Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 Page 6 of 9 Figure 4 Intracellular acidification by cis-UCA activates the ERK and JNK signalling pathways. (A.) The 5637 cells were treated with 0-3% cis-UCA at pH 6.5 for 2 h after which the culture medium was changed and the MTS/PMS reagent added to cells. Absorbance (490 nm) was measured hourly up to 4 h (Mean ± SEM; n = 3, representative of two independent triplicate experiments). (B.) The 5637 cells were treated with 2% cis-UCA at pH 6.5. After harvesting, whole cell lysates were prepared, and ERK and JNK activity was monitored by Western blotting against phospho-ERK, ERK, phospho- JNK, and JNK. Actin expression was measured as loading control. (C.) The cells were treated with JNK inhibitor SP600125 or ERK inhibitor PD98059 for 40 min before addition of 2% cis-UCA at pH 6.5 for the following 2 h. Twenty hours later, cell viability was monitored by the MTS assay (Mean ± SD; n = 3). a very low degree (data not shown), and the phosphory- lation of Akt was not affected by cis-UCA (data not shown). It is well known that the small-molecule inhibitors SP600125 and PD98059 are able to prevent the activity of JNK and ERK, respectively. To investigate if we could counteract the effect of cis-UCA on cell viability by inhibiting JNK and ERK1/2, the 5637 cells were incu- bated with cis-UCA in the presence of SP600125 and PD98059, after which cellular viability was measured by MTS assay. Surprisingly, the inhibitors were unable to prevent cis-UCA-mediated loss of cellular viability (Fig- ure 4C). The inhibitors alone were non-toxic to the cells (Figure 4C and data not shown). Moreover, SP600125 and PD98059 did not affect the cis-UCA- mediated increase in phosphorylation of ERK and JNK (data not shown). Together, these data indicate that cis-UCA is not capable of directly activating these signalling pathways. cis-UCA inhibits cellular phosphatase activity in 5637 bladder carcinoma cells Thus far we have demonstrated that the action of cis-UCA in 5637 cells is associated with increased phos- phorylation of ERK1/2 and JNK2, but the inhibition of these kinase pathways did not prevent cis-UCA-induced decrease in cell survival (Figure 4C). To provide an explanation for these unexpected data, we hypothesized that the cis-UCA-induced intracellular acidification would negatively regulate the phosphatase activity in cancer cells, which would then lead to enhanced protein phosphorylation of ERK1/2 and JNK2. To investigate this possibility, we analysed the activity of protein tyro- sine phosphatases as well as serine/threonine phospha- tase 2A in 5637 cell lysates prepared immediately after the 2-h cis-UCA pulse. We detected a significant decrease in serine/threonine phosphatase 2A activity with 1-2% cis-UCA as well as a decreased activity towards the two tested tyrosine phosphatase substrate peptides with 0.5-2% cis-UCA (Figure 5A-C). These results suggest that cis-UCA indeed inhibits serine/ threonine and protein tyrosine phosphatase activity. It is therefore likely that the reduced phosphatase activity indirectly promotes the major MAPK signalling path- ways. Discussion In the present study, we aimed at characterizing the cell signalling processes that contribute to the previously reported cytotoxic effects of cis-UCA in cultured bladder cancer cells [3]. The 2-h pulse treatment model in the