932). Positive correlations were also obtained in cases of advanced grade or stage, larger, recurrent and multiple tumours. Positive correlation between BLCA-4 and MMP-9 was also found in papillary urothelial neoplasm of low malignant potential (PUNLMP).. BLCA-4 may not effect pro-angiogenic pathways in bladder cancer, it can however interact with IL-1α, IL-8, VEGF and MMP-9 to enhance tumourigenesis and tumour invasiveness.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Squamous Cell; Eye Proteins; Female; Humans; Immunohistochemistry; Interleukin-1alpha; Interleukin-8; Male; Matrix Metalloproteinase 9; Middle Aged; Neoplasm Invasiveness; Neovascularization, Pathologic; Nerve Growth Factors; Nuclear Proteins; Serpins; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Young Adult

Bladder cancer is one of the most common tumors of the genitourinary tract; however, the molecular events underlying growth and invasion of the tumor remain unclear. Here, role of the CXCR7 receptor in bladder cancer was further explored. CXCR7 protein expression was examined using high-density tissue microarrays. Expression of CXCR7 showed strong epithelial staining that correlated with bladder cancer progression. In vitro and in vivo studies in bladder cancer cell lines suggested that alterations in CXCR7 expression were associated with the activities of proliferation, apoptosis, migration, invasion, angiogenesis and tumor growth. Moreover, CXCR7 expression was able to regulate expression of the proangiogenic factors IL-8 or VEGF, which may involve in the regulation of tumor angiogenesis. Finally, we found that signaling by the CXCR7 in bladder cancer cells activates AKT, ERK and STAT3 pathways. The AKT and ERK pathways may reciprocally regulate, which are responsible for in vitro and in vivo epithelial to mesenchymal transition (EMT) process of bladder cancer. Simultaneously targeting the two pathways by using U0126 and LY294002 inhibitors or using CCX733, a selective CXCR7 antagonist drastically reduced CXCR7-induced EMT process. Taken together, our data show for the first time that CXCR7 plays a role in the development of bladder cancer. Targeting CXCR7 or its downstream-activated AKT and ERK pathways may prove beneficial to prevent metastasis and provide a more effective therapeutic strategy for bladder cancer.

Topics: Animals; Butadienes; Chromones; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; MAP Kinase Signaling System; Mice; Mice, SCID; Morpholines; Neovascularization, Pathologic; Nitriles; Proto-Oncogene Proteins c-akt; Receptors, CXCR; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Current urine-based assays for bladder cancer (BCa) diagnosis lack accuracy, so the search for improved biomarkers continues. Through genomic and proteomic profiling of urine, we have identified a panel of biomarkers associated with the presence of BCa. In this study, we evaluated the utility of three of these biomarkers, interleukin 8 (IL-8), Matrix metallopeptidase 9 (MMP-9) and Syndecan in the diagnosis of BCa through urinalysis.. Voided urines from 127 subjects, cancer subjects (n = 64), non-cancer subjects (n = 63) were analyzed. The protein concentrations of IL-8, MMP-9, and Syndecan were assessed by enzyme-linked immunosorbent assay (ELISA). Data were also compared to a commercial ELISA-based BCa detection assay (BTA-Trak©) and urinary cytology. We used the area under the curve of a receiver operating characteristic (AUROC) to compare the performance of each biomarker.. Urinary protein concentrations of IL-8, MMP-9 and BTA were significantly elevated in BCa subjects. Of the experimental markers compared to BTA-Trak©, IL-8 was the most prominent marker (AUC; 0.79; 95% confidence interval [CI], 0.72-0.86). Multivariate regression analysis revealed that only IL-8 (OR; 1.51; 95% CI, 1.16-1.97, p = 0.002) was an independent factor for the detection of BCa.. These results suggest that the measurement of IL-8 in voided urinary samples may have utility for urine-based detection of BCa. These findings need to be confirmed in a larger, prospective cohort.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Carcinoma, Transitional Cell; Case-Control Studies; Cohort Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Matrix Metalloproteinase 9; Middle Aged; ROC Curve; Sensitivity and Specificity; Syndecans; Urinary Bladder Neoplasms

Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their inhibitors. The purpose of this study was to investigate whether the expression of MMP-9, MMP-2 and its specific inhibitors, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis in Bladder Cancer (BC).. MMP-9, MMP-2 and its specific inhibitors expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in fresh-frozen malignant tissue collected from 40 patients with BC submitted to transurethral resection of bladder. The control group consisted of normal bladder tissue from five patients who had undergone retropubic prostatectomy to treat benign prostatic hyperplasia.. MMP-9 was overexpressed in 59.0 % of patients, and MMP-2, TIMP-1, TIMP-2, MMP-14, RECK and IL-8 was underexpressed in most of the patients. Regarding prognostic parameters we observed that high-grade tumors exhibited significantly higher levels of MMP-9 and IL-8 (p = 0.012, p = 0.003). Invasive tumors (pT1-pT2) had higher expression levels of MMP-9 than superficial tumors (pTa) (p = 0.026). The same was noted for IL-8 that was more expressed by invasive tumors (p = 0.015, p = 0.048). Most importantly tumor recurrence was related with higher levels of both MMP-9 (p = 0.003) and IL-8 (p = 0.005).. We have demonstrated that the overexpression of MMP-9 and higher expression of IL-8 are related to unfavorable prognostic factors of urothelial bladder cancer and tumor recurrence and may be useful in the follow up of the patients.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Transitional Cell; Case-Control Studies; Cohort Studies; Female; Gene Expression Profiling; GPI-Linked Proteins; Humans; Interleukin-8; Male; Matrix Metalloproteinase 14; Matrix Metalloproteinase 9; Middle Aged; Prognosis; Real-Time Polymerase Chain Reaction; Tissue Inhibitor of Metalloproteinases; Urinary Bladder Neoplasms

Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These data show that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.

Topics: Adult; Aged; Aged, 80 and over; Apolipoproteins E; Biomarkers, Tumor; Carcinoma; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-8; Male; Middle Aged; Multivariate Analysis; Neoplasm Grading; Neoplasm Staging; ROC Curve; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Sensitive and specific urinary biomarkers can improve patient outcomes in many diseases through informing early diagnosis. Unfortunately, to date, the accuracy and translation of diagnostic urinary biomarkers into clinical practice has been disappointing. We believe this may be due to inappropriate standardization of diagnostic urinary biomarkers. Our objective was therefore to characterize the effects of standardizing urinary levels of IL-6, IL-8, and VEGF using the commonly applied standards namely urinary creatinine, osmolarity and protein. First, we report results based on the biomarker levels measured in 120 hematuric patients, 80 with pathologically confirmed bladder cancer, 27 with confounding pathologies and 13 in whom no underlying cause for their hematuria was identified, designated "no diagnosis". Protein levels were related to final diagnostic categories (p = 0.022, ANOVA). Osmolarity (mean = 529 mOsm; median = 528 mOsm) was normally distributed, while creatinine (mean = 10163 µmol/l, median = 9350 µmol/l) and protein (0.3297, 0.1155 mg/ml) distributions were not. When we compared AUROCs for IL-6, IL-8 and VEGF levels, we found that protein standardized levels consistently resulted in the lowest AUROCs. The latter suggests that protein standardization attenuates the "true" differences in biomarker levels across controls and bladder cancer samples. Second, in 72 hematuric patients; 48 bladder cancer and 24 controls, in whom urine samples had been collected on recruitment and at follow-up (median = 11 (1 to 20 months)), we demonstrate that protein levels were approximately 24% lower at follow-up (Bland Altman plots). There was an association between differences in individual biomarkers and differences in protein levels over time, particularly in control patients. Collectively, our findings identify caveats intrinsic to the common practice of protein standardization in biomarker discovery studies conducted on urine, particularly in patients with hematuria.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Carcinoma, Transitional Cell; Case-Control Studies; Creatinine; Hematuria; Humans; Interleukin-6; Interleukin-8; Male; Middle Aged; Reference Standards; Sensitivity and Specificity; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

