Purpose: Interleukin (IL)-8 is an important mediator of angiogenesis, tumorigenicity, and metastasis in transitional cell carcinoma (TCC) of the bladder. Nuclear factor κB (NF-κB)/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-κB, and whether inhibition of NF-κB function by mutant IκB-α prevents induction of IL-8 expression.

Experimental Design: 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% O2) conditions. The involvement of NF-κB 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 IκB-α.

Results: 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-κB, 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 IκB-α transfectants. Tumor growth and lymph node metastasis were inhibited in UM-UC-14 mutant IκB-α transfectants compared with UM-UC-14 controls. This effect was associated with the inhibition of IL-8 production, cellular proliferation, and angiogenesis.

Conclusions: These results suggest that TCCs of the bladder have heterogenic responses to physicochemical changes in the microenvironment and identify NF-κB as a potential molecular target for therapy.

IL-8,3 a member of the superfamily of CXC chemokines, has a wide range of proinflammatory effects. It was initially described as a neutrophil and lymphocyte chemoattractant (1, 2) but has subsequently been identified as a proangiogenic agent and a modulator of collagenase secretion (3, 4, 5, 6, 7, 8, 9, 10). Recently, it has been appreciated that IL-8 regulates angiogenesis in a wide range of human malignancies (2, 11, 12, 13), including TCC of the bladder (14), and that the level of expression directly correlates with the metastatic potential of TCC (15) Whether IL-8 acts as an autocrine growth factor or an angiogenic factor in TCC is unclear, but in any event, several biological functions of IL-8 are of significance to the pathology and treatment of this disease (13, 15).

Two important promoter regions have been identified for the transcriptional regulation of IL-8, a distal promoter element composed of an AP-1-binding site and a proximal promoter element containing binding sites for nuclear factor-IL-6 and NF-κB (16). NF-κB is a dimeric transcription factor composed of five members of the NF-κB/relA family. In nonlymphoid mammalian cells, NF-κB exists predominantly as a heterodimer composed of RelA (p65) and NF-κB1 [p50 (17, 18)]. Different neoplasms have variable constitutive and inducible levels of NF-κB expression (19, 20, 21). NF-κB regulates the expression of proangiogenic molecules, including IL-8 (20), and in turn is regulated by a family of inhibitory proteins, IκBs, which sequester NF-κB in the cytosol (21, 22, 23, 24). Certain stimuli trigger a cascade of events leading to the phosphorylation of IκB-α, its polyubiquitination, and subsequent degradation by the 26S proteasome (25). Thus, NF-κB is liberated, allowing translocation to the nucleus and transcription of target genes including IL-8 (26). IκB-α degradation requires phosphorylation of specific serine residues at sites 32 and 36. Substitution of these serine residues interferes with IκB-α phosphorylation, polyubiquitination, and degradation and therefore inhibits the transcriptional activity of NF-κB (27, 28, 29, 30). Recently, Huang et al.(31, 32) reported that blockade of NF-κB by mutant IκB-α reduced constitutive expression of vascular endothelial growth factor and IL-8 and subsequently decreased angiogenesis, invasion, and metastasis in human ovarian and prostate cancer models.

The regulation of IL-8 by NF-κB in human TCC in response to physicochemical changes in the microenvironment (acidosis and hypoxia) has not previously been evaluated. We performed the studies below to assess the regulation of IL-8 by NF-κB induced by acidosis and hypoxia. We found that NF-κB has a central role in regulating the expression of IL-8 in human TCC.

Cell Lines and Culture Conditions.

The human bladder carcinoma cell lines UM-UC-14 (33), HTB-9 (34), RT-4 (35), 253J B-V (10, 36), and KU-7 (37) were grown as a monolayer in modified Eagle’s MEM supplemented with 10% fetal bovine serum, vitamins, sodium pyruvate, l-glutamine, nonessential amino acids, and penicillin-streptomycin (CMEM).

Acidic and Hypoxic Conditions.

Cells were plated in culture dishes containing CMEM 48 h before incubation under normal, acidic, or hypoxic conditions (described below). When the cultures were 70–80% confluent, fresh medium with 1% fetal bovine serum was added, and cultures were incubated for 24 h. The dishes were then incubated at 37°C under acidic conditions for 6 h or hypoxic conditions for 12–24 h. Stabilization of pH during acidosis was evaluated using three media conditions: (a) CMEM (pH 7.35); for acidosis, initial pH (7.0, 6.5, 6.0) was adjusted by adding the appropriate amount of 20 mm 2-[N-morpholino]ethanesulfonic acid (Fig. 1,A); (b) MEM without bicarbonate (pH 5.5); for acidosis, initial pH (7.0, 6.5, 6.0) was adjusted by adding appropriate amount of 20 mm Tris (hydroxymethyl) aminomethane (Fig. 1,B); and (c) mixture of CMEM/MEM without bicarbonate as buffer medium; for acidosis, an appropriate amount of MEM without bicarbonate (pH 5.5) was added to a fixed amount of CMEM (pH 7.35) to achieve initial pH [7.0, 6.5, 6.0 (Fig. 1,C)]. During preliminary studies, we established that maintenance of the pH stability was optimized in the CMEM/MEM buffer medium, and thus this was used for the experimental studies. For hypoxia experiments, regulation of pH was tested in the three different media described above. We found that the maintenance of pH stability was optimal in CMEM (Fig. 1,D) or in the CMEM/MEM without bicarbonate buffered medium (Fig. 1,F) but unsatisfactory in the MEM without bicarbonate alone (Fig. 1 E). Thus, the hypoxia experiments were performed in CMEM at 37°C for 12–24 h using 5% CO2-95% air (control) or a hypoxic incubator (Precision Scientific, Winchester, VA) with 1% O2 balanced with 5% CO2 and nitrogen (hypoxia). For all experiments, the pH of the medium was measured at both the initial and final point of incubation. During hypoxia studies in CMEM, the pH of the medium increased by a maximum of 0.25 unit during the experiments. During acidosis studies, the pH of the CMEM/MEM buffered medium remained stable except with severe acidosis, during which the pH increased by a maximum of 0.25 unit. Thus, the experiments described were performed with pH monitoring at the start and end of each experiment under very stringent conditions that were achieved using the appropriately adjusted medium. Hence, the effects of hypoxia and acidosis of transcription could be studied independently.

