Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 Background absorbance (medium without cells) was sub- tracted from the measurements. For measurement of metabolic activity, the MTS assay was done in triplicate samples on 24-well plates. After a 2-h cis-UCA pulse at pH 6.5, the cell medium was chan- ged and the assay reagent was added. The absorbance was measured hourly at 490 nm up to 4 h of incubation at 37°C. Background absorbance (medium without cells) was subtracted from the measurements. Cell cycle analysis For cell cycle analysis, the cells were synchronized by serum starvation for 48 h, treated with cis-UCA for 2 h and let to recover for the next 24 h. After collection, the cells were disrupted and the nuclei labelled for DNA content with PI by resuspension to sodium citrate buffer (40 mM Na-citrate, 0.3% Triton X-100, 50 μg/ml PI). After incubation at room temperature for 10 min, the samples were immediately analysed by flow cytometry. Western blotting Whole-cell lysates were prepared by lysing the cells in Laemmli sample buffer and boiling for 10 minutes after which proteins were separated by SDS-PAGE. Western blotting was performed using antibodies against p44/42 ERK1/2, phosphorylated Thr202/Tyr204 ERK1/2, p46/54 JNK1/2, phosphorylated Thr183/Tyr185 JNK1/2 (all from Cell Signaling Technologies), and actin (Sigma-Aldrich). Secondary antibodies were purchased from Promega. Measurement of phosphatase activity Analysis of serine/threonine phosphatase 2A and tyro- sine phosphatase activity was conducted with assay kits (product no. V2460 and V2471, respectively; Promega) according to manufacturer’s protocol. In this assay, cell lysate is added on artificial phosphorylated substrates, and the generation of free phosphate is determined by measuring the absorbance of a complex formed by phosphate, molybdate, and malachite green [10-12]. Statistical analysis The statistical significance of differences in the data was calculated with a two-way Student’s t-test, with p < 0.05 considered to indicate significant differences. Results cis-UCA induces intracellular acidification in 5637 bladder carcinoma cells According to the protodynamic concept, the prerequisite of the anticancer action of a cell-permeable weak acid such as cis-UCA is the induction of intracellular acidifi- cation. We have recently reported that cis-UCA impairs the survival of 5637 bladder cancer cells at pH 6.5 but not at pH 7.4 in vitro [3]. To demonstrate that cis-UCA Page 3 of 9 is also able to decrease the intracellular pH, the 5637 cells were labelled with a pH sensitive fluorescent probe BCECF and treated with various concentrations of cis- UCA. In these experiments, dose-dependent acidifica- tion by up to around 0.8 pH units was seen within 1 h after the addition of cis-UCA at pH 6.5 but only by less than 0.2 units at pH 7.4 (Figure 1), confirming the pH- lowering effect of cis-UCA in bladder cancer cells. All subsequent experiments in this study were performed at pH 6.5 unless otherwise indicated. cis-UCA induces both apoptotic and necrotic cell death in 5637 bladder carcinoma cells Next, we examined the mechanisms of cell death in cis- UCA-treated 5637 cells. A 2-h pulse treatment model, which we have previously established [3], was used in these studies. Briefly, 5637 cells were first incubated for 2 h in the presence of 0-4% cis-UCA, followed by a 20-h recovery without cis-UCA. We were able to detect for- mation of cytoplasmic DNA-histone complexes indicative of apoptotic cell death after 2% cis-UCA treatment at pH 6.5, but not at pH 7.4 (Figure 2A). In the mildly acidic extracellular pH 6.5, also mitochondrial depolarization (Figure 2B) and activation of the caspase-3 (Figure 2C) were detected in cis-UCA-treated cells. These events are typical of apoptotic cell death. However, cell permeabili- zation indicated by propidium iodide labelling of the cells occurred with 2% cis-UCA, suggesting that cis-UCA also induces necrotic cell death (Figure 2D). This result was supported by the observation that the amount of Figure 1 cis-UCA induces intracellular acidification in 5637 bladder carcinoma cells. 5637 cells were labelled with BCECF prior to treatment with cis-UCA at indicated concentrations in buffer solutions adjusted to pH 7.4 or pH 6.5. After one hour the samples were analysed by flow cytometry for intracellular pH.