Pigment epithelium-derived factor (PEDF) is an antiangiogenic factor which is effective in tumour inhibition in a variety of tumours and has not yet been studied in bladder tumour before. In this study the expression of PEDF, interleukin-1α (IL-1α) and -8 (IL-8) in bladder tumours was investigated. Immunohistochemistry was performed on 64 bladder tumour and 23 normal uroepithelium samples. Expression change of the factors was compared with clinicopathological parameters. Correlations between PEDF, IL-1α and IL-8 were analyzed. None of the factors was in relation to gender, tumour occurrence, and size or onset pattern. PEDF (P=0.014) and IL-1α (P=0.049) expression was down-regulated with grade progression. PEDF expression was lower in normal uroepithelium than in papillary urothelial neoplasm of low malignant potential (PUNLMP) (P=0.000) and carcinoma (P=0.009) whilst IL-1α (P=0.000 and P=0.000 respectively) and IL-8 (P=0.000 and P=0.023 respectively) expression was higher in the same grouping. PEDF expression had a negative correlation with IL-8 in PUNLMP (P=0.049, r=-0.578) as well as in tumour grouping (P=0.033, r=-0.276). Deranged expressional change of PEDF, IL-1α and IL-8 could be in relation to loss of differentiation from normal uroepithelium to papillary lesion and eventually to carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Papillary; Eye Proteins; Female; Humans; Interleukin-1alpha; Interleukin-8; Male; Middle Aged; Nerve Growth Factors; RNA, Messenger; Serpins; Urinary Bladder Neoplasms; Young Adult

Chemokines and transcription factor NF-kappaB play a pivotal role in development of carcinoma of the bladder (CaB). The present study was conducted to analyze the association of chemokines IL-8 -251 T>A and +678 C>T and NF-kappaB -94 (ATTG) insertion/deletion polymorphisms with the risk of CaB and outcome after bacillus Calmette-Guerin (BCG) immunotherapy in a cohort of northern India.. Histologically confirmed 205 CaB cases and 270 controls were included. Of these, 71 patients were treated with BCG immunotherapy. Genotyping was done using allele-specific PCR methodology.. The variant genotype (AA) of IL-8 -251 polymorphism was associated with more than 2-fold risk of CaB (OR 2.12; p = 0.003; 95% CI 1.28-3.52). None of the other genotypes showed association with CaB risk. Subsequently, the diplotype -251A/+678T demonstrated a 1.8-fold increased risk for CaB (OR 1.84, 95% CI 1.37-2.47). Furthermore, -251 AA genotypes reduced the risk of recurrence after BCG immunotherapy (AA; HR 0.12; 95% CI 0.04-0.41). Subsequently, improved recurrence-free survival (mean recurrence-free survival for GG, GA and AA genotypes was 24, 39 and 53 months respectively). Similarly, NF-kappaB ATTG Ins/Ins genotype was at reduced risk of recurrence after BCG treatment compared to Del/Del genotype, which exhibited a 2.5-fold increased risk of recurrence in patients treated with BCG immunotherapy (HR, 2.53; 95% CI 1.00-6.36). Subsequently, mean recurrence-free survival (Ins/Ins, 41; Ins/Del, 44 and Del/Del, 10 months; log rank, 0.030).. Our results suggested that the IL-8 -251 T>A polymorphism may be a relevant host susceptibility factor for bladder carcinoma development and may influence outcome after BCG immunotherapy. Similarly, NF-kappaB ATTG polymorphism may also modify risk-free survival of BCG-treated patients.

Topics: Aged; BCG Vaccine; Female; Genetic Predisposition to Disease; Humans; Immunotherapy; India; Interleukin-8; Kaplan-Meier Estimate; Male; Middle Aged; NF-kappa B; Nicotiana; Polymorphism, Genetic; Recurrence; Treatment Outcome; Urinary Bladder Neoplasms

Bladder cancer is a major health problem in Egypt as it represents the most common malignancy.. Evaluation of the potential usefulness of serum IL-8 and IGF-1 in Egyptian bladder cancer patients.. Serum levels of IL-8 and IGF-1 were determined in 51 bladder cancer patients and 45 controls using a chemiluminescence enzyme immunometric assay.. Serum IL-8 was significantly higher in cancer patients than in controls (P < 0.0001). It was significantly higher in patients with invasive cancer than those with superficial cancer (P < 0.01). Also, IL-8 showed a significant elevation in relation to schistosomal infection (P = 0.02) however, it did not differ in relation to either pathological type of tumor or its grade (P > 0.05). Receiver operating characteristic (ROC) curve indicated that IL-8 cut-off value of 35 pg/mL yielded the best combination of sensitivity (71%) and specificity (98%) for differentiating patients with bladder cancer from control subjects. Serum IGF-1 levels showed no significant difference between bladder cancer patients and controls (P > 0.05). There was no relationship between IGF-1 levels and clinicopathological parameters.. In Egyptian patients with bladder cancer, serum IL-8 is significantly elevated and its level is related to tumor invasion and associated schistosomal infection. Moreover, serum IGF-1 level does not help as a serum tumor marker in these patients.

Topics: Aged; Biomarkers, Tumor; Egypt; Female; Humans; Insulin-Like Growth Factor I; Interleukin-8; Male; Middle Aged; Neoplasm Invasiveness; Schistosomiasis; Urinary Bladder Neoplasms

This study was designed to determine the relative activity of angiogenesis-related genes in the regulation of tumorigenicity and subsequent metastases of urothelial cell carcinomas (UC) of the urinary bladder.. We selected the clones with the highest and lowest expression level of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)/vascular permeability factor or interleukin-8 (IL-8) in the highly tumorigenic and metastatic human UC cell line 253J B-V. Tumorigenicity and production of spontaneous lymph node metastases were evaluated 1, 2, 4, 8 and 12 weeks after orthotopic implantation of each specific expression clone into the urinary bladder of athymic nude mice. Moreover, the transitional changes in the expression of angiogenesis-related genes and neovascularization were determined in tumors and metastases.. At the early stage of tumor growth following orthotopic implantation, tumorigenicity and metastases were significantly increased in the clones with the highest expression of bFGF and IL-8, while they were significantly inhibited in the clones with the lowest expression of bFGF and IL-8 compared to parental 253J B-V. In the tumors, specific expression of angiogenesis-related genes and intratumor neovascularity of each clone were gradually regulated to the same level as parental 253J B-V. In metastasized tumors of the highest and lowest IL-8-expressing clones, IL-8 expression was consistently high and low, respectively.. These findings indicate that at the early stage of tumor growth, bFGF and IL-8 expression play important roles in the regulation of angiogenesis, tumorigenicity and subsequent metastases of human bladder cancer.

Topics: Animals; Carcinoma; Cell Line, Tumor; Disease Progression; Fibroblast Growth Factor 2; Gene Expression; Humans; Interleukin-8; Lymphatic Metastasis; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

The molecular and pharmacological properties of adenosine receptors in the T24 human bladder epithelial carcinoma cell line were assessed by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Ca2+ Flux, cAMP production and interleukin-8 measurements. RT-PCR experiments detected the presence of transcripts for the adenosine A1, A2A and A2B receptors but not for the adenosine A3 subtype. Application of specific adenosine receptor ligands resulted in concentration-dependent increases in intracellular calcium ([Ca2+]i) with the following order of potency and EC50 values: 5'-N-Ethylcarboxamidoadenosine (NECA) (1153+/-214)>5'-(N-Cyclopropyl)carboxamidoadenosine (CPCA) (1436+/-186)>adenosine (4823+/-932). This rank order of potency is typical of adenosine A2B receptors. In addition, select adenosine receptor antagonists N-(4-acetylphenyl)-2-[4-(2,3,6,7-tetrahydro-2,6 dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]acetamide (MRS 1706), 8-[4-[((4-Cyano[2,6-]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)-xanthine (MRS 1754), 1,3-Diethyl-8-phenylxanthine (DPCPX), 1,3-Diethyl-8-phenylxanthine (DPX), Alloxazine, 8-(3-Chlorostyryl)caffeine (CSC), and Theophylline blocked the NECA-induced calcium responses. Additionally, NECA, CPCA, and adenosine stimulated cAMP formation with a rank order of potency characteristic of adenosine A2B receptors. The select adenosine A2A antagonist, 5-amino-7-(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH 58261) failed to antagonize the NECA response, whereas the potent and highly selective adenosine A2B antagonists MRS 1754 and MRS 1706 inhibited NECA-stimulated cAMP production. Lastly, NECA-induced interleukin-8 secretion was inhibited by MRS 1754. Taken together, these data indicate that [Ca2+]i accumulation and cAMP production as well as interleukin-8 secretion is mediated through the adenosine A2B receptor in the T24 cell line.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcium; Cell Line, Tumor; Cyclic AMP; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Intracellular Fluid; Protein Isoforms; Purines; Receptors, Purinergic P1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Urinary Bladder Neoplasms; Xanthines