Northern Blot Analysis.

Total RNA was extracted directly from each cell line using TRI Reagent (Invitrogen, San Diego, CA), electrophoresed on 1% denatured formaldehyde-agarose gel, electrotransferred to a Genescreen nylon membrane (DuPont, Boston, MA), and cross-linked with a UV Stratalinker 1800 (Stratagene, La Jolla, CA) at 120,000 mJ/cm2. Filters were washed, and the membranes were hybridized and probed for IL-8; the presence of GAPDH was used to control for loading. The cDNA probes used in this study were a 0.5-kb EcoRI cDNA fragment corresponding to human IL-8 [a gift of Dr. K. Matsushima, Kanazawa, Japan (38)] and a 1.28-kb fragment from GAPDH for standardizing IL-8 expression level (39). The cDNA probes were radiolabeled by a random primer technique using a commercial kit (Boehringer Mannheim, Indianapolis, IN) and [α-32P]dCTP (Amersham, Arlington Heights, IL).

IL-8 ELISA.

Viable cells (5 × 104) were seeded in 96-well plates, and conditioned medium was removed after 24 h. The cells were washed and then incubated with 200 μl of medium under hypoxic or acidotic conditions as described above. For in vivo experiments, blood was collected from the tail vein of each mouse at necropsy. IL-8 protein in cell-free culture supernatants and in mouse serum was determined using the commercial Quantakine ELISA kit (R&D Systems, Minneapolis, MN). The protein concentration of IL-8 was determined by comparison of the absorbance with the standard curve. Results were corrected for cell number or tumor weight.

Transfection and Selection of Clones Expressing Mutant IκB-α.

UM-UC-14 cells were transfected with pLXSN-IκB-α-mutant (pLXSN-IκB-α-M; CLONETECH, Palo Alto, CA) or pLXSN control vector using a lipofection reagent (Life Technologies, Inc., Gaithersburg, MD). Mutant IκB-α contains mutations at residues 32 and 36 of the NH2 terminus (S32A and S36A) and a COOH-terminal PEST sequence mutation. The cultures were placed in a 37°C incubator for 12 h, after which the medium was replaced. After 24 h, 800 μg/ml G418 sulfate (Life Technologies, Inc.) was added. The CMEM/G418 medium was replaced every 3 days until individual resistant colonies were isolated and established in culture as individual lines. All of the lines were maintained in CMEM/G418. The expression of exogenous pLXSN-IκB-α-M was verified by Western immunoblot analysis. Two clones (M1 and M2) were selected for in vitro and in vivo studies.

Nuclear and Cell Extracts.

Whole-cell lysate was prepared in Triton X-100 lysis buffer at 4°C. The supernatants were cleared by centrifugation. For nuclear protein extracts, the cells were washed with cold PBS and harvested by scraping. After centrifugation, the pellet was resuspended in cytoplasmic lysis buffer and incubated on ice for 10 min. For nuclear protein extract from tumor, tissues were mechanically dissociated, homogenized, and resuspended in NP40-based cytoplasmic lysis buffer and incubated on ice for 20 min. Cell- and tumor-derived specimens were centrifuged at 8000 rpm for 2 min at 4°C. Supernatants containing cytoplasmic protein were discarded, and the pellets were resuspended in nuclear lysis buffer for 30 min on ice. Protein concentration was measured using the Bradford assay.

Western Immunoblot Analysis.

Equal amounts of protein were boiled in Laemmli SDS sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose, and probed with rabbit anti-IκB-α (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit anti-β-actin (Sigma, St. Louis, MO) at 4°C overnight. After washing, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated antirabbit secondary antibody (Amersham). Signals were detected by the enhanced chemiluminescence detection system (Amersham). The rabbit anti-IκB-α detects both mutant and wild-type IκB-α; however, the former migrates faster in the gel (21).

In Vitro DNA Fragmentation Analysis.

UM-UC-14 parent cells (UM-UC-14), pLXSN-NEO control vector-transfected cells (UM-UC-14-NEO), and mutant IκB-α-transfected cells (UM-UC-14-M1 and UM-UC-14-M2) were treated with TNF-α for 24 h. Cells were harvested and pelleted by centrifugation and resuspended in PBS containing 50 μg/ml propidium iodide, 0.1% Triton X-100, and 0.1% sodium citrate. Propidium iodide incorporation as a measure of in vitro DNA fragmentation was measured by fluorescence-activated cell-sorting analysis (37). Cells in the sub-G1 population were assumed to be apoptotic (FACScan; Becton Dickinson, Mountain View, CA).

Electrophoretic Mobility Gel Shift Assay.

EMSA was performed using nuclear extracts prepared from UM-UC-14 cells cultured for various times under normal, acidic, and hypoxic conditions and from tumor tissue. For EMSA experiments, the following double-stranded oligonucleotides were used: NF-κB; AP-1; and nonspecific oligonucleotide SP-1. The oligonucleotides were annealed and 5′-end-labeled with [32P]ATP with T4 polynucleotide kinase using standard procedures. The binding reaction was carried out by preincubating nuclear extract protein (5 μg) in 20 ml of HEPES (pH 7.9), 50 ml of NaCl, 5% glycerol, 0.1 ml of DTT, and 1 μg of poly(deoxyinosinic-deoxycytidylic acid) at room temperature for 15 min followed by the addition of the double-stranded [32P]ATP. For labeled competition assays, a 50-fold molar excess of labeled oligonucleotide was added to the binding reaction. Where indicated, antibodies to the specific transcription factors were added for the supershift assays, including anti-p50 and anti-p65 for NF-κB and anti-jun and anti-fos for AP-1 binding. Samples were loaded onto a 5% polyacrylamide gel. Electrophoresis was performed at room temperature for 3 h at 100 V. The gels were dried and exposed to Kodak film at −70°C

Animals.

