T24 cells were purchased from the Wuhan Cell Bank of China (Wuhan, Hubei, China) and maintained in RPMI-1640 culture medium (Invitrogen Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen Life Technologies), 100 U/ml penicillin and 100 U/ml streptomycin, in a humidified atmosphere of 5% CO₂ at 37°C. UM-UC-3 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in minimum essential medium (Invitrogen Life Technologies). KT5720 and UO126 cells (Calbiochem Bioscience, La Jolla, CA, USA) were dissolved in dimethyl sulfoxide (Sigma, St. Louis, MO, USA) and cholera toxin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile ultra-purified water. The cells were plated and allowed to grow overnight in medium containing 10% FBS. The medium was then replaced with serum-free medium during the treatment.

Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma). The optical density (OD) was determined at 570 nm using an EXL800 microimmunoanalyzer (Bio-Tek Instruments, Burlington, VT, USA). The cell viability rate was calculated using the following formula: OD_experiment/OD_control × 100%.

The cells were placed in a six-well plate at a density of 500 cells/well and maintained with or without 10 ng/ml cholera toxin in normal medium containing 10% FBS for 10 days. Colonies were fixed with methanol and stained with 0.1% crystal violet in 20% methanol for 15 min and images of the representative colonies were captured.

For Hoechst 33342 staining, the cells were incubated with Hoechst 33342 (5 μg/ml; Sigma) for 20 min in the dark and then observed using a fluorescent microscope (Olympus, Melvie, NY, USA) with a 340 nm excitation filter.

Cytotoxicity of diazepam was evaluated using the LDH release assay. LDH release was quantified using a CytoTox 96^® nonradioactive cytotoxicity assay kit (Promega Corporation, Madison, WI, USA) in accordance with the manufacturer’s instructions. Absorbance was measured at 490 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA). Lysis solution was used as a positive control for maximum LDH release. To calculate the %_Cytotoxicity of each treatment group, the following formula was used, in accordance with the manufacturer’s instructions: %_Cytotoxicity = Experimental LDH release (OD490)/Maximum LDH release (OD490). The LDH release (%_control) was calculated as the %_Cytotoxicity of the treatment group over the %_Cytotoxicity of the vehicle control (0 μM) group.

The cells were seeded in six-well culture plates at a density of 8×10⁵ cells/well and incubated overnight. Cholera toxin (0–50 ng/ml) was added to the serum-free medium for 0–24 h. The cells were harvested by trypsinization, suspended in 100 μl medium and then fixed in 75% dehydrated alcohol overnight in 4°C. The cells were subsequently incubated with the DNA-binding dye propidium iodide (PI; 50 mg/ml), RNase (4 Ku/ml), NaF (0.3 mg/ml) and sodium citrate (1 mg/ml) for 1–2 h at room temperature in the dark. Red fluorescence from 488 mm laser-excited PI in every cell was analyzed using an EPICS Altra flow cytometer (Beckman Coulter, Fullerton, CA, USA) using a peak fluorescence gate to discriminate aggregates. The percentage of cells in the G1, S and G2/M phases were determined from the DNA content histograms using MultiCycle for windows software (Phoenix Flow Systems, San Diego, CA, USA).

The Raf kinase assay kit was purchased from Upstate Biotechnology (Charlottesville, VA, USA) and used in accordance with the manufacturer’s instructions. Western blot analysis was performed as previously described and the blots were visualized by peroxidase reaction using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA) (15). The following antibodies were used: cyclin D1, cyclin D3, Cdk4, Cdk6, p-Rb^{Ser807/Ser811}, c-Raf, p-c-Raf^Ser259, B-Raf, p-B-Raf^Ser445, p-Mek1/2^Ser217/221, Mek, p-Erk1/2^{Thr202/Tyr204}, Erk and GAPDH (Cell Signaling Technology, Beverly, MA, USA); cyclin E, Cdk2 and Rb (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and p-c-Raf^Ser43 and p-c-Raf^Ser621 (Abcam, Cambridge, MA, USA); β-actin (Neomarker, Fremont, CA, USA) and α-tubulin (Sigma). The blots are representative of three independent experiments.

Statistical analysis was performed using PASS 11 (NCSS, LLC, Kaysville, Utah, USA) using one way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The effect of cholera toxin on human bladder TCC cell growth was investigated. As shown in Fig. 1A, the viability of T24 and UM-UC-3 cells decreased following incubation with the cholera toxin (between 2 and 50 ng/ml) for 48 h. The inhibitory effect of cholera toxin (10 ng/ml) increased time dependently between 24 and 72 h (Fig. 1B) in the two cell lines. However, cell apoptosis and cell necrosis were not detected at 72 h (Fig. 1C). In combination with the result that fewer and smaller colonies are observed when cells are incubated with 10 ng/ml cholera toxin for 10 days (Fig. 1D), these results indicated that cholera toxin significantly inhibited T24 and UM-UC-3 cell proliferation in a dose- and time-dependent manner.

To further investigate the mechanism underlying the anti-proliferative activity of cholera toxin, its effect on cell cycle distribution was investigated using flow cytometry. In cholera toxin-treated T24 cells there was an accumulation of 93.4, 94.5 and 94.5% cells, respectively, in the G1 phase following 24 h incubation with cholera toxin at 2, 10 and 50 ng/ml, respectively, compared with the control group comprising of 78.5% cells. This was accompanied by a decrease in the proportion of cells in the S phase (Fig. 2A). In the time-response study, an increase in the percentage of cells in the G1 phase was observed with 10 ng/ml cholera toxin treatment for 12 (P<0.05) and 24 h (P<0.001) compared with the control cells (Fig. 2B).