The role of polymorphonuclear neutrophil granulocytes (PMN) in antitumoral immune responses displays a striking dichotomy. Under inflammatory conditions, PMN may promote tumor growth and progression. In contrast, especially in the context of therapeutic interventions, PMN can exert important antitumor functions. However, until now, the mechanisms of PMN-mediated activation of tumor immunity are poorly defined. Based on a murine model of Bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer, we provide evidence for a novel immunoregulatory role of this leukocyte subset. PMN immigrate into the bladder after intravesical BCG instillation and depletion of PMN from tumor-bearing mice completely abrogated antitumor efficacy of BCG. PMN stimulated with BCG in vitro as well as PMN isolated from the urine of BCG-treated patients were a major source of the chemokines interleukin-8, growth-related oncogene-alpha, macrophage inflammatory protein-1 alpha and of the inflammatory cytokine migration inhibitory factor. In vitro, BCG-stimulated PMN indirectly induced T-cell chemotaxis via the accessory function of activated monocytes. In vivo, depletion of PMN from BCG-treated mice significantly impaired CD4(+) T-cell trafficking to the bladder. These data show that PMN direct the migration of effector cells to the bladder and by this means are indispensable for effective tumor immunotherapy. Thus, our findings provide evidence for a novel early immunoregulatory role of these innate immune cells in local antitumor immunity.

Topics: Animals; BCG Vaccine; Chemokine CCL4; Chemokine CXCL1; Chemokines, CXC; Granulocytes; Immunosuppression Therapy; Immunotherapy; Interleukin-8; Macrophage Inflammatory Proteins; Mice; Neutrophils; Reference Values; Urinary Bladder Neoplasms

In a recent study, we showed that the proteasome inhibitor bortezomib sensitizes human bladder cancer cells to IFN-induced cell death. Here, we characterized the molecular mechanisms underlying the antitumoral effects of the combination in more detail. Bortezomib synergized with IFN-alpha to promote apoptosis via a tumor necrosis factor-related apoptosis-inducing ligand-associated mechanism but did not inhibit production of proangiogenic factors (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) in human UM-UC-5 cells. In contrast, exposure to the combination did not increase the levels of apoptosis in human UM-UC-3 cells but did inhibit the production of basic fibroblast growth factor and vascular endothelial growth factor. Studies with tumor xenografts confirmed that combination therapy with bortezomib plus IFN-alpha was effective in both models but that the effects were associated with differential effects on tumor necrosis factor-related apoptosis-inducing ligand-associated apoptosis (predominant in UM-UC-5) versus inhibition of angiogenesis (predominant in UM-UC-3). Together, our results show that combination therapy with IFN-alpha plus bortezomib is effective but can work via different mechanisms (apoptosis versus angiogenesis inhibition) in preclinical models of human bladder cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Cell Growth Processes; Fibroblast Growth Factor 2; Humans; Interferon-alpha; Interleukin-8; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Pyrazines; TNF-Related Apoptosis-Inducing Ligand; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

Since chronic inflammation contributes to tumorigenesis, we hypothesized that the risk and clinical outcome of bladder cancer (BC) might be modulated by genetic variations in inflammation genes.. Using the TaqMan method, we genotyped single nucleotide polymorphisms in interleukin (IL) -6 (-174 G-->C), IL-8 (-251 T-->A), tumor necrosis factor-alpha (TNF-alpha; -308 G-->A), and peroxisome proliferator-activated receptor gamma (PPARG; Pro12Ala), and determined their associations with BC initiation and clinical outcome.. We found that the IL-6 variant genotype (C/C) was associated with an increased BC risk (OR, 1.77; 95% CI, 1.25 to 2.51). There were joint effects between the variant IL-6 genotypes and smoking status, and between the variant genotypes of IL-6 and other genes. To assess effect on recurrence, we grouped non-muscle-invasive BC patients according to intravesical Bacillus Calmette-Guerin (BCG) treatment status: no BCG, induction BCG (iBCG), and maintenance BCG (mBCG). In the Cox proportional hazards model, the variant IL-6 genotype was associated with an increased recurrence risk (hazard ratio [HR], 4.60; 95% CI, 1.24 to 17.09) in patients receiving mBCG. The variant PPARG genotype was associated with a reduced recurrence risk (HR, 0.41; 95% CI, 0.20 to 0.86) among untreated patients. In patients with non-muscle-invasive BC, the variant IL-6 genotype was associated with an increased progression risk (HR, 1.88; 95% CI, 0.80 to 4.11). In patients with invasive BC, variant IL-6 was associated with improved 5-year overall and disease-specific survival (HR, 0.43; 95% CI, 0.19 to 0.94 and HR, 0.39; 95% CI, 0.15 to 1.00, respectively).. Inflammation gene polymorphisms are associated with modified BC risk, treatment response, and survival.

Topics: Aged; Case-Control Studies; Chi-Square Distribution; Disease Progression; Female; Genotype; Humans; Inflammation; Interleukin-6; Interleukin-8; Logistic Models; Male; Middle Aged; Neoplasm Recurrence, Local; Polymorphism, Genetic; PPAR gamma; Proportional Hazards Models; Risk Factors; Survival Analysis; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms

Bortezomib (PS-341, Velcade) is a dipeptidyl boronic acid inhibitor of the 20S proteasome that was developed as a therapeutic agent for cancer. Here, we investigated the effects of bortezomib on the growth of human 253JB-V bladder cancer cells. Although the drug did not stimulate significant increases in levels of apoptosis, it inhibited cell growth in a concentration-dependent fashion and augmented the growth inhibitory effects of gemcitabine in vitro. These effects were associated with accumulation of p53 and p21 and suppression of cyclin-dependent kinase 2 activity. Bortezomib also inhibited secretion of the proangiogenic factors matrix metalloproteinase-9, interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF). In vivo studies with 253JB-V tumors growing in nude mice demonstrated that bortezomib (1 mg/kg) did not inhibit tumor growth when it was delivered as a single agent, although it reduced tumor microvessel density and inhibited expression of VEGF and IL-8. However, combination therapy with bortezomib plus gemcitabine produced synergistic tumor growth inhibition associated with strong suppression of tumor cell proliferation. Together, our results demonstrate that bortezomib has significant antiproliferative activity in aggressive bladder cancer cells, which is best exploited within the context of combination chemotherapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; CDC2-CDC28 Kinases; Cell Death; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Deoxycytidine; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Gemcitabine; Humans; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-8; Male; Matrix Metalloproteinase 9; Mice; Mice, Nude; Multienzyme Complexes; Neoplasm Transplantation; Neovascularization, Pathologic; Proteasome Endopeptidase Complex; Pyrazines; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A

This study was planned to evaluate the feasibility of the assay of leukocyte arylsulfatase-A (AS-A) activity, and some urinary cytokine levels (tumor necrosis factor-alpha [TNF-alpha] and interleukin-8 [IL-8]), as noninvasive diagnostic tools in different stages of bladder cancer patients.. Blood and urine samples of 79 subjects were analyzed, including 28 healthy volunteers, 27 patients with superficial bladder cancer (SBC), and 24 patients with muscle invasive bladder cancer (MIBC).. In SBC patients, the mean leukocyte AS-A activity was slightly higher (11.4%) than healthy subjects without reaching statistical significance. On the other hand, the enzyme activity in MIBC patients was significantly higher than those of controls (38.9%) and SBC patients (18.3%). Urinary TNF-alpha levels in both cancer groups were not significantly different from the control group. Urinary IL-8 levels of MIBC patients were significantly increased when compared with the levels of SBC patients and healthy subjects (P < 0.001).. Based on our results, it may be concluded that measurement of leukocyte AS-A activity is not a sufficiently reliable noninvasive diagnostic test in distinguishing early stage bladder cancer from healthy subjects as well as detecting disease progression. Whereas measurement of urinary IL-8 may be valuable as a noninvasive diagnostic and prognostic test especially in patients with advanced bladder cancer. It also appears that complementary biochemical information may be obtained about the prognosis of the disease by monitoring urinary IL-8 profile. However, further confirmatory clinical trials about the prognostic value of the measurement of urinary IL-8 are needed.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Cerebroside-Sulfatase; Female; Humans; Interleukin-8; Leukocytes; Male; Middle Aged; Prognosis; ROC Curve; Sensitivity and Specificity; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms

Interleukin (IL)-8 is one of several angiogenic factors produced in bladder cancer cell lines. Although many studies have indicated that IL-8 is increased in infectious/inflammatory processes, elevated levels of IL-8 in urine also may be indicative of active transitional cell carcinoma (TCC).. Urinary IL-8 levels were measured with an enzyme-linked immunosorbent assay in subjects with TCC (n = 51), prostate cancer (n = 17), or a history of successfully treated TCC (n = 23) and in healthy subjects (n = 49). In addition, urinary IL-8 levels were measured in 21 subjects before, during, and 1 month after intravesical therapy with bacille Calmette-Guérin or mitomycin C.. The median urinary IL-8 levels were greater in subjects with TCC (64 pg/mL urine) than in healthy subjects (6 pg/mL urine), subjects with prostate cancer (0.5 pg/mL urine), or subjects with a history of successfully treated TCC (5.0 pg/mL urine). Urinary IL-8 levels were greater in subjects with invasive (high-stage) TCC than in those with low-stage TCC. Furthermore, the postintravesical instillation levels of urinary IL-8 levels were greater in patients whose TCC recurred compared with those whose TCC was in remission.. IL-8 levels were greater in subjects with TCC compared with those with successfully treated TCC. IL-8 levels increased with TCC stage, indicating that they are greater in more invasive (ie, angiogenic) tumors. A reduction in urinary IL-8 levels after treatment with bacille Calmette-Guérin or mitomycin C may reflect a decrease in bladder cancer cells and/or in other cells that produce IL-8.

Topics: Adenocarcinoma; Administration, Intravesical; Aged; Antineoplastic Agents, Alkylating; BCG Vaccine; Biomarkers, Tumor; Carcinoma in Situ; Carcinoma, Transitional Cell; Female; Humans; Immunotherapy; Interleukin-8; Male; Middle Aged; Mitomycin; Prospective Studies; Prostatic Neoplasms; Urinary Bladder Neoplasms

Interleukin (IL)-8 is an important mediator of angiogenesis, tumorigenicity, and metastasis in transitional cell carcinoma (TCC) of the bladder. Nuclear factor kappaB (NF-kappaB)/relA regulates IL-8 expression in several neoplasms. The purpose of this study was to determine whether the organ microenvironment (hypoxia, acidosis) regulates the expression of IL-8 in TCC via NF-kappaB, and whether inhibition of NF-kappaB function by mutant IkappaB-alpha prevents induction of IL-8 expression.. IL-8 mRNA expression and protein production by human TCC cell lines (UM-UC-14, HTB-9, RT-4, KU-7 and 253J B-V) were measured by Northern blot analysis and ELISA under acidic (pH 7.35-6.0) and hypoxic (1.0% O(2)) conditions. The involvement of NF-kappaB and activator protein 1 in the regulation of IL-8 production was evaluated by electrophoretic mobility shift assay. Furthermore, the tumorigenicity and metastatic potential of UM-UC-14 cells were determined after transfection with mutant IkappaB-alpha.. We found that acidic and hypoxic conditions increased IL-8 mRNA expression and protein production by several, but not all, TCC cell lines evaluated. NF-kappaB, but not activator protein 1, was inducibly activated in UM-UC-14 under both acidic and hypoxic conditions, but not in UM-UC-14 mutant IkappaB-alpha transfectants. Tumor growth and lymph node metastasis were inhibited in UM-UC-14 mutant IkappaB-alpha transfectants compared with UM-UC-14 controls. This effect was associated with the inhibition of IL-8 production, cellular proliferation, and angiogenesis.. These results suggest that TCCs of the bladder have heterogenic responses to physicochemical changes in the microenvironment and identify NF-kappaB as a potential molecular target for therapy.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Division; Cell Line, Tumor; Cell Nucleus; DNA Fragmentation; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Humans; Hydrogen-Ion Concentration; Hypoxia; I-kappa B Proteins; In Situ Hybridization; Interleukin-8; Lymphatic Metastasis; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutation; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; Protein Binding; RNA, Messenger; Transfection; Up-Regulation; Urinary Bladder Neoplasms

We previously demonstrated that overexpression of interleukin 8 (IL-8) in human transitional cell carcinoma (TCC) resulted in increased tumorigenicity and metastasis. This increase in tumor growth and metastasis can be attributed to the up-regulation in the expression and activity of the metalloproteinases MMP-2 and MMP-9.. To investigate whether targeting IL-8 with a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy for controlling TCC growth, we studied its effects on TCC growth in vitro and in an in vivo mouse model. Human TCC cell lines 253J B-V and UM UC3 (high IL-8 producers), 253J (low IL-8), and 253J transfected with the IL-8 gene (high producer) were used.. ABX-IL8 had no effect on TCC cell proliferation in vitro. However, in the orthotopic nude mouse model, after 4 weeks of treatment (100 micro g/week, i.p.), a significant decrease in tumor growth of both cell lines was observed. IL-8 blockade by ABX-IL8 significantly inhibited the expression, activity, and transcription of MMP-2 and MMP-9, resulting in decreased invasion through reconstituted basement membrane in vitro. The down-regulation of MMP-2 and MMP-9 in these cells could be explained by the modulation of nuclear factor-kappaB expression and transcriptional activity by ABX-IL8.. Our data point to the potential use of ABX-IL8 as a modality to treat bladder cancer and other solid tumors, either alone or in combination with conventional chemotherapy or other antitumor agents.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Carcinoma, Transitional Cell; Cell Division; Cell Line, Tumor; Cell Nucleus; Collagen; Down-Regulation; Drug Combinations; Humans; Interleukin-8; Laminin; Luciferases; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; NF-kappa B; Nucleic Acid Hybridization; Promoter Regions, Genetic; Proteoglycans; RNA, Messenger; Time Factors; Transcription, Genetic; Up-Regulation; Urinary Bladder Neoplasms

The angiogenic inhibitor TNP-470 (AGM-1470, O-chloracetyl-carbamoyl fumagillol) has been reported to inhibit the growth of human transitional cell carcinoma (TCC) in the urinary bladder. However, it is still unknown whether TNP-470 inhibits metastasis of TCC. Here, we identify an efficient protocol using TNP-470, and optimize its antitumor and antimetastatic effects on human TCC in the urinary bladder.. In vitro, the human metastatic TCC cell line 253J B-V and human umbilical vascular endothelial cells were treated with TNP-470, and examined for cell growth and protein production of angiogenic factors. To study in vivo effects of TNP-470, 253J B-V cells were implanted orthotopically into athymic nude mice. TNP-470 was administered in several dosing and scheduling regimens, and its effects on tumor growth, metastasis, intratumor neovascularization, and mRNA expression of angiogenic factors were determined in both nonestablished and established tumors.. In vitro treatment with TNP-470 inhibited cell growth and production of basic fibroblast growth factor protein in 253J B-V and human umbilical vascular endothelial cells in a dose-dependent manner. In vivo daily administration of TNP-470 significantly inhibited tumor growth (P < 0.001), metastasis (P < 0.05), intratumor neovascularization (P < 0.005), and mRNA expression of basic fibroblast growth factor and MMP-9 (P < 0.005), in both nonestablished and established tumors. Increasing the daily dose did not increase the effect on tumor growth, metastasis, and angiogenesis; however, the drug-induced toxicity did increase in a dose-dependent manner.. Frequent administration of TNP-470 at an optimal biological dose provided maximal antitumor and antimetastatic effects of human TCC of the urinary bladder. It may function by down-regulating angiogenic factors.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Transitional Cell; Cell Division; Cyclohexanes; Dose-Response Relationship, Drug; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Humans; Immunoenzyme Techniques; In Situ Hybridization; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Interleukin-8; Lymphatic Metastasis; Lymphokines; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; O-(Chloroacetylcarbamoyl)fumagillol; Platelet Endothelial Cell Adhesion Molecule-1; RNA, Messenger; Sesquiterpenes; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