Male athymic BALB/c nude mice were obtained from the Animal Production Area of the National Cancer Institute, Frederick Cancer Research Facility (Frederick, MD). The mice were maintained in a laminar airflow cabinet under pathogen-free conditions and used at 8–12 weeks of age. All facilities are approved by the American Association for Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the United States Department of Agriculture, the Department of Health and Human Services, and the NIH.

Orthotopic Implantation of Tumor Cells.

UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells (60–70% confluent) were prepared for injection as described previously (10). Mice were anesthetized with i.p. sodium pentobarbital. For orthotopic implantation, a lower midline incision was made, and viable tumor cells were injected into the bladder wall. The formation of a bulla indicated a satisfactory injection. The bladder was returned to the abdominal cavity, and the abdominal wall was closed with a single layer of metal clips.

ISH Analysis.

A specific antisense oligonucleotide DNA probe was designed complementary to the mRNA transcripts based on published reports of the cDNA sequences of IL-8 (38, 40), and the specificity was confirmed by Northern blot analysis (40). A poly(dT)20 oligonucleotide was used to verify the integrity and lack of degradation of the mRNA in each sample.

ISH was performed as described previously, using the Microprobe Manual Staining System [Fisher Scientific, Pittsburgh, PA (40, 41)]. To check the specificity of the hybridization signal, the following controls were performed: (a) RNase pretreatment of sections; and (b) substitution of a biotin-labeled sense probe for the antisense probe. No hybridization signal was observed under either of these conditions. Control for endogenous alkaline phosphatase included treatment of the sample in the absence of the biotinylated probe and the use of chromogen alone (40, 41). The color reaction was quantified with a Zeiss photomicroscope (Carl Zeiss, Thornwood, NY) equipped with a three-chip, charge-coupled device color camera (model DXC-969 MD; Sony Corp., Tokyo, Japan), and the images were analyzed using Optimas image analysis software (version 6.2; Media Cybernetics, Silver Spring, MD). The intensity of staining was determined by comparison with the integrated absorbance of poly(dT)20. The results were presented as the ratio of the expression of each gene indexed against controls, which were arbitrarily set at 100 (42).

IHC.

For IHC analysis, frozen tissue sections (8 μm) were fixed in cold acetone. Formalin-fixed, paraffin-embedded specimens (5 μm) were deparaffinized in xylene and rehydrated in graded ethanol. Antigen retrieval was performed using pepsin for 12 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Specimens were blocked with 5% normal horse serum plus 1% normal goat serum in PBS. The samples were incubated for 18 h at 4°C with one of the following: (a) a 1:800 rat monoclonal anti-CD31 antibody [PharMingen, San Diego, CA (43)]; (b) a 1:50 dilution of a rabbit polyclonal anti-IL-8 antibody (Biosource International, Camarillo, CA); or (c) a 1:100 dilution of mouse monoclonal anti-PCNA antibody (DAKO, Carpinteria, CA).

The samples were then rinsed four times with PBS before incubation with the appropriate secondary antibody: (a) peroxidase-conjugated antirat IgG (H+L; Jackson ImmunoResearch Laboratory, West Grove, PA); (b) antirabbit IgG; (c) F(ab)2 fragment (Jackson ImmunoResearch Laboratory); (d) antimouse IgG1 (PharMingen); or (e) antimouse IgG (Jackson ImmunoResearch Laboratory). The specimens were again rinsed with PBS, incubated with diaminobenzidine (Research Genetics), and mounted using universal mount (Research Genetics).

Quantification of IHC for IL-8, MVD, and Cellular Proliferation.

The intensity of IL-8 immunostaining was quantified in each sample by image analysis using the Optimas software program (Bioscan, Edmonds, WA). Five different areas in each sample were evaluated to yield an average measurement of intensity of immunostaining. The results were presented as a ratio between the expression by the tumor and normal mucosa, which was arbitrarily set at 100 (42) MVD was determined by light microscopy after immunostaining of sections with anti-CD31 antibodies according to the procedure of Weidner et al.(43, 44), and cell proliferation was determined by immunohistochemical staining of tissue sections with anti-PCNA antibody. Tissue images were recorded using a cooled charge-coupled device Optronics Tec 470 camera (Optronics Engineering, Goletha, CA) linked to a computer and digital printer (Sony Corp.). The MVD and density of proliferative cells were expressed as the average number of five highest areas identified within a single ×100 field (42).

Statistical Analysis.

Tumor weights and staining intensities are expressed as median ± SD. Differences in the number of blood vessels, proliferative cells, and staining intensity for IL-8 in the bladder tumors between treatment groups were analyzed using the Mann-Whitney U test. Incidences of tumors and metastases were analyzed by the χ2 test. P < 0.05 was considered significant.

Heterogeneity of Induction of IL-8 in Human Bladder Cancer Cells under Acidic and Hypoxic Conditions.

In the present study, we used stringent conditions and careful pH monitoring at the beginning and end of each experiment to control for changes in pH over the course of the experiments to independently study the effects of hypoxia and acidosis on IL-8 transcription. We were able to keep the pH within a range of 6.8–7.1 to prevent cytotoxic effects that occur once the pH is lowered below 6.7, and we were able to evaluate the effects of hypoxia on IL-8 expression independent of the confounding effects of acidosis that can accompany hypoxia (17). We then determined whether IL-8 mRNA expression and protein production were induced by acidic (Fig. 2,A) and hypoxic conditions (Fig. 2,B). GAPDH mRNA expression was used as a control for loading (data not shown). Cells were incubated under normal pH (7.35) or acidic pH (7.0, 6.5, 6.0) conditions for 6 h. IL-8 mRNA expression (Northern blotting) and protein production (ELISA) were significantly up-regulated at pH 7.0 (acidic conditions) in UM-UC-14, HTB-9, and RT-4 cells. IL-8 was not detected in KU-7 cells under normal or acidic conditions. 253J B-V cells constitutively expressed high levels of IL-8 under normal conditions, which were not up-regulated under acidic conditions. Further reductions in pH to 6.5 and 6.0 suppressed IL-8 in all of the cell lines tested (Fig. 2 A).

