Molecular targets for the protodynamic action of cis-urocanic acid in human bladder carcinoma cells

5637 bladder carcinoma cell were cultured as monolayers in logarithmic growth phase in Dulbecco's Modified Eagle medium (Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in a humidified incubator at 37°C and 5% CO₂. The medium was adjusted to pH 6.5 or 7.4 with NaOH after the addition of cis-UCA (BioCis Pharma, Turku, Finland) [3] or PIPES (Sigma-Aldrich) as osmotic control. For inhibition of the JNK and ERK kinase pathways, the cells were incubated with the JNK inhibitor SP600125 (10 μM; Sigma-Aldrich) or the MAPKK inhibitor PD98059 (40 μM; Sigma-Aldrich) for 40 min before cis-UCA treatment. In the protein phosphatase activity measurements, calyculin A (Calbiochem/Merck, Darmstadt, Germany) was used at 20 nM and sodium orthovanadate (New England Biolabs) at 1 mM for 2 h during the cis-UCA pulse to inhibit phosphatases.

Two million cells were incubated in 1 ml of Hank's balanced salt solution (HBSS; Sigma-Aldrich), pH 7.4, containing 0.35 μM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF, acetoxymethyl ester; Molecular Probes) at room temperature for 30 min, washed twice in HBSS, and resuspended in 0.5 ml of physiological saline. Various concentrations of cis-UCA were added to HBSS buffer, the solutions were adjusted to pH 6.5 or 7.4 and 300 μl of each solution was mixed with 15 μl of the BCECF-labelled cell suspension. The cells were analysed by flow cytometry (FACScan, BD Pharmingen) within 1 h. Fluorescence intensities were calibrated by analysing BCECF-labelled cells in 50 mM potassium phosphate buffers, pH 6.2-7.8, containing 43 mM KCl and 10 μM nigericin (Molecular Probes).

After the 2-h cis-UCA pulse in indicated concentrations and a subsequent recovery time of 20 h without cis-UCA, both the floating and attached cells were collected by trypsinization. Caspase-3 activation was detected according to manufacturer's protocol (BD Pharmingen). Briefly, the cells were washed, fixed, permeabilized and labelled with phycoerythrin-conjugated anti-active caspase-3 antibody. After labelling, the cells were washed and analysed by flow cytometry (FacsCalibur, BD Pharmingen). Mitochondrial membrane potential was measured by sequestering of tetramethyl rhodamine methyl ester (TMRM) to cells. The cells were incubated in 50 nM TMRM (Invitrogen) in PBS for 15 min at 37°C, washed and analysed by flow cytometry. The TMRM-negative population was gated as cells with lost mitochondrial membrane potential. Formation of cytoplasmic and released histone-associated DNA fragments was assayed in triplicate cell lysates and cell culture supernatants, respectively, with a photometric enzyme immunoassay (Cell Death Detection ELISA^PLUS, Roche, Mannheim, Germany) according to the manufacturer's instructions. Background absorbance of the reaction buffers at the corresponding pH without cells and the absorbance values of each well at a non-specific reference wavelength were subtracted. For detection of necrotic cell death, the cells were placed on ice and incubated with 50 μg/ml propidium iodide (PI; Sigma-Aldrich) in PBS for 10 min. The samples were analysed directly by flow cytometry for PI labelling indicative of cellular permeabilization. Nuclear morphology of the cells was investigated by labelling with DAPI. The cells were fixed with 3% paraformaldehyde for 15 min at room temperature and resuspended in PBS. Cytospin preparates were made and the samples were mounted with DAPI Vectashield (Vector Laboratories). The samples were analysed with Zeiss LSM confocal microscope (405 nm excitation, 420 nm long-pass emission filtering, 40× oil immersion objective).

The survival of cells was quantified as the number of viable cells by using a colorimetric method (CellTiter 96 AQueous One Solution Cell Proliferation Assay; Promega) that quantifies the formation of a formazan product from 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) in live cells. The absorbance at 490 nm was read in flat-bottom 96-well plates at 4 h after the addition of the assay reagent. Background absorbance (medium without cells) was subtracted 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 changed 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.