T24 human bladder carcinoma cells reveal a high locomotor activity (70% locomoting cells) within a 3-dimensional collagen matrix. This high migratory activity is induced by an autocrine engagement of the interleukin-8 receptor A, as was shown by antibodies neutralizing the secreted interleukin-8. Treatment of the cells with these specific antibodies reduced the locomotor activity by half. The intracellular signal transduction underlying the interleukin-8-induced T24 locomotion involves the activity of protein tyrosine kinases (PTKs), the phospholipase Cgamma (PLCgamma) and the protein kinase C (PKC), as proven by the use of specific enzyme inhibitors. These results suggest the following model for the regulatory signal transduction of interleukin-8-induced human T24 bladder carcinoma cell migration: The engagement of the interleukin-8-receptor, a receptor of the serpentine family, leads to the beta-arrestin-mediated activation of PTKs. These kinases phosphorylate the PLCgamma, which generates the second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP(3)). DAG activates the PKC, whereas IP(3) mediates the release of calcium from the endoplasmatic reticulum. By means of confocal laser microscopy, we observed an oscillation of the cytosolic calcium concentration in migrating T24 cells, which were loaded with the calcium-dye fluo-3/AM. Here, we report on a new autocrine function of secreted interleukin-8 and the intracellular signal transduction leading to the regulation of cytosolic calcium and to a migratory tumor cell phenotype.

Topics: Antibodies; Arrestins; beta-Arrestins; Calcium; Cell Movement; Diglycerides; Enzyme Activation; Humans; Inositol 1,4,5-Trisphosphate; Interleukin-8; Ionomycin; Isoenzymes; Kinetics; Microscopy, Confocal; Phospholipase C gamma; Protein Kinase C; Protein-Tyrosine Kinases; Receptors, Interleukin-8A; Second Messenger Systems; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Type C Phospholipases; Urinary Bladder Neoplasms

Studies on solid cancer(such as breast cancer, hepatocellular cancer, pancreatic cancer) indicated that the abnormal expression of nuclear transcription factor Kappa B (NF-kappa B) regulates angiogenesis and cyclin-related genes. This study was designed to investigate the expression differences of NF-kappa B and its regulated genes between human primary transitional cell carcinoma(TCCs) of bladder and non-cancer bladder mucosa and its clinical significance.. Forty-three frozen sections including 30 bladder cancer and 13 non-cancer bladder mucosa were subjected to immunohistochemistry and nucleus staining for determining levels of NF-kappa B family and I kappa B alpha; Five paired cancer and non-cancer specimens were subjected to Western blot for analysis p65, an important subtype of NF-kappa B; Thirteen paired specimens were subjected to RT-PCR for determination mRNA levels of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8.. Expressions of p50, p52, p65, c-Rel, RelB, I kappa B alpha, CyclinD1, IL-8 mRNAs in bladder cancer were higher than that in non-cancer bladder mucosa (P < 0.01, P < 0.05, P < 0.01, P < 0.01, P < 0.05, P < 0.05, P < 0.05 and P < 0.05, respectively). Nucelus stainings of p50, p52, p65, c-Rel, RelB were also stronger in bladder cancer(P < 0.01, P < 0.01, P < 0.01, P < 0.01 and P < 0.01, respectively). Moreover, p65 was expressed more in cancer tissue than that in non-cancer mucosa evidenced by Western blot. In bladder cancer, the ranking score differences of p52, p65, c-Rel protein between lymphatic metastasis group and non-lymphatic metastasis group were statistically significant (P < 0.01, P < 0.01, P < 0.05, respectively).. Compared to noncancer bladder mucosa, expressions of NF-kappa B family and its regulated genes in bladder cancer are markedly higher. NF-kappa B may be related to lymphatic metastasis.

Topics: Adult; Aged; Aged, 80 and over; Blotting, Western; Cyclin D1; Female; Humans; I-kappa B Proteins; Immunohistochemistry; Interleukin-8; Male; Middle Aged; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-rel; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Transcription Factor RelB; Transcription Factors; Urinary Bladder Neoplasms

We determined whether the IFN-beta gene could suppress angiogenesis, tumor growth, and metastasis of human bladder transitional cell carcinoma. The highly tumorigenic and metastatic 253J B-V(R) human bladder transitional cell carcinoma (TCC) cell line (resistant to the antiproliferative effects of IFN-beta) was infected in vitro with adenoviral beta-galactosidase (Ad-LacZ), murine adenoviral IFN-beta (Ad-mIFN-beta), or human adenoviral IFN-beta (Ad-hIFN-beta) and implanted into the bladders of athymic nude mice. Ad-mIFN-beta and Ad-hIFN-beta were used because of the species specificity of IFN-beta. The transient production of mIFN-beta and hIFN-beta from the infected 253JB-V(R) tumor cells significantly inhibited tumorigenicity and spontaneous lymph node metastasis. Subsequently, the 253J B-V(R) cells were implanted into the subcutis of athymic nude mice, and established tumors were treated by direct intratumoral injection with Ad-mIFN-beta, Ad-hIFN-beta, Ad-LacZ, or PBS. By in situ hybridization (ISH) and immunohistochemical analysis (IHC), expression of hIFN-beta and mIFN-beta mRNA and protein within the tumors was demonstrated after Ad-hIFN-beta and Ad-mIFN-beta gene therapy, respectively. The therapy also induced necrosis in both the Ad-mIFN-beta- and Ad-hIFN-beta-treated tumors. IHC revealed decreased tumor cell proliferation and the sequestration of activated macrophages within the tumors after Ad-mIFN-beta therapy. In addition, the expression of the proangiogenic factors bFGF, and MMP-9 protein (by IHC) was significantly down-regulated by Ad-hIFN-beta gene therapy. Double-immunofluorescent IHC for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and CD-31 demonstrated tumor and endothelial cell apoptosis in those tumors treated with Ad-hIFN-beta gene therapy. Tumor-induced angiogenesis, as determined by the microvessel density, was decreased in tumors treated with both Ad-mIFN-beta and Ad-hIFN-beta. These data suggest that the inhibition of tumorigenicity and the metastasis of the 253J B-V(R) cells after infection with Ad-IFN-beta is caused by the inhibition of angiogenesis and the activation of host effector cells.

Topics: Animals; Apoptosis; Endothelial Growth Factors; Endothelium, Vascular; Fibroblast Growth Factor 2; Genetic Therapy; Genetic Vectors; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Interferon-beta; Interleukin-8; Lymphokines; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Proliferating Cell Nuclear Antigen; Transfection; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

Anti-angiogenesis is likely to develop into a novel therapeutic approach for patients with solid malignancies. Most current clinical trials evaluate anti-angiogenic drugs aimed primarily against single angiogenesis stimulators. Here, we show that a single solid malignancy, i.e., a human embryonal rhabdomyosarcoma, produces in vivo at least three biologically active angiogenesis stimulators (vascular endothelial growth factor, basic fibroblast growth factor and interleukin-8). This suggests that tumour angiogenesis results from the activity of multiple, rather than a single angiogenesis stimulator(s). We, furthermore, show that a combination of anti-angiogenic drugs is more effective in inhibiting tumour-induced endothelial cell growth than a single agent. Our results imply that clinical anti-angiogenic strategies for the treatment of solid malignancies may be most effective when multiple rather than single antiangiogenic drugs are used.