Under conditions of hypoxia, IL-8 mRNA and protein production in UM-UC-14 cells was minimally increased at 12 h but substantially increased at 24 h compared with normoxic conditions (Fig. 2 B). Similar induction of IL-8 was noted in HTB-9 cells under hypoxic conditions. Conversely, hypoxia reduced IL-8 expression in RT-4 cells but had no effect on the constitutive expression of IL-8 in 253J B-V cells. IL-8 was not detected in KU-7 cells under normal or hypoxic conditions.

Acidic and Hypoxic Conditions Induce NF-κB in UM-UC-14 Cells.

EMSA was performed to evaluate the response of the transcription-regulatory DNA-binding proteins NF-κB and AP-1 to acidic and hypoxic conditions. [γ-32P]ATP-labeled NF-κB and AP-1 oligonucleotide probes were incubated with nuclear protein extracted from UM-UC-14 cells. Under normoxic conditions, low level binding of nuclear protein to NF-κB was observed. Acidosis (pH 7.0) increased the binding activity of NF-κB accompanied by p50 and p65 supershift bands that identified the NF-κB subunits involved in DNA binding (Fig. 3,A). Similarly, during hypoxia, increased binding activity of NF-κB accompanied by p50 and p65 supershift bands was observed (Fig. 3 B). The DNA binding activity of AP-1 was unaltered under acidic and hypoxic conditions compared with normal conditions (data not shown). The highly metastatic 253J B-V cells demonstrate constitutive NF-κB binding activity that did not increase upon exposure to acidic or hypoxic conditions (data not shown).

Mutant IκB-α Prevented Induction of NF-κB DNA Binding Reaction and IL-8 Expression by Acidosis and Hypoxia.

UM-UC-14 cells were transfected with the pLXSN-IκB-α-M vector. Mutation of Ser32 and Ser36 in IκB-α prevents Iκκ-mediated phosphorylation of the protein, which is required for ubiquitination and degradation by the proteasome. Immunoblot analysis confirmed that the mutant IκB-α protein expressed by the transfectants was resistant to TNF-induced degradation compared with UM-UC-14 cells, which are sensitive to relatively low doses of TNF-α, as shown in Fig. 4.

We evaluated whether mutant IκB-α suppressed inducible NF-κB DNA binding under acidic and hypoxic conditions. Cells were incubated under acidic conditions (pH 7.0) for 6 h or under hypoxic conditions (1% O2) for 24 h. After incubation, nuclear extracts were harvested and examined by EMSA. NF-κB DNA-binding function in UM-UC-14-M1 and UM-UC-14-M2 cells was suppressed during acidosis and hypoxic conditions compared with UM-UC-14 and UM-UC-14-NEO cells. The extent of suppression of NF-κB correlated with the expression level of exogenous mutant IκB-α (Fig. 5). The presence of endogenous IκB-α in cells expressing the mutant IκB-α protein is consistent with the presence of a subpopulation of cells not expressing the mutant IκB-α protein.

We evaluated whether overexpression of mutant IκB-α blocked IL-8 mRNA expression and protein production induced by acidic and hypoxic conditions in UM-UC-14-M (UM-UC-14-M1 and UM-UC-14-M2) cells. Under homeostatic conditions, the expression of IL-8 by UM-UC-14-M1 and UM-UC-14-M2 cells was equivalent to that of UM-UC-14-NEO controls (Fig. 5). The induction in IL-8 mRNA (Northern blotting) and protein (ELISA) observed in UM-UC-14 and UM-UC-14-NEO cells under acidic (Fig. 5,A) and hypoxic conditions (Fig. 5 B) was not observed in UM-UC-14-M1 and UM-UC-14-M2 cells under similar conditions.

Inhibition of Tumorigenicity and Metastasis of Orthotopic Xenografts Expressing Mutant IκB-α.

We implanted UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells into the bladder wall of athymic nude mice. Eight weeks after tumor implantation, mice were killed, bladder tumors were removed and weighed, and retroperitoneal lymph nodes were assessed for metastasis (Table 1). Tumors in the UM-UC-14-M1 and UM-UC-14-M2 cells were smaller than those in the UM-UC-14 cells (UM-UC-14-M1, P < 0.005; UM-UC-14-M2, P < 0.05) or UM-UC-14-NEO cells (UM-UC-14-M1, P < 0.005; UM-UC-14-M2, P = 0.05). In keeping with the expression pattern of mutant IκB, the UM-UC-14-M1 tumors were significantly smaller than the UM-UC-14-M2 tumors (P < 0.05). The incidence of lymph node metastasis in UM-UC-14-M1 tumors was significantly lower compared with that for either UM-UC-14 or UM-UC-14-NEO tumors (P < 0.05). The incidence of lymph node metastasis in UM-UC-14-M2 tumors was lower than that in controls, but the difference was not statistically significant.

Inhibition of NF-κB-DNA Binding, Down-Regulation of IL-8, MVD, and Cell Proliferation by Mutant IκB-α Transfection in UM-UC-14 Orthotopic Tumors.

NF-κB-DNA binding was suppressed in nuclear extracts prepared from in vivo tumors derived from UM-UC-14-M1 and UM-UC-14-M2 cells compared with the tumors derived from UM-UC-14-NEO cells (Fig. 6). The degree of suppression of NF-κB DNA binding activity was proportionate to the expression of mutant IκB by the UM-UC-14-M1 and UM-UC-14-M2 cells.

