Peuhu et al. BMC Cancer 2010, 10:521 http://www.biomedcentral.com/1471-2407/10/521 R E S E A R C H A R T I C L E Open Access Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells Emilia Peuhu1,2†, Aura Kaunisto1,2†, Jarmo K Laihia3, Lasse Leino3*, John E Eriksson1,2* Abstract Background: cis-urocanic acid (cis-UCA) is an endogenous amino acid metabolite capable of transporting protons from the mildly acidic extracellular medium into the cell cytosol. The resulting intracellular acidification suppresses many cellular activities. The current study was aimed at characterizing the molecular mechanisms underlying cis-UCA-mediated cytotoxicity in cultured cancer cells. Methods: 5367 bladder carcinoma cells were left untreated or treated with cis-UCA. Cell death was assessed by measuring caspase-3 activity, mitochondrial membrane polarization, formation and release of cytoplasmic histone- associated DNA fragments, and cellular permeabilization. Cell viability and metabolic activity were monitored by colorimetric assays. Nuclear labelling was used to quantify the effects of cis-UCA on cell cycle. The activity of the ERK and JNK signalling pathways was studied by immunoblotting with specific antibodies. Phosphatase activity in cis-UCA-treated cells was determined by assay kits measuring absorbance resulting from the dephosphorylation of an artificial substrate. All statistical analyses were performed using the two-way Student’s t-test (p < 0.05). Results: Here we report that treatment of the 5637 human bladder carcinoma cells with 2% cis-UCA induces both apoptotic and necrotic cell death. In addition, metabolic activity of the 5637 cells is rapidly impaired, and the cells arrest in cell cycle in response to cis-UCA. Importantly, we show that cis-UCA promotes the ERK and JNK signalling pathways by efficiently inhibiting the activity of serine/threonine and tyrosine phosphatases. Conclusions: Our studies elucidate how cis-UCA modulates several cellular processes, thereby inhibiting the proliferation and survival of bladder carcinoma cells. These anti-cancer effects make cis-UCA a potential candidate for the treatment of non-muscle invasive bladder carcinoma. Background cis-urocanic acid [cis-UCA; 3-(1H-imidazol-4-yl)prop-2- enoic acid] is an endogenous metabolite of amino acid histidine found in the mammalian skin after exposure to ultraviolet radiation. cis-UCA is known to induce immu- nosuppression in animals [1,2]. We have recently dis- covered that cis-UCA is capable of transporting protons from the mildly acidic extracellular medium into the cytosol of cancer cells, thereby inhibiting the prolifera- tion of several human tumour cell lines [3,4], and indu- cing apoptosis in vitro and in tumour xenografts in vivo [4]. Intracellular acidification is known to affect cell * Correspondence: lasse.leino@biocis.com; john.eriksson@abo.fi † Contributed equally 1Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, BioCity, FI-20520 Turku, Finland 3BioCis Pharma Ltd., Itäinen Pitkäkatu 4 B, FI-20520 Turku, Finland Full list of author information is available at the end of the article proliferation [5,6] and to promote apoptosis [7,8]. This new mode of action of cis-UCA, which we have named as the protodynamic action, constitutes a novel concept of anticancer therapy. The transportation and release of protons into the cytosol is based on the unique second acid dissociation constant (pKa2) of cis-UCA [9], and it is inherently independent of enzymes and membrane receptors. The use of cis-UCA as an anticancer agent against non-muscle-invasive bladder cancer has previously been evaluated in cultured moderately and poorly differen- tiated human transitional bladder cancer cells using a short term (up to 2 h) pulse treatment model in vitro, which mimics the clinical intravesical chemotherapy regimen [3]. In the current study, we aimed at charac- terizing the molecular mechanisms underlying cis-UCA- mediated cytotoxicity to cultured cancer cells. Here we show that cis-UCA extends a wide range of effects on © 2010 Peuhu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.