Topics: Angiogenesis Inhibitors; Animals; Cattle; Chromatography, Affinity; Endothelial Growth Factors; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Humans; Interleukin-8; Lymphokines; Neovascularization, Pathologic; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Urinary tract epithelial cells (T 24/83) are able to express interleukin (IL)-6, IL-8, platelet-derived growth factor (PDGF) and tumour necrosis factor-alpha, but not IL-1 beta, IL-2, IL-4 and IL-10 in response to an infection with uropathogenic bacteria. The process of cytokine secretion is time dependent, with a significant increase in the cytokine activity after 60 min. The expression of virulence factors of the bacteria does not seem to play a role. The interaction between bacterial products (e.g. lipopolysaccharide) and/or bacterial adhesion mediated by adhesins and specific receptor molecules of cell surfaces may be responsible for the activity of mediator protein expression in the epithelial cells. The release of PDGF and IL-8 was found to be higher when due to Escherichia coli HB 101 (rough form) than that caused by other bacterial strains. Citrobacter CB 3009 provoked the highest level of IL-6. The PDGF level correlated significantly with IL-6 and IL-8 values (P<0.001). There was a significant correlation between the time-dependent release of IL-6 and IL-8 (P<0.05). In epithelial cytokine response to bacterial infection, the reaction of the epithelial cells may modify themselves (e.g. internalization of bacteria) and the immuno-regulatory processes that are caused by infection and responsible for parenchymal injury.

Topics: Citrobacter; Cytokines; Enterobacteriaceae Infections; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Humans; Interleukin-6; Interleukin-8; Platelet-Derived Growth Factor; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms; Urinary Tract Infections; Urogenital System; Urothelium; Virulence

Tumor invasion and metastasis are regulated by the expression of genes such as E-cadherin, which regulates cell adhesion, and matrix metalloproteinase-9 (MMP-9), which alters the integrity of the extracellular matrix. Both up-regulation of MMP-9 and down-regulation of E-cadherin correlate with bladder cancer metastasis. The purpose of this study was first to determine whether an imbalance between MMP-9 and E-cadherin expression correlates with metastasis from human transitional cell carcinoma (TCC) of the bladder after therapy with neoadjuvant chemotherapy and radical cystectomy and then to determine whether treatment of human TCC xenografts growing in nude mice with interferon (IFN)-alpha would restore this balance, thereby limiting tumor invasion and metastasis. We used in situ hybridization to evaluate the expression of several metastasis-related genes, including MMP-9 and E-cadherin, in paraffin-embedded biopsy specimens from 55 patients with muscle-invasive TCC treated with neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin chemotherapy and radical cystectomy. By multivariate analysis, an MMP-9:E-cadherin ratio of >1.8 was an independent prognostic factor for disease progression. In vitro incubation of an IFN-resistant, highly metastatic human TCC cell line, 253J B-V(R) with noncytostatic concentrations of IFN-alpha down-regulated the activity of MMP-9, up-regulated E-cadherin, and inhibited in vitro invasion. 253J B-V(R) cells were implanted into the bladders of athymic nude mice. Systemic therapy with IFN-alpha (10,000 units s.c. daily) decreased the expression of MMP-9, increased expression of E-cadherin, reduced tumor volume, and inhibited metastasis. The MMP-9:E-cadherin ratio was 4.5 in untreated controls and 1.1 after IFN-alpha treatment. Moreover, systemic low-dose daily IFN-alpha potentiated the efficacy of paclitaxel. These studies indicate that in addition to its antiproliferative and antiangiogenic effects, IFN-alpha limits tumor invasion by restoring the normal balance between MMP-9 and E-cadherin and enhances the activity of systemic chemotherapy.

Topics: Adult; Aged; Animals; Antineoplastic Agents, Phytogenic; Biopsy; Blood Vessels; Blotting, Northern; Cadherins; Carcinoma, Transitional Cell; Cell Movement; Collagen; Collagenases; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Interferon-alpha; Interleukin-8; Laminin; Lymphokines; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Neovascularization, Pathologic; Paclitaxel; Prognosis; Proteoglycans; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

Interleukin 8 (IL-8) is mitogenic and chemotactic for endothelial cells. Within a neoplasm, IL-8 is secreted by inflammatory and neoplastic cells. The highly tumorigenic and highly metastatic human transitional cell carcinoma (TCC) cell line 253J B-V overexpresses IL-8 relative to the nontumorigenic and nometastatic 253J-P cell line. To determine whether IL-8 expression regulates tumorigenicity and metastasis in human TCC, 253J B-V cells were transfected with the full-sequence antisense (AS) cDNA for IL-8, whereas 253J-P cells were transfected with the full-length IL-8 cDNA, and control cells for each were transfected with the neomycin resistance (Neo) gene. In vitro, sense-transfected 253J-P cells overexpressed IL-8-specific mRNA and protein, whereas both of these were markedly reduced in AS-IL-8-transfected 253J B-V cells relative to controls. Moreover, sense-transfected cells showed up-regulation in matrix metalloproteinase type 9 mRNA, collagenase activity, and increased invasiveness through Matrigel-coated filters, whereas these measures were lower in AS-transfected cells relative to controls. After implantation into the bladders of athymic nude mice, the sense-transfected 253J-P cells acquired increased tumorigenicity and metastasis, whereas the AS-transfected cells significantly inhibited tumorigenicity and metastases in the 253J B-V cell lines. This effect was accompanied by reduced IL-8 expression and microvessel density. These studies demonstrate that IL-8 expression enhances angiogenic activity through the induction of matrix metalloproteinase type 9 and subsequently regulates the tumorigenesis and production of spontaneous metastases of human TCC.

Topics: Animals; Carcinoma, Transitional Cell; Collagen; Collagenases; Drug Combinations; Endothelial Growth Factors; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Interleukin-8; Laminin; Lymphatic Metastasis; Lymphokines; Matrix Metalloproteinase 9; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Neovascularization, Pathologic; Promoter Regions, Genetic; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA, Antisense; RNA, Messenger; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Vascular endothelial cell growth factor (VEGF) regulates angiogenesis and metastasis of bladder cancer (transitional cell carcinoma, TCC) through binding to VEGF receptor-2 (VEGFR-2). In this study, we evaluated whether the anti-VEGFR monoclonal antibody (Mab) DC101 in combination with paclitaxel inhibited tumorigenesis, angiogenesis, and metastasis of human TCC growing within the bladder of athymic nude mice. In vivo therapy with Mab DC101 and paclitaxel induced significant regression of bladder tumors compared with either agent alone. Median bladder weights were reduced from 601 mg in untreated controls, 422 mg in mice treated with paclitaxel alone (P < 0.005), 361 mg in mice treated with DC101 alone (P < 0.005), and 113 mg in mice that received combination therapy (P < 0.0005). Only one of nine mice developed spontaneous lymph node metastasis after combined treatment, compared with seven of seven untreated controls (P < 0.0005), six of eight after DC101 (P < 0.01), and five of eight mice after paclitaxel (P < 0.05). Combined treatment with both paclitaxel and DC101 inhibited tumor-induced neovascularity compared with all other groups (P < 0.005), without altering the expression of VEGF or flk1. Mab DC101 and paclitaxel combined enhanced apoptosis in the tumor and endothelial cells compared with other treatment (P < 0.005). These studies indicate that Mab DC101, which blocks VEGFR-2 function, has significant efficacy against human TCC, especially when combined with the chemotherapeutic agent paclitaxel. The antitumor effect was mediated by inhibition of angiogenesis and induction of both tumor cell and endothelial cell apoptosis.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Apoptosis; Carcinoma, Transitional Cell; Cell Division; Endothelial Growth Factors; Fibroblast Growth Factor 2; Humans; Interleukin-8; Lymphokines; Male; Matrix Metalloproteinase 9; Mice; Mice, Nude; Microcirculation; Neovascularization, Pathologic; Paclitaxel; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Xenograft Model Antitumor Assays