IL-8 mRNA expression was determined by ISH using an antisense oligonucleotide probe. IL-8 protein expression, cell proliferation, and MVD were determined by IHC using anti-IL-8, anti-PCNA, and anti-CD31 antibodies, respectively (Table 2 and Fig. 7). IL-8 mRNA and protein expression, cell proliferation, and MVD were significantly lower within UM-UC-14-M1 tumors than in either the UM-UC-14 or UM-UC-14-NEO tumors (P < 0.05). Similar differences in these factors were observed between UM-UC-14-M2 and UM-UC-14 and UM-UC-14-NEO tumors. However the differences in the expression of IL-8 protein and MVD between UM-UC-14-M2 and UM-UC-14-NEO tumors were not statistically significant.

Previously, we demonstrated that the level of IL-8 expression correlated with the metastatic potential of human TCC (15). In this study, we report for the first time that conditions in the host microenvironment (acidosis and hypoxia) alter IL-8 expression by human TCC via NF-κB. We studied a panel of human TCC cells that differed in their metastatic potential and discovered a heterogenic response in NF-κB activation and IL-8 expression in response to hypoxia and acidosis. We observed that highly metastatic TCC constitutively expressed high levels of NF-κB and IL-8, whereas less aggressive TCC cells expressed lower constitutive levels of NF-κB activity that were inducible after exposure to hypoxia or acidosis and led to the up-regulation of IL-8 expression. Blockade of NF-κB by mutant IκB-α prevented the induction of IL-8 and resulted in inhibition of angiogenesis and metastasis in human TCC xenografts growing orthotopically in the bladder of nude mice. These experiments identify NF-κB as a promising target for novel therapeutic strategies being designed by us for the treatment of advanced human bladder cancer.

Human tumors, including TCCs, are heterogeneously oxygenated due to functional and structural abnormalities of the vasculature. As a consequence, hypoxia is a common feature of bladder cancer, and this hypoxia in turn increases anaerobic metabolism and leads to increased production of acidic metabolites (46, 47, 48, 49, 50, 51, 52, 53). The resulting low extracellular pH in association with hypoxia can promote malignant progression by altering the expression of IL-8 and many other genes, including cell cycle-regulatory proteins, metabolic enzymes, metastasis-regulatory proteins, transcription factors, and angiogenesis-regulatory proteins (45, 54, 55, 56).

In clinical studies, high IL-8 expression has been reported to correlate with clinical stage and grade in human tumors (57, 58, 59, 60). In the present study, we demonstrated that TCCs of different phenotypes have heterogenous expression patterns of IL-8 expression under control conditions and variable responses to environmental stress. Neither IL-8 mRNA nor protein was detectable in KU-7 cells (superficial papillary TCC cells) under control conditions, and neither was induced by hypoxia or acidosis. The well-differentiated grade 1 TCC cells (RT-4) and grade 2 TCC cells (HTB-9) expressed low and moderate levels of IL-8, respectively, under homeostatic conditions. In both cell lines, IL-8 expression was inducible under stressful conditions (hypoxia or acidosis). In contrast, 253J B-V cells (high-grade TCC) constitutively expressed high levels of IL-8 and NF-κB activity, which did not increase under acidic or hypoxic conditions. Paradoxically, under homeostatic conditions, UM-UC-14 cells (high-grade TCC) expressed low levels of IL-8 that were inducible by acidosis and hypoxia due to an increase in NF-κB binding activity. Although UM-UC-14 cells were derived from a high-grade TCC, they demonstrate morphological and histological features characteristic of low-grade TCC and have lower metastatic potential in comparison with the highly metastatic 253J B-V cells (10). Thus, these data suggest that the heterogeneity of constitutive NF-κB activity and induction observed in this study might correlate with the histological grade of TCC cells: well- to moderately differentiated TCC cells may express low basal NF-κB activity and IL-8 expression (which is inducible in response to appropriate stimuli); whereas poorly differentiated TCC cells may express IL-8 constitutively due to constitutively active NF-κB. Interestingly, these observations are in contrast to findings in human melanoma cells, in which IL-8 mRNA and protein are inducible in the highly aggressive and metastatic cells, but not in the poorly aggressive cells (61).

The IL-8 promoter region –133 to –70 contains binding sites for NF-κB, AP-1, and nuclear factor-IL-6 and is required for IL-8 transcription. IL-8 transcription is regulated by cytokines, growth factors, irradiation, cytotoxic agents, nitric oxide, and physical changes in the microenvironment such as acidosis and hypoxia. In melanoma (62), ovarian tumors (63, 64), and pancreatic tumors (25), constitutive or inducible expression of IL-8 is controlled via NF-κB and/or AP-1. However, the relative importance of these transcription elements is stimulus specific and cell type specific (16). In the present study, we demonstrated that the induction of IL-8 in TCC is regulated through NF-κB, but not AP-1: specifically, the dimer of p50 and p65 regulated IL-8 expression during acidosis and hypoxia. Under basal conditions, NF-κB is sequestered in the cytoplasm via association with IκB, which prevents NF-κB translocation to the nuclei. The IκB family consists of seven IκBs, IκB-α-β-ε, Bcl-3, p100, and p105 (65). These IκBs preferentially associate with NF-κB/Rel family protein dimers. Specifically, IκB-α predominantly associates with p65/p50 heterodimers. In response to a wide variety of extracellular and intracellular signals including acidosis and hypoxia, IκBs are phosphorylated by IκB kinase and subsequently polyubiquitinated and proteolytically degraded. This liberates NF-κB, allowing it to translocate to the nucleus and function as a transcription factor. Recently, detailed structures and functions of IκB-α have been elucidated. IκB-α is a cytoplasmic protein distinguished by the presence of six ankyrin-like repeats (30, 66, 67, 68). Phosphorylation of serine 32 and serine 36 residues on the NH2 terminus in response to certain stimuli, including TNF-α, IL-1, and 12-O-tetradecanylphorbol-13-acetate, precedes its degradation and dissociation from NF-κB (69, 70, 71). One of the striking structural features of IκB-α is the presence of a COOH-terminal region that is rich in the amino acids proline, glutamic acid, serine, and threonine [PEST (72)]. The presence of such PEST sequences in many other proteins predicts rapid protein turnover (72). The PEST sequence of IκB-α lies downstream of the sixth ankyrin, beginning at amino acid 281. There are phosphoacceptor sites in the PEST region of IκB-α, with serines 283, 289, and 293 and threonines 291 and 299 all reported to be at least partially phosphorylated (73, 74, 75, 76). It has been suggested that constitutive phosphorylation of COOH-terminal phosphoacceptors is necessary for efficient turnover of free IκB-α in unstimulated cells (77). For the experiments described above, we chose the pLXSN-IκB-α-M (mutant), which contains substitutions at the serine 32 and serine 36 residues in the NH2-terminal and deletion of the PEST sequence in the COOH-terminal. Transfection of ovarian and prostate cancer cells with similar mutants of IκB-α decreased tumorigenicity by reducing the expression of proangiogenic molecules (IL-8 and vascular endothelial growth factor) that are regulated by NF-κB (31, 32). In a murine tumor model with murine lung alveolar cells expressing dominant negative IκB-α, the incidence of metastasis was reduced via inhibitory effects on matrix metalloproteinase 9, urinary plasminogen activator, and heparanase production independent of primary tumor size (59).

