Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 bladder carcinoma cells. Treatment of 5637 bladder carcinoma cells with 2% cis-UCA induced a combina- tion of apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells was rapidly impaired, and cell cycle arrest was induced at 1-3% cis-UCA. Importantly, we were able to show that cis-UCA pro- motes the ERK and JNK signalling pathways. Rather than due to increased kinase activation, the observed effect seemed to be caused by a decrease in both tyro- sine and serine/threonine phosphatase activities that act as critical regulators of the ERK and JNK signalling pathways. Within the scope of the protodynamic therapy concept, our study suggests a novel mechanism for cis- UCA-induced inhibition of cancer cell survival, based upon compromised signalling and metabolic functions and cell cycle arrest leading to apoptotic and necrotic cell death. Our observations give incentive to further studies addressing the efficacy of cis-UCA in intravesical treatment of bladder carcinoma. Methods Cell culture and treatments 5637 bladder carcinoma cell were cultured as monolayers in logarithmic growth phase in Dulbecco’s Modified Eagle medium (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin- streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator at 37°C and 5% CO2. The medium was adjusted to pH 6.5 or 7.4 with NaOH after the addi- tion of cis-UCA (BioCis Pharma, Turku, Finland) [3] or PIPES (Sigma-Aldrich) as osmotic control. For inhibition of the JNK and ERK kinase pathways, the cells were incu- bated with the JNK inhibitor SP600125 (10 μM; Sigma- Aldrich) or the MAPKK inhibitor PD98059 (40 μM; Sigma-Aldrich) for 40 min before cis-UCA treatment. In the protein phosphatase activity measurements, calyculin A (Calbiochem/Merck, Darmstadt, Germany) was used at 20 nM and sodium orthovanadate (New England Biolabs) at 1 mM for 2 h during the cis-UCA pulse to inhibit phosphatases. Measurement of intracellular pH Two million cells were incubated in 1 ml of Hank’s balanced salt solution (HBSS; Sigma-Aldrich), pH 7.4, containing 0.35 μM 2’,7’-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein (BCECF, acetoxymethyl ester; Mole- cular Probes) at room temperature for 30 min, washed twice in HBSS, and resuspended in 0.5 ml of physiologi- cal saline. Various concentrations of cis-UCA were added to HBSS buffer, the solutions were adjusted to pH 6.5 or 7.4 and 300 μl of each solution was mixed with 15 μl of the BCECF-labelled cell suspension. The cells were analysed by flow cytometry (FACScan, BD Page 2 of 9 Pharmingen) within 1 h. Fluorescence intensities were calibrated by analysing BCECF-labelled cells in 50 mM potassium phosphate buffers, pH 6.2-7.8, containing 43 mM KCl and 10 μM nigericin (Molecular Probes). Detection of cell death After the 2-h cis-UCA pulse in indicated concentrations and a subsequent recovery time of 20 h without cis-UCA, both the floating and attached cells were collected by trypsinization. Caspase-3 activation was detected accord- ing to manufacturer’s protocol (BD Pharmingen). Briefly, the cells were washed, fixed, permeabilized and labelled with phycoerythrin-conjugated anti-active caspase-3 anti- body. After labelling, the cells were washed and analysed by flow cytometry (FacsCalibur, BD Pharmingen). Mito- chondrial membrane potential was measured by seques- tering of tetramethyl rhodamine methyl ester (TMRM) to cells. The cells were incubated in 50 nM TMRM (Invitro- gen) in PBS for 15 min at 37°C, washed and analysed by flow cytometry. The TMRM-negative population was gated as cells with lost mitochondrial membrane poten- tial. Formation of cytoplasmic and released histone-asso- ciated DNA fragments was assayed in triplicate cell lysates and cell culture supernatants, respectively, with a photometric enzyme immunoassay (Cell Death Detection ELISAPLUS, Roche, Mannheim, Germany) according to the manufacturer’s instructions. Background absorbance of the reaction buffers at the corresponding pH without cells and the absorbance values of each well at a non- specific reference wavelength were subtracted. For detec- tion of necrotic cell death, the cells were placed on ice and incubated with 50 μg/ml propidium iodide (PI; Sigma-Aldrich) in PBS for 10 min. The samples were analysed directly by flow cytometry for PI labelling indi- cative of cellular permeabilization. Nuclear morphology of the cells was investigated by labelling with DAPI. The cells were fixed with 3% paraformaldehyde for 15 min at room temperature and resuspended in PBS. Cytospin preparates were made and the samples were mounted with DAPI Vectashield (Vector Laboratories). The sam- ples were analysed with Zeiss LSM confocal microscope (405 nm excitation, 420 nm long-pass emission filtering, 40× oil immersion objective). Detection of cell survival and metabolic activity The survival of cells was quantified as the number of viable cells by using a colorimetric method (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega) that quantifies the formation of a formazan product from 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) in live cells. The absorbance at 490 nm was read in flat-bottom 96-well plates at 4 h after the addition of the assay reagent.