We evaluate the predictive value of urinary cytokine levels of interleukin (IL) 8 and 18 for response in patients receiving intravesical bacillus Calmette-Guerin (BCG) for prevention of recurrences of superficial bladder cancer and treatment of carcinoma in situ.. In 28 patients with superficial bladder cancer treated with BCG IL-8 expression in the urine during the first 6 hours after the first BCG instillation was determined. In 17 patients IL-18 levels were also evaluated during the first 12 hours after BCG instillation. IL-8 and 18 levels were determined by solid phase double ligand enzyme-linked immunosorbent assay.. In 12 of the 28 patients assessed for IL-8 expression disease recurred after a median followup of 66 months. Median IL-8 expression during the first 6 hours for these patients was 851 ng. (range 232 to 8,497). Median IL-8 expression during the first 6 hours in patients without recurrence was 4,200 ng. (range 432 to 12, 232). Of 8 patients with a followup of greater than 36 months 7 (88%) had no recurrent disease and IL-8 levels greater than 4,000 ng. Patients secreting more than 4,000 ng. IL-8 into the urine after BCG have a significantly higher chance of remaining disease-free (p <0.05), and those with elevated IL-18 expression have a significantly longer disease-free survival (p <0.05). After a median followup of 23 months (range 7 to 93) 6 of the 17 patients assessed for IL-18 expression had treatment failure. Median IL-18 expression in those patients during the first 12 hours was 2,632 pg. (range 860 to 8,298). Median IL-18 expression during the first 12 hours in patients without recurrence was 12,258 pg. (range 1,727 to 151,495).. In this study we confirmed the value of quantitative IL-8 expression in the urine during the first 6 hours after BCG instillation for superficial bladder cancer to predict freedom of disease. Furthermore, to our knowledge we report for the first time the potential value of IL-18 expression in the urine during the first 12 hours after BCG to predict freedom from disease. These findings may help improve the treatment of patients with superficial bladder cancer, especially by identifying those with a high risk of disease recurrence and progression after BCG therapy.

Topics: Administration, Intravesical; Aged; Aged, 80 and over; BCG Vaccine; Carcinoma, Transitional Cell; Enzyme-Linked Immunosorbent Assay; Female; Follow-Up Studies; Humans; Immunotherapy; Interleukin-18; Interleukin-8; Male; Middle Aged; Neoplasm Recurrence, Local; Predictive Value of Tests; Sensitivity and Specificity; Treatment Outcome; Urinary Bladder Neoplasms

Thymidine phosphorylase (TP) (E.C. 2.4.2.4), also known as platelet-derived endothelial cell growth factor, is a potent angiogenic factor. The expression of TP correlates with poor prognosis in a range of tumor types. 2-Deoxy-D-ribose-1-phosphate, a product of thymidine catabolism by TP, is a strongly reducing sugar that generates oxygen radical species during the early stages of protein glycation. We show that thymidine induces oxidative stress in TP-overexpressing carcinoma cells, promoting secretion of the stress-induced angiogenic factors vascular endothelial growth factor and interleukin-8, and inducing matrix metalloproteinase-1. Our findings outline a putative mechanism for TP-induced angiogenesis and identify novel targets for intervention.

Topics: Carcinoma; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Humans; Interleukin-8; Lymphokines; Matrix Metalloproteinase 1; Neovascularization, Pathologic; Oxidative Stress; Thymidine; Thymidine Phosphorylase; Transfection; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

To determine the prognostic value of angiogenesis factor expression for patients with muscle-invasive transitional cell carcinoma (TCC) of the bladder treated with neoadjuvant methotrexate, vinblastine, doxorubicin, and cisplatin (M-VAC) chemotherapy and radical cystectomy, we evaluated the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and interleukin 8 (IL-8) by in situ hybridization, and we determined microvessel density (MVD) by immunohistochemistry. These factors were evaluated in 55 biopsy specimens prior to therapy and in the cystectomy specimens of 51 patients after completion of therapy. By univariate analysis, VEGF expression and MVD in the biopsy specimens were significant predictors of disease recurrence. By multivariate analysis, only VEGF expression was an independent prognostic factor. Pathological stage, bFGF expression, and MVD in the cystectomy specimens after therapy were all independent prognostic factors for disease recurrence. The results of this exploratory study indicate that the expression levels of VEGF and bFGF as indicated by in situ hybridization and MVD as indicated by immunohistochemistry identify patients with muscle-invasive TCC who are at high risk of developing metastasis after aggressive therapy with systemic M-VAC chemotherapy and radical cystectomy.

Topics: Adult; Aged; Angiogenesis Inducing Agents; Antineoplastic Combined Chemotherapy Protocols; Biopsy; Carcinoma, Transitional Cell; Chemotherapy, Adjuvant; Cisplatin; Cystectomy; Disease-Free Survival; Doxorubicin; Endothelial Growth Factors; Female; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; In Situ Hybridization; Interleukin-8; Lymphokines; Male; Methotrexate; Microcirculation; Middle Aged; Multivariate Analysis; Muscle Neoplasms; Neoplasm Metastasis; Prognosis; Recurrence; RNA, Messenger; Time Factors; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vinblastine

Previously we reported that when cells from the human transitional cell carcinoma cell line 253J B-V growing orthotopically within the bladder of athymic nude mice were treated with the anti-epidermal growth factor receptor monoclonal antibody C225, angiogenesis was inhibited, resulting in regression of the primary tumor and inhibition of metastasis. In this study, we evaluated whether paclitaxel enhanced this therapeutic effect of C225. In vitro, the proliferation of 253J B-V cells was inhibited more by the combination of C225 and paclitaxel than with either agent alone. In vivo therapy with C225 and paclitaxel resulted in significantly greater regression of tumors compared with either agent alone. Median bladder tumor weight was 85 mg (range, 69-133 mg) compared with 168 mg (range, 72-288 mg) after C225 alone (P < 0.05), and 273 mg (range, 83-563 mg) after paclitaxel alone (P < 0.005). The incidence of spontaneous lymph node metastasis was also reduced by the combination of C225 with paclitaxel, although this result did not significantly differ from results after the use of C225 alone. Treatment with paclitaxel and C225 down-regulated the expression of basic fibroblast growth factor, vascular endothelial cell growth factor, interleukin-8, and matrix metalloproteinase type 9 and inhibited tumor-induced neovascularity compared with untreated controls (P < 0.005). Moreover, the combination of C225 and paclitaxel enhanced apoptosis in tumor and endothelial cells compared with either agent alone (P < 0.005). These studies indicate that therapy with paclitaxel increases the ability of C225 to inhibit tumorigenicity and metastasis. This effect is mediated by inhibition of angiogenesis and induction of apoptosis.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Transitional Cell; Cell Division; Cetuximab; Combined Modality Therapy; Dose-Response Relationship, Drug; Dose-Response Relationship, Immunologic; Down-Regulation; Endothelial Growth Factors; Endothelium; ErbB Receptors; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Inhibitory Concentration 50; Interleukin-8; Lymphatic Metastasis; Lymphokines; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Microscopy, Fluorescence; Neoplasm Transplantation; Neovascularization, Pathologic; Organ Size; Paclitaxel; RNA, Messenger; Time Factors; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Epidermal growth factor receptor (EGFR) regulates the growth and progression of human transitional cell carcinoma (TCC) of the bladder. We have shown that therapy targeting EGFR inhibited the growth of human TCC established orthotopically in nude mice. The purpose of this study was to evaluate whether EGFR-directed therapy affects angiogenesis associated with the growth and metastasis of human TCC. We determined the cytostatic effect and the effect on production of angiogenic factors after in vitro treatment of the human TCC cell line 253J B-V with MAb C225, a chimerized monoclonal anti-EGFR antibody. The 253J B-V cells were implanted orthotopically into athymic nude mice, and established tumors (4 weeks) were treated with i.p. MAb C225. Expression of the angiogenic factors vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) was evaluated by immunohistochemistry and in situ mRNA hybridization analyses and correlated with microvessel density evaluated after immunohistochemical staining with anti-CD31. In vitro treatment with MAb C225 inhibited mRNA and protein production of VEGF, IL-8, and bFGF by 253J B-V cells in a dose-dependent manner. MAb C225 therapy of nude mice with established TCCs growing orthotopically resulted in inhibition of growth and metastasis compared with controls (P <0.0005). VEGF, IL-8, and bFGF expression was significantly lower in treated tumors than in controls. The down-regulation of these angiogenic factors preceded the involution of blood vessels. These studies indicate that therapy with anti-EGFR MAb C225 has a significant antitumor effect mediated, in part, by inhibition of angiogenesis.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Division; Cetuximab; Down-Regulation; Endothelial Growth Factors; ErbB Receptors; Fibroblast Growth Factor 2; Humans; Interleukin-8; Lymphokines; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Neoplasm Transplantation; Neovascularization, Pathologic; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

In intravesical therapy for superficial bladder carcinoma urothelial cells may, through the production of cytokines, contribute to the bacillus Calmette-Guerin (BCG)-induced local immunological reaction and associated antitumor efficacy. The aim of this study was to investigate such a role for the neutrophil-attracting cytokine interleukin-8 (IL-8). The appearance of IL-8 in patients urine after BCG therapy was compared with BCG-induced IL-6 and IL-2 and the stability of IL-8 in urine was tested. Compared to IL-6 and IL-2, a rapid induction of IL-8 was observed, occurring after the first BCG instillation. Urinary IL-8 was highly stable, even after 24 h incubation at 37 degrees C. The IL-8 concentration after the first instillation seemed to be associated with subsequent development of an immune response. Consequently, IL-8 seems an attractive candidate for investigation of its prognostic value for a clinical response to BCG therapy.