In the current study, mutant IκB-α-transfection of UM-UC-14 TCC cells prevented the rapid TNF-α-induced degradation of IκB-α (Fig. 4,A) and increased TNF-α-induced DNA fragmentation (Fig. 4 B). TNF-α induces apoptosis by activating caspases 8 and 10, but it can also induce the expression of inhibitors of apoptosis via NF-κB (78, 79). The TNF-α-induced DNA fragmentation in UM-UC-14-M1 and UM-UC-14-M2 cells was greatly increased compared with UM-UC-14 or UM-UC-14-NEO cells, suggesting a relative excess of proapoptotic stimuli over antiapoptotic stimuli.

During hypoxia and acidosis, DNA binding by NF-κB in the mutant IκB-α-transfected cells (UM-UC-14-M1 and UM-UC-14-M2) was inhibited, and therefore IL-8 expression in vitro was reduced. Similarly, DNA binding by NF-κB was inhibited in vivo in the mutant IκB-α-transfected cells that produced less IL-8 in vivo. Furthermore, the growth and metastatic potential of mutant IκB-α-transfected cells in vivo was reduced proportionate to the level of expression mutant IκB-α. In keeping with the previously described antiangiogenic effects of IL-8, MVD was reduced in tumors arising from mutant IκB-α-transfected cells compared with controls. Other studies have demonstrated that inhibition of NF-κB function by the proteasome inhibitor PS-341 reduces tumor growth and enhances chemo- and radiosensitivity by inhibiting chemotherapy- and radiotherapy-induced NF-κB activation in colorectal (80, 81), squamous cell (82), and pancreatic cancers (83). In our studies, the reduction in lymph node metastasis correlated with the degree of inhibition of NF-κB function in the transfected cell lines UM-UC-14-M1 and UM-UC-14-M2. This finding, coupled to the known IL-8 transcription-regulatory activity of NF-κB, supports a role for IL-8 as a mediator of metastasis in bladder cancer. We and others have previously reported proposed this role for IL-8 in bladder cancer (15), melanoma (4), and prostate cancer (13). Considering the present data, blockade of NF-κB translocation via inhibition of the NF-κB-IκB complex dissociation has potential as a therapeutic intervention in bladder cancer due to its inhibitory effects on IL-8 expression and subsequent inhibition of angiogenesis, tumor growth, and metastasis (15). We are planning additional studies to evaluate the potential of mutant IκB-α as a novel gene therapy strategy in combination with chemotherapy and radiotherapy.

In summary, our experiments demonstrate heterogeneity in constitutive and inducible NF-κB activity in human TCC. Moreover, blockade of NF-κB by mutant IκB-α inhibits IL-8 induction by acidosis and hypoxia and inhibits angiogenesis, growth, and metastasis of human TCC growing within the bladder wall of athymic nude mice.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Specialized Programs of Research Excellence Grant in Genitourinary Cancer CA91846 and National Cancer Institute Core Grant CA16672.

3

The abbreviations used are: IL, interleukin; TCC, transitional cell carcinoma; CMEM, complete Eagle’s MEM; NF-κB, nuclear factor κB; TNF, tumor necrosis factor; AP-1, activator protein 1; EMSA, electrophoretic mobility shift assay; ISH, in situ mRNA hybridization; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen; MVD, microvessel density; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase.

Fig. 1.

Stabilization of the pH of the medium during acidosis and hypoxia. Preliminary studies were performed to establish optimal medium conditions to stabilize pH during the hypoxia and acidosis experiments. The pH was measured at the initial and final points in all experiments. For acidosis, most stable conditions were achieved with CMEM/MEM without bicarbonate buffer medium (C). CMEM or MEM without bicarbonate produced large variations in pH during the acidosis experiments (A and B). For hypoxia, stable conditions were achieved with CMEM or CMEM/MEM without bicarbonate buffer media (D and F). Large variations in pH occurred with MEM without bicarbonate during hypoxia (E).

Fig. 1.

Stabilization of the pH of the medium during acidosis and hypoxia. Preliminary studies were performed to establish optimal medium conditions to stabilize pH during the hypoxia and acidosis experiments. The pH was measured at the initial and final points in all experiments. For acidosis, most stable conditions were achieved with CMEM/MEM without bicarbonate buffer medium (C). CMEM or MEM without bicarbonate produced large variations in pH during the acidosis experiments (A and B). For hypoxia, stable conditions were achieved with CMEM or CMEM/MEM without bicarbonate buffer media (D and F). Large variations in pH occurred with MEM without bicarbonate during hypoxia (E).

Close modal
Fig. 2.