Incubation with cholera toxin at 2, 10 and 50 ng/ml for 24 h, resulted in an increase in the number of UM-UC-3 cells in the G1 phase to 75.5, 76.9 and 77.3%, respectively, compared with 65.1% observed in the control cells (Fig. 2A). However, the effect of cholera toxin was weaker in UM-UC-3 cells compared with T24 cells. Similar to T24 cells, the effect of cholera toxin on G1 arrest in UM-UC-3 cells was also largely accompanied by a decrease in S phase cells (Fig. 2B). Overall, these results demonstrated that cholera toxin induced a significant G1 arrest of the cell cycle in T24 and UM-UC-3 cells.

Based on the G1 phase arrest induced by cholera toxin in T24 and UM-UC-3 cells, the expression of cell cycle regulatory molecules involved in G1 phase progression was then investigated. Cell cycle progression through the G1 phase is primarily regulated by the sequential activation of the cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes, which induce Rb phosphorylation and E2F release. However, Cdk1 (e.g. p27^Kip1) is capable of deactivating the cyclin D-Cdk4/6 and cyclin E-Cdk2 complexes and thus decreases the phosphorylation of Rb (16). In the present study, it was found that cholera toxin was able to notably reduce the protein expression levels of cyclin D1, Cdk4 and Cdk6 in a dose- and time-dependent manner. In addition, Rb phosphorylation markedly decreased in the two cell lines (Fig. 2A and B). However, cholera toxin was not observed to have an effect on other G1 regulators, including cyclin D3, cyclin E, Cdk2 and P27^Kip1 (data not shown). These results suggested that cholera toxin reduces the activation of Rb by downregulating the expression of cyclin D1, Cdk4 and Cdk6 thus inducing G1 phase arrest.

Our previous studies confirmed that PKA activators, cholera toxin or forskolin, repressed cell growth via cell cycle arrest in the G1 phase in malignant glioma cells via PKA activation (13,14). To examine whether PKA activation is also involved in the G1 growth arrest of cholera toxin in T24 and UM-UC-3 cells, a specific pharmacological inhibitor of PKA, KT5720, was used. As shown in Fig. 3A, pretreatment with KT5720 (1 μM) for 2 h eradicated cholera toxin-induced G1 phase arrest in the two cell lines. Decreased expression levels of cyclin D1, Cdk4 and Cdk6, as well as the reduced phosphorylation of Rb due to cholera toxin, were rescued by KT5720 pretreatment (Fig. 3B). This indicated that in TCC cell lines T24 and UM-UC-3, PKA stimulation was responsible for the cholera toxin-induced downregulation of G1-S transition promoters and G1 phase arrest.

A number of pathways are involved in the regulation of cell proliferation, including the c-Raf/Mek/Erk pathway, and functions via activation of multiple transcription factors, which switch on their target genes, for example cyclin D1 (7). To further investigate the mechanism by which cholera toxin inhibits proliferation and arrests cells in the G1 phase in the two TCC cell lines, the level of c-Raf phosphorylation was assessed at serine 43, 259 and 621 following cholera toxin treatment for 24 h. The result demonstrated that the phosphorylation of serine 43 by c-Raf was elevated by cholera toxin in a time-dependent manner (Fig. 4A).

The activity of the c-Raf/Mek/Erk pathway was then detected using the Raf kinase assay and western blot analysis. As shown in Fig. 4B, c-Raf activity was attenuated in cholera toxin-treated T24 and UM-UC-3 cells. Among the four major MAPK modules, ERK5/p38/JNK/Erk1/2, the one converging on the activation of ERK and its upstream activator MAPK and ERK kinase (MEK) is the most extensively studied and perhaps the most relevant to cancer pathogenesis and therapy. p-Mek1/2^Ser217/221 and p-Erk1/2^{Thr202/Tyr204} are markers of activated Mek1/2 and Erk1/2 and were found to be time-dependently downregulated in response to 10 ng/ml cholera toxin. Furthermore, KT5720 significantly inhibited c-Raf phosphorylation at serine 43 and upregulated the expression of p-Mek^Ser217/221 and p-Erk^{Thr202/Tyr204} in T24 and UM-UC-3 cells (Fig. 4C). These results demonstrated that PKA activation was required for the inactivation of the c-Raf/Mek/Erk pathway by cholera toxin.

To further identify the role of the c-Raf/Mek/Erk pathway in G1-S phase transition and cell proliferation, UO126, a specific inhibitor of Mek activity, was used. Treatment with 1 μM UO126 for 24 h arrested the cell cycle in the G1 phase, accompanied by a decrease in cell population in the S phase in T24 and UM-UC-3 cells (Fig. 4D). In addition, the expression of p-Erk^{Thr202/Tyr204}, cyclin D1, Cdk4, Cdk6 and p-Rb was downregulated in UO126-treated cells (Fig. 4E). The effect of UO126 on the two cell lines is consistent with cholera toxin. This suggested that the inactivation of the c-Raf/Mek/Erk pathway is responsible for cholera toxin-induced decreases in cyclin D1, Cdk4 and Cdk6 and results in G1 phase arrest.