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.

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.

Analysis of serine/threonine phosphatase 2A and tyrosine 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].

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.

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 acidification. 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 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 acidification 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.

Bild vergrößern

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 formation 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 permeabilization 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 cell-released DNA-histone complexes, measured in the extracellular medium, increased with 2% and 4% cis-UCA (data not shown). To eliminate the possibility that these effects were caused by increased extracellular osmolality due to high concentrations of cis-UCA, cells were treated with an equimolar concentration of PIPES, which is otherwise nontoxic to cells. In these experiments, PIPES affected neither cell viability nor cellular markers of apoptosis and necrosis (Figure 2).

Bild vergrößern

To further analyse the biochemical events that take place after 2% cis-UCA treatment, the nuclei were labelled with DAPI and imaged by confocal microscopy (Additional file 1: Figure S1). The results showed that a large fraction of the cis-UCA-exposed 5637 cells displayed nuclei with characteristic apoptotic features, including nuclear condensation and fragmentation. To investigate caspase signalling upstream of caspase-3, the activation-induced cleavage of caspase-8 was monitored by Western blotting. Interestingly, we were unable to detect caspase-8 cleavage, indicating that caspase-8 is not activated in response to cis-UCA (data not shown). This suggests that the extrinsic apoptotic pathway is probably not involved in cis-UCA-induced cell death. A modest cleavage of poly(ADP-ribose) polymerase-1 (PARP1) after treatment with 2% cis-UCA was detected (data not shown), suggesting that in our experimental setup, the cleavage of PARP1 is more likely to indicate general cellular stress than progression of classical apoptotic cell death.

As cis-UCA was able to trigger cell death, we sought to determine whether cell cycle progression of the 5637 cells was also affected by cis-UCA. For this purpose, the cells were synchronized to G0/G1 by serum starvation, and the nuclei were analysed after the 2-h cis-UCA pulse followed by 24-h recovery for DNA content by PI labelling and flow cytometry. Interestingly, already 1% cis-UCA induced G2/M block, as the cells accumulated in this phase (Figure 3). Higher concentrations of cis-UCA slightly increased the fraction of cells in the G0/G1 phase, potentially indicating a block at the cell cycle entry after synchronization. In addition, the higher concentrations are also likely to induce a general cell cycle block due to short-circuits in cell cycle control. Western blotting studies showed that cis-UCA does not cause the accumulation of the cyclin-dependent kinase inhibitors p21(WAF1/Cip1) and p27(Kip1), which were highly expressed already in untreated 5637 cells (data not shown), indicating that the arrest is a consequence of general disturbances in cell control mechanisms rather than specific triggering of emergency break mechanisms, such as expression of p21 or p27.

Bild vergrößern

The metabolic activity of the 5637 cells was followed for 4 h after the 2-h cis-UCA pulse using a MTS assay. A clear inhibition of the cumulative cellular reducing power was monitored by hourly measurements after 2% and 3% cis-UCA treatments (Figure 4A) without any visible effects on cell viability at these early time points. To study whether the observed metabolic inhibition was associated with activation of any major signalling pathways, we measured the effects of cis-UCA on stress and mitogen-inducible protein kinases. For this purpose, we collected whole-cell lysates immediately after the 2-h pulse with 2% cis-UCA and analysed the phosphorylation and overall expression levels of ERK1/2 and JNK1/2 by Western blotting. Interestingly, we detected a marked increase in the phosphorylated forms of ERK1/2 and JNK2 in response to cis-UCA treatment (Figure 4B), suggesting strong activation of these pathways. In contrast, the phosphorylation of JNK1 was only increased to a very low degree (data not shown), and the phosphorylation of Akt was not affected by cis-UCA (data not shown).

Bild vergrößern

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 incubated 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 (Figure 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.

Thus far we have demonstrated that the action of cis-UCA in 5637 cells is associated with increased phosphorylation 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 tyrosine phosphatases as well as serine/threonine phosphatase 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.

Bild vergrößern