Bladder cancer cells were incubated in normal and acidic (pH 7.0 to 6.0) medium for 6 h (A) or under control (using nonadjusted pH of medium), normoxia, and hypoxia conditions for 12 and 24 h (B). IL-8 protein expression in cell culture supernatants was measured by ELISA assay (bar graph). The concentration of IL-8 was corrected for cell number. IL-8 mRNA expression was measured by Northern blotting. A probe for GAPDH was used as a loading control (data not shown). IL-8 protein and mRNA expression in UM-UC-14, HTB-9, and RT-4 cells was significantly increased under acidic conditions (pH 7.0). IL-8 mRNA and protein were undetectable in KU-7 cells. Acidic pH decreased IL-8 protein and mRNA levels in 253J B-V cells (A). With more severe acidosis (pH < 6.7), IL-8 production was decreased in all cells, which may reflect cytotoxicity. The IL-8 protein and mRNA expression of UM-UC-14 and HTB-9 cells was significantly increased under hypoxia conditions compared with the cells incubated under either basal or normoxia conditions. KU-7 cells expressed undetectable levels of IL-8. 253J B-V cells constitutively expressed high levels of IL-8, which were not altered by hypoxia (B).

Fig. 2.

Bladder cancer cells were incubated in normal and acidic (pH 7.0 to 6.0) medium for 6 h (A) or under control (using nonadjusted pH of medium), normoxia, and hypoxia conditions for 12 and 24 h (B). IL-8 protein expression in cell culture supernatants was measured by ELISA assay (bar graph). The concentration of IL-8 was corrected for cell number. IL-8 mRNA expression was measured by Northern blotting. A probe for GAPDH was used as a loading control (data not shown). IL-8 protein and mRNA expression in UM-UC-14, HTB-9, and RT-4 cells was significantly increased under acidic conditions (pH 7.0). IL-8 mRNA and protein were undetectable in KU-7 cells. Acidic pH decreased IL-8 protein and mRNA levels in 253J B-V cells (A). With more severe acidosis (pH < 6.7), IL-8 production was decreased in all cells, which may reflect cytotoxicity. The IL-8 protein and mRNA expression of UM-UC-14 and HTB-9 cells was significantly increased under hypoxia conditions compared with the cells incubated under either basal or normoxia conditions. KU-7 cells expressed undetectable levels of IL-8. 253J B-V cells constitutively expressed high levels of IL-8, which were not altered by hypoxia (B).

Close modal
Fig. 3.

Electrophoretic gel mobility shift assay. Nuclear proteins were extracted from UM-UC-14 cells incubated under normal (pH 7.35) and acidic conditions (pH 7.0 to 6.0) for 3 h (A) or under normoxic and hypoxic (1% O2) conditions for 12 h (B). Binding reactions were performed as described in “Materials and Methods.” All lanes contain labeled NF-κB probe. Under acidic conditions (pH 7.0; A) and hypoxic conditions (B), enhanced NF-κB protein-DNA binding function accompanied by p50 and p65 supershifted bands (but not c-Rel) was observed compared with normal conditions.

Fig. 3.

Electrophoretic gel mobility shift assay. Nuclear proteins were extracted from UM-UC-14 cells incubated under normal (pH 7.35) and acidic conditions (pH 7.0 to 6.0) for 3 h (A) or under normoxic and hypoxic (1% O2) conditions for 12 h (B). Binding reactions were performed as described in “Materials and Methods.” All lanes contain labeled NF-κB probe. Under acidic conditions (pH 7.0; A) and hypoxic conditions (B), enhanced NF-κB protein-DNA binding function accompanied by p50 and p65 supershifted bands (but not c-Rel) was observed compared with normal conditions.

Close modal
Fig. 4.

Suppression of acidosis- and hypoxia-induced NF-κB protein binding and up-regulation of IL-8 mRNA and protein in UM-UC-14 cells after transfection with pLXSN-IκB-α-M (mutant). UM-UC-14 cells were transfected with mutant IκB-α. After stimulation with 50 ng/ml TNF-α for 30 min, cytosolic protein was extracted from UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells. Endogenous IκB-α (top band) and exogenous IκB-αM (bottom band) were detected by Western immunoblotting using rabbit anti-IκB-α polyclonal antibody. Equal loading was confirmed with anti-β-actin antibody. Endogenous IκB-α in UM-UC-14 and UM-UC-14-NEO cells was rapidly degraded after TNF-α stimulation. In contrast, in UM-UC-14-M1 and UM-UC-14-M2 cells, exogenous mutant IκB-α was not degraded by TNF-α stimulation.

Fig. 4.

Suppression of acidosis- and hypoxia-induced NF-κB protein binding and up-regulation of IL-8 mRNA and protein in UM-UC-14 cells after transfection with pLXSN-IκB-α-M (mutant). UM-UC-14 cells were transfected with mutant IκB-α. After stimulation with 50 ng/ml TNF-α for 30 min, cytosolic protein was extracted from UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells. Endogenous IκB-α (top band) and exogenous IκB-αM (bottom band) were detected by Western immunoblotting using rabbit anti-IκB-α polyclonal antibody. Equal loading was confirmed with anti-β-actin antibody. Endogenous IκB-α in UM-UC-14 and UM-UC-14-NEO cells was rapidly degraded after TNF-α stimulation. In contrast, in UM-UC-14-M1 and UM-UC-14-M2 cells, exogenous mutant IκB-α was not degraded by TNF-α stimulation.

Close modal
Fig. 5.

Suppression of acidosis- and hypoxia-induced up-regulation of IL-8 mRNA and protein and NF-κB protein-DNA binding in UM-UC-14 cells after transfection with pLXSN-IκB-α-M (mutant). Total RNA, cell culture supernatant, and nuclear protein were extracted from these transfected and control cells. IL-8 mRNA (bottom panel) and protein (middle panel) and NF-κB binding function (top panel) of UM-UC-14 cells transfected with pLXSN-NEO vector were up-regulated under acidic (pH 7.0; A) and hypoxic (B) conditions. These effects were not observed in the IκB-α mutant transfectants under similar conditions.

Fig. 5.

