Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 cytotoxic effects of cis-UCA. However, inhibitors of the ERK and JNK pathways failed to block the effect of cis- UCA on cell viability. This indicated that cis-UCA might not promote ERK and JNK by activating their upstream regulators, but indirectly by inhibiting the phosphatases that inactivate these kinases. Remarkably, our measurements showed that the activity of protein tyrosine phosphatases and type 2A serine/threonine phosphatases was significantly decreased by 1-2% cis- UCA. Therefore, inhibition of phosphatase activity represents a novel mechanism for cis-UCA-mediated, intracellular acidification-dependent cytotoxic effects. The idea of using a phosphatase-inhibiting agent as a means to kill cancer cells is relatively uncommon, as synthetic phosphatase inhibitors are known for their high, unspecific toxicity and tumour-promoting poten- tial [13]. Nevertheless, natural products such as the pro- tein phosphatase 2A inhibitor cantharidin have been shown to display anti-tumour effects [14]. Mutations of the H-Ras proto-oncogene increase phosphatase 2A activity and sensitize cancer cells to okadaic acid- induced apoptosis [15], the effects of cis-UCA might not be linked to these mutations, as the H-Ras mutation seems to be rather rare in bladder cancer [16]. However, transitional cell carcinoma of human urinary bladder has been reported to harbour H-Ras mutations [17], leaving the potential role of H-Ras in this context open. In this study, we have characterized the molecular mechanisms underlying the anticancer effects of cis-UCA. In addition to causing apoptotic and necrotic cell death, cis-UCA also interferes with the metabolic activity and cell cycle progression of cancer cells. It is evident that the pro- todynamic action of cis-UCA has no selectivity against tumour cells versus normal cells. Instead, the potential of cis-UCA as an anticancer drug is based on the fact that solid tumour cells typically form a transmembrane pH gradient by acidifying the extracellular microenvironment, usually to around pH 6.7, while maintaining normal, slightly alkaline intracellular pH [18]. In these conditions, the tumour can be killed by intracellular acidification- induced injury caused by a protodynamic agent transport- ing hydrogen ions into the cytosol in a pH-sensitive manner. In the normal tissues, including the blood, the cytotoxicity of cis-UCA would be “neutralized”, because no hydrogen ion transportation would occur in the absence of the transmembrane pH gradient. Extensive preclinical evidence has confirmed that cis- UCA is neither mutagenic nor locally irritating. A single intravesical dose of up to high concentrations of 6% (60 mg/ml; 19) and 12% (120 mg/ml; Laihia et al. unpublished) cis-UCA instilled in the rat urinary bladder for 1 or 2 h, respectively, is well tolerated with no signs of clinical abnormality or macroscopic bladder damage. In addition, there is no apparent local or systemic Page 8 of 9 toxicity in dogs treated with 2-12% cis-UCA intravesi- cally twice a week for 1 h per instillation for 4 weeks (Laihia et al. unpublished). In light of the insignificant toxicity of cis-UCA, our cellular data, demonstrating marked cytotoxicity in bladder carcinoma cells, suggest that protodynamic therapy with cis-UCA could be a fea- sible intravesical treatment for bladder cancer. To con- clude, our results elucidate how cis-UCA modulates numerous key cellular processes, thereby compromising the viability of bladder carcinoma cells. The detailed mechanisms behind phosphatase inactivation and mito- chondrial permeabilization by cis-UCA treatment will be topics of our future studies. Conclusions In conclusion, our study describes the cytotoxic and cytostatic effects of cis-UCA in bladder carcinoma cells, and elucidates the underlying molecular mechanisms. The protodynamic action of cis-UCA leads to decreased intracellular pH, impaired metabolic activity and increased in cell death. Importantly, cis-UCA inhibits the activity of serine/threonine and tyrosine phospha- tases, thereby indirectly promoting ERK and JNK activ- ity. In the future, the protodynamic action of cis-UCA could be utilized in intravesical therapy to compromise the viability of bladder carcinoma cells. Additional material Additional file 1: Figure S1: Apoptotic nuclear morphology following 2% cis-UCA treatment. The control cells and the cells treated with 2% cis-UCA (pH 6.5) were fixed, labelled with DAPI and analysed by confocal microscopy for nuclear morphology (40× objective). The pictures are representative of two independent experiments. Abbreviations cis-UCA: cis-urocanic acid; ERK: extracellular signal-regulated kinase; JNK: c- Jun N-terminal kinase; MAPK: mitogen-activated protein kinase; PARP1: poly (ADP-ribose) polymerase-1. Acknowledgements Ms. Katarina Himberg is thanked for valuable technical assistance. Author details 1Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, BioCity, FI-20520 Turku, Finland. 2Department of Biosciences, Åbo Akademi University, FI-20520 Turku, Finland. 3BioCis Pharma Ltd., Itäinen Pitkäkatu 4 B, FI-20520 Turku, Finland. Authors’ contributions EP, AK, JL, LL and JE planned the study. EP, AK and JL carried out the experimental work, and EP, AK, JL, LL and JE analyzed the results. EP, AK and JL wrote the manuscript, and LL and JE edited the manuscript. All authors read and approved the final manuscript. Competing interests Jarmo Laihia and Lasse Leino are employees, board members, stock holders and patent inventors for BioCis Pharma Ltd. This study has been partially funded by BioCis Pharma Ltd.