Topics: Administration, Intravesical; BCG Vaccine; Carcinoma, Transitional Cell; Humans; Interleukin-2; Interleukin-6; Interleukin-8; Prognosis; Time Factors; Urinary Bladder Neoplasms

We describe the cellular infiltrate and cytokine profile in sequential synovial membrane biopsies from a patient with acute followed by chronic synovitis after intravesical bacillus Calmette-Guérin (BCG) therapy for an in situ transitional cell carcinoma of the bladder. Histological and immunohistochemical analysis of 3 synovial biopsies were done sequentially over a 9 month period. The patient was HLA-B27 positive, but HLA-DR4 negative, and did not have the "shared epitope." Unlike other cases, this patient's arthritis did not respond initially to nonsteroidal antiinflammatory drugs and was exacerbated by corticosteroid therapy. The synovitis took a neutrophilic form, with marked synovial membrane content of interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). It subsequently developed into chronic lymphoplasmacytoid synovitis, similar to rheumatoid arthritis (RA), with decreased IL-8 but continuing IL-1 and TNF-alpha production in the synovial membrane. The synovitis resolved to a fibrotic synovium with residual thickening of the synovial lining layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial membrane yielded a cellular infiltrate and cytokine content that had marked similarities with that seen in RA; however, the arthritis eventually remitted spontaneously.

Topics: Aged; Arthritis, Reactive; BCG Vaccine; Carcinoma, Transitional Cell; Cytokines; Female; HLA-B Antigens; HLA-DR Antigens; Humans; Immunohistochemistry; Interleukin-1; Interleukin-8; Synovial Membrane; Synovitis; Time Factors; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms

To determine the effects of isoniazid (isonicotinic acid hydrazide), used to reduce the serious side-effects of immunotherapy of superficial bladder cancer with bacille Calmette-Guérin (BCG), on the proliferation and constitutive BCG-induced synthesis of interleukins 6 (IL6) and 8 (IL8) in human bladder cancer cells cultured in vitro.. Three poorly differentiated human cell lines, T24, TCC-SUP and BT-B, were used to study the effect of isoniazid on cell proliferation. Cells were inoculated in tissue culture plates and various concentrations of isoniazid added to the medium. Cell density was then monitored for up to 6 days using a colorimetric assay. To determine the effects of isoniazid on constitutive and BCG-induced cytokine synthesis, cells were cultured in medium containing no additions, BCG, isoniazid or BCG with isoniazid, at various concentrations. Samples of medium were collected regularly for 6 h and the cytokine content (IL6 and IL8) determined using enzyme-linked immunosorbent assays.. Continuous incubation of proliferating T24, TCC-SUP and BT-B cells with isoniazid at concentrations of 0-100 micrograms/mL did not affect the rate of proliferation. Unlike TCC-SUP and BT-B cells, T24 cells released more IL6 and IL8 during incubation with BCG. At 6 h after the addition of BCG, the cumulative mean (SD) IL6 and IL8 production of T24 cells was 2.6 (0.1) and 2.3 (0.4) ng per 3 x 10(5) cells, compared with a constitutive level of 0.1 (0.0) and 1.3 (0.2) ng, respectively. There was no significant effect of isoniazid (1-100 micrograms/mL) on either the constitutive or BCG-induced synthesis of IL6 and IL8 in T24 cells.. Assuming an essential role of (tumour) epithelial cells in the local immune response induced by BCG, these in vitro results suggest that the administration of isoniazid does not interfere with this part of the mechanisms by which BCG operates.

Topics: Antitubercular Agents; BCG Vaccine; Cell Division; Humans; Immunotherapy; Interleukin-6; Interleukin-8; Isoniazid; Neoplasm Proteins; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Chronic inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. We have shown that acute and chronic inflammation induced by intravesical instillations of killed Escherichia coli strikingly enhances N-methyl-N-nitrosourea (MNU)-initiated rat bladder carcinogenesis. To test the hypothesis that cytokines released during inflammation may be involved in the enhancement of bladder carcinogenesis, we conducted an in vitro experiment. Using soft agar growth as an index of transformation, we examined the effect of inflammation-associated cytokines on the enhancement of MNU-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. In the first experiment, after 1-h exposure to MNU (50 micrograms/ml), cells (5 x 10(4)) were grown in soft agar in the presence of interleukin (IL)-1 alpha, IL-6, IL-8, or tumor necrosis factor-alpha (10 to 100 ng/ml). Colonies consisting of more than 20 cells were counted 4 weeks later. Among the cytokines tested, IL-6 (100 ng/ml) significantly increased colony counts over those for the untreated controls (P < 0.001). In the second experiment, the cells treated with MNU similarly as in the first experiment were cultured with or without IL-6 (100 ng/ml) for 1 week before the cells (5 x 10(4)) were grown in soft agar in the presence or absence of IL-6. IL-6 pretreatment increased colony counts irrespective of subsequent IL-6 treatment (P < 0.05). Moreover, IL-6-stimulated anchorage-dependent growth of MNU transformants far exceeded that of the parental MYP3. However, among the transformants, there was no parallel relationship in response to IL-6 between anchorage-dependent and -independent growth. Our results suggest that IL-6 may provide a selective growth advantage to MNU-initiated bladder epithelial cells in vitro and that it may be a factor accounting for the marked enhancement of inflammation-associated rat bladder carcinogenesis.

Topics: Animals; Antigens, CD; Carcinogens; Carcinoma; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Epithelial Cells; Gene Expression Regulation, Neoplastic; In Vitro Techniques; Interleukin-1; Interleukin-6; Interleukin-8; Methylnitrosourea; Rats; Receptors, Interleukin; Receptors, Interleukin-6; RNA, Messenger; RNA, Neoplasm; Tumor Necrosis Factor-alpha; Urinary Bladder; Urinary Bladder Neoplasms

Neutrophil-activating peptide-1/interleukin-8 (NAP-1/IL-8), secreted by monocytes, macrophages and a number of other cells, acts as a chemoattractant for neutrophil leukocytes and stimulates them to produce a series of responses such as shape change, adherence, exocytosis and respiratory burst, events that are of importance in inflammation. To study the release of NAP-1/IL-8, two human models of inflammation were chosen: transurethral resection of superficial bladder cancer and the subsequent instillation of bacillus Calmette-Guérin (BCG), performed in order to reduce the recurrence rate of papillary bladder tumors. As the secretions of the bladder wall are retained in the urine, patients' urine was collected during 4-hour periods. These urine samples were chromatographed on phosphocellulose. In the elution fractions NAP-1/IL-8 was quantified by a bioassay that measured the elastase release by human neutrophils. The neutrophil-stimulating activity was further purified by reverse phase high performance liquid chromatography. Although no NAP-1/IL-8 activity could be detected in normal individuals, formation of this inflammatory cytokine was observed in patients after transurethral resection and after BCG treatment. The significance and possible use of this secretion are discussed.

Topics: Administration, Intravesical; Aged; Aged, 80 and over; BCG Vaccine; Biological Assay; Chromatography, Liquid; Combined Modality Therapy; Cystitis; Humans; Interleukin-8; Middle Aged; Postoperative Complications; Urethra; Urinary Bladder Neoplasms