Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 Page 7 of 9 Figure 5 cis-UCA inhibits phosphatase activity in 5637 bladder carcinoma cells. To measure the effect of cis-UCA on phosphatase activity, the 5637 cells were treated with 0.5-2% cis-UCA or phosphatase inhibitors, 20 nM calyculin A and 1 mM orthovanadate (Na2VO4), at pH 6.5 for 2 h. The cells were subsequently lysed, incubated with the substrate peptide for Ser/Thr phosphatase 2A (RRA(pT)VA; A.), or Tyr phosphatases (END(pY)INASL; B. and DADE (pY)LIPQQG; C.) for 30 min, and the absorbance was measured 20 min after the end of the substrate reaction (Mean ± SD; n = 3; Student’s t-test * p < 0.05, ** p < 0.01, *** p < 0.001). Ser/Thr phosphatase 2A activity (A.) was measured in a reaction buffer without MgCl2 to inhibit phosphatase 2C activity. moderately differentiated human transitional bladder cancer cells was chosen to mimic conventional clinical intravesical instillation chemotherapy treatment of patients with non-muscle-invasive bladder cancer. We found that the protodynamic use of cis-UCA initiates intracellular acidification in bladder carcinoma cells, followed by modulation of several key processes includ- ing cell cycle, metabolic activity, and cell death. A 2-h pulse with 2% cis-UCA approximately triples the amount of apoptosis of the control level, but also promotes non- apoptotic cell death. The continuum between cell death responses can be both a consequence of secondary necrosis due lost selective membrane permeability in apoptotic cells (upon prolonged incubation), as well as the triggering of unspecific cell death within the cell population. When we analysed caspase activity and other markers of apoptosis, we found that cis-UCA induces the activation of caspase-3, causes mitochondrial depolariza- tion and formation of cytoplasmic DNA-histone com- plexes, while caspase-8 activation is not affected. In addition, 2-3% cis-UCA treatment results in PARP1 clea- vage. These data suggest that cis-UCA-mediated apopto- sis occurs via the intrinsic apoptotic pathway, concurring with the idea of the mitochondrial pathway as the major mediator of stress-induced apoptosis. Importantly, cis-UCA not only caused cell death, but also had cytostatic effects. We found that 2-3% cis-UCA severely impaired the metabolic activity of the 5637 bladder carcinoma cells. This effect could be detected already 1 h after the cis-UCA pulse, and the relative dif- ference compared to the untreated cells grew over time. The induction of both apoptotic and partly necrotic responses in the bladder carcinoma cells reflect the effects of cis-UCA treatment on cellular metabolism, as mitochondrial functions play a central role in triggering of both apoptotic and necrotic cell death. Moreover, cell cycle progression was disturbed by cis-UCA, but it did not seem to be mediated by p53-dependent mechan- isms, as we found that the compound had no effect on the cyclin-dependent kinase inhibitors p27 and p21, both of which are important mediators of p53-depen- dent cell cycle arrest. However, as the untreated 5637 bladder carcinoma cells expressed high levels of the p27 and p21 in the absence of cell cycle arrest, these inhibi- tory pathways may be impaired in this cancer cell type. In addition to clarifying which cellular processes are targeted by cis-UCA, we were also interested in analys- ing the signalling pathways contributing to these effects. We discovered that the 5637 bladder carcinoma cells show very low endogenous activities of ERK, JNK, and Akt. Interestingly, cis-UCA remarkably increased the activity of both ERK and JNK, whereas the levels of phosphorylated Akt remained unchanged. This suggests that ERK and JNK pathways may mediate some of the