Suppression of acidosis- and hypoxia-induced up-regulation of IL-8 mRNA and protein and NF-κB protein-DNA binding in UM-UC-14 cells after transfection with pLXSN-IκB-α-M (mutant). Total RNA, cell culture supernatant, and nuclear protein were extracted from these transfected and control cells. IL-8 mRNA (bottom panel) and protein (middle panel) and NF-κB binding function (top panel) of UM-UC-14 cells transfected with pLXSN-NEO vector were up-regulated under acidic (pH 7.0; A) and hypoxic (B) conditions. These effects were not observed in the IκB-α mutant transfectants under similar conditions.

Close modal
Fig. 6.

In vivo suppression of NF-κB DNA binding in nuclear extracts from tumors derived from UM-UC-14-M1 and UM-UC-14-M2 cells (containing mutant IκB) compared with tumors derived from controls (UM-UC-14-NEO cells). The degree of suppression of NF-κB DNA binding activity was proportionate to the expression of mutant IκB by the UM-UC-14-M1 and UM-UC-14-M2 cells; more NF-κB DNA binding activity exists in the UM-UC-14-M2 cells. Each lane represents nuclear extracts derived from a single tumor.

Fig. 6.

In vivo suppression of NF-κB DNA binding in nuclear extracts from tumors derived from UM-UC-14-M1 and UM-UC-14-M2 cells (containing mutant IκB) compared with tumors derived from controls (UM-UC-14-NEO cells). The degree of suppression of NF-κB DNA binding activity was proportionate to the expression of mutant IκB by the UM-UC-14-M1 and UM-UC-14-M2 cells; more NF-κB DNA binding activity exists in the UM-UC-14-M2 cells. Each lane represents nuclear extracts derived from a single tumor.

Close modal
Fig. 7.

IL-8 mRNA expression, IL-8 protein expression, cell proliferation, and MVD in orthotopic xenografts. Tumors from IκB-α-M-transfected cells expressed lower IL-8 mRNA and protein and demonstrated reduced proliferation and decreased neovascularization compared with controls (UM-UC-14-P or NEO).

Fig. 7.

IL-8 mRNA expression, IL-8 protein expression, cell proliferation, and MVD in orthotopic xenografts. Tumors from IκB-α-M-transfected cells expressed lower IL-8 mRNA and protein and demonstrated reduced proliferation and decreased neovascularization compared with controls (UM-UC-14-P or NEO).

Close modal
Table 1

Tumorigenicity of UM-UC-14 cells growing orthotopically in athymic nude mice after transfection with mutant IκBα

Mice were implanted with 5 × 105 UM-UC-14, UM-UC-14-NEO, UM-UC-14-M1, and UM-UC-14-M2 cells. All mice were killed 8 weeks after implantation of tumor cells. UM-UC-14-M1 tumors were significantly smaller (P < 0.005) and had fewer lymph node metastases (P < 0.05) compared with either UM-UC-14 or UM-UC-14-NEO tumors. UM-UC-14-M2 tumors were significantly smaller than either UM-UC-14 (P < 0.05) or UM-UC-14-NEO (P = 0.05), and the incidence of lymph node metastasis was reduced. The UM-UC-14-M1 tumors were significantly smaller than UM-UC14-M2 tumors (P < 0.05). This is one representative experiment of two.

GroupTumorigenicityTumor weightLymph node metastasis
Median (mg)Range
UM-UC-14 10/10 141 45–236 5/10 
UM-UC-14-NEO 10/10 113 49–210 4/10 
UM-UC-14-M1 10/10 43a 30–57 0/10b 
UM-UC-14-M2 10/10 57c,d 38–164 1/10 
GroupTumorigenicityTumor weightLymph node metastasis
Median (mg)Range
UM-UC-14 10/10 141 45–236 5/10 
UM-UC-14-NEO 10/10 113 49–210 4/10 
UM-UC-14-M1 10/10 43a 30–57 0/10b 
UM-UC-14-M2 10/10 57c,d 38–164 1/10 
a

P < 0.005 compared with UM-UC-14 and UM-UC-14-NEO.

b

P < 0.05 compared with UM-UC-14 and UM-UC-14-NEO (χ2 test).

c

P < 0.05 compared with UM-UC-14 and UM-UC-14-M1

d

P = 0.05 compared with UM-UC-14-NEO (Mann-Whitney U test).

Table 2

In vivo IL-8 mRNA and protein expression, cell proliferation, and MVD of UM-UC-14 cells growing orthotopically in athymic nude mice after transfection with mutant IκBα

GroupIL-8 mRNA indexaIL-8 protein indexbProliferation indexc (mean ± SD)MVDd (mean ± SD)
UM-UC-14 100 100 142 ± 45 57 ± 9 
UM-UC-14-NEO 92 89 118 ± 26 55 ± 10 
UM-UC-14-M1 61e,f 63e,f 68 ± 15e,f 30 ± 2e,f 
UM-UC-14-M2 64e,f 70e 81 ± 25e,f 41 ± 8e 
GroupIL-8 mRNA indexaIL-8 protein indexbProliferation indexc (mean ± SD)MVDd (mean ± SD)
UM-UC-14 100 100 142 ± 45 57 ± 9 
UM-UC-14-NEO 92 89 118 ± 26 55 ± 10 
UM-UC-14-M1 61e,f 63e,f 68 ± 15e,f 30 ± 2e,f 
UM-UC-14-M2 64e,f 70e 81 ± 25e,f 41 ± 8e 
a

The intensity of the cytoplasmic color reaction obtained by ISH was quantified using image analysis and compared with the maximal intensity of the poly (dT)20 color reaction in each sample. The results are presented for each cell type indexed to the controls, which were arbitrarily defined as 100.

b

The intensity of the cytoplasmic immunostaining was quantified using image analysis in five different areas of each sample to yield an average measurement that is indexed to the control tumors, which were arbitrarily defined as 100.

c

The density of cell proliferation was measured by IHC with anti-PCNA antibodies and is expressed as the average of the five highest areas identified within a single ×100 field.

d

MVD was expressed as an average number of CD31-positive cells in the five highest areas identified within a single ×100 field.

e

P < 0.05 compared with UM-UC-14.

f

P < 0.05 compared with UM-UC-14-NEO.

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