Cytotoxic effects exerted by pentachlorophenol by targeting nodal pro-survival signaling pathways in human pancreatic cancer cells

Abstract

Pancreatic adenocarcinoma is one of the deadliest human solid tumors in the developed countries characterized by high resistance toward chemotherapeutic treatment. We have previously shown that silencing of the pro-survival protein kinase CK2 by RNA interference contributes to enhance the cytotoxicity of the chemotherapeutic agent 2',2'-difluoro 2'-deoxycytidine (gemcitabine). Initial experiments showed that pentachlorophenol (PCP) inhibits CK2 and induces cell death in human pancreatic cancer cell lines. We report here evidence that exposure of this type of cells to PCP induces caspase-mediated apoptosis, inhibition of the lysosome cysteine protease cathepsin B and mitochondrial membrane depolarization. Beside cellular inhibition of CK2, the analysis of signaling pathways deregulated in pancreatic cancer cells revealed that PCP causes decreased phosphorylation levels of NF-κB/p65, suppresses its nuclear translocation and leads to activation of JNK-mediated stress response. Surprisingly, exposure to PCP results in increased phosphorylation levels of AKT at the canonical S473 and T308 activation sites supporting previous data showing that AKT phosphorylation is not predictive of tumor cell response to treatment. Taken together, our study provides novel insights into the effects induced by the exposure of pancreatic cancer cells to chlorinated aromatic compounds posing the basis for more advanced studies in vivo.

Keywords: CK2; Caspase-mediated apoptosis; Pancreatic cancer cells; Pentachlorophenol; Signaling pathways.

Fig. 1

PCP is the active component of C11 exerting ATP site-directed inhibition of CK2. (a) Chemical structures of PCP and DMA present in C11 in a 1:1 molar ratio. (b) The IC₅₀ values for the CK2α subunit and the CK2 holoenzyme were determined by a dose-response curve for each individual compound/mixture, respectively. Inhibition is expressed in percentage relative to the activity of the enzyme measured in the absence of compound/mixture (i.e. control experiment). Results are mean values ± standard deviation (S.D.) from assays run in triplicates. *p < 0.005, **p < 0.001 denote statistical significant differences for measurements performed with C11 and PCP with respect to control values. (c) Kinetic analysis of enzyme inhibition by PCP. Upper bar-graphs refer to Michaelis–Menten kinetics while lower bar-graphs to Lineweaver–Burk plots. Enzyme activity was determined at various ATP concentrations in the absence or presence of two fixed concentrations of PCP, i.e. 1 μM and 10 μM, respectively. Results are mean values ± S.D. from experiments run in triplicates.

Fig. 2

Cytotoxic effects of PCP on human pancreatic cancer cells. (a) Panc-1 and MIA Paca-2 cells were incubated with increasing concentrations of C11, PCP, DMA for 48 h, respectively. PCP+DMA refers to the two individual compounds added to the cells in a 1:1 molar ratio. Cells incubated with solvent DMSO (0 μM) were used as a control. Cell viability was determined by the WST-1 assay as described in Section 2. Viability was determined as a difference in absorbance measured at 450 nm and 690 nm (reference) wavelengths, respectively. Values were expressed in percentage relative to control assay. (b) Flow cytometry analysis of CD44 and CD24 expression in Panc-1 cells after enrichment. Two phenotypic subpopulations were collected. CD24-depleted cells and CD24-enriched cells. Percentage refers to the amount of CD44⁺/CD24⁺ cells in whole CD44⁺-cell population. (c) CD24-depleted and CD24-enriched cell populations were treated with C11 or PCP for 48 h. Cytotoxicity was determined as described above. Control refers to the cell population before the enrichment procedure. Experiments were repeated three times obtaining similar results. Data from a representative experiment (mean values ± S.D. of eight replicates) are shown.

Fig. 3

Cell incubation with PCP induces apoptotic cell death in human pancreatic cancer cells. (a) Cells were incubated with DMSO or exposed to 100 μM C11, PCP and DMA, respectively, for variable incubation times. Cell cycle analysis was performed by flow cytometry. The distribution of cells in the various phases of the cell cycle is expressed in percentage. Cell death is indicated by the percentage of cells in the sub-G1 phase. (b) Western blot analysis of markers for apoptotic cell death from cells treated with 100 μM C11 and PCP, respectively, for 48 h. Control experiments are represented by the analysis of whole lysates from cells treated with DMSO. β-actin detection was performed for verifying equal loading. Experiments shown here, were performed three times obtaining similar results.

Fig. 4

Cathepsin B activity in whole cell lysates after treatment of cells with C11 and PCP reveals decreased enzyme activity. Cells were treated with DMSO (control experiment), C11 or PCP as indicated in the figure for 48 h. A positive control experiment was carried out by incubating cells with 150 μM temozolomide (TMZ) for 48 h. A negative control experiment was performed by adding 2 μl CB inhibitor to whole cell lysate (Cathepsin B assay kit, BioVision) for 2 h. The released fluorochrome AFC is expressed as percentage of fluorescence emitted/sample relative to release of reaction product in DMSO-treated cells. *p < 0.005 indicates values with statistically significant difference with respect to numbers obtained from DMSO-treated cells. Experiments were performed two times with four replicates each.

Fig. 5

Cytochrome c release following C11 or PCP treatment is cell type specific. (a) The content of isolated mitochondria from cells treated as indicated in the figure for 48 h was analyzed by Western blot using an antibody directed against cytochrome c. The detection of ATP synthase subunit β (ATP5B) was carried out as a control for equal loading. (b) Mitochondrial membrane potential was analyzed by cell staining with JC-1 reagent after the indicated treatments for 48 h. JC-1 monomeric form and aggregates in the mitochondria were detected using a BP 525/20; 635/40 filter allowing the simultaneous detection of green and red fluorescence emission. Cell images were taken at 400× magnification. (c) Quantification of the red fluorescence emission was carried out by flow cytometry and expressed in percentage relative to DMSO-treated cells. Results are presented as the average of three independent experiments. *p < 0.005 denotes statistical significant difference for measurements performed with cells treated with PCP with respect to control values.

Fig. 6

Analysis of cell signaling pathways de-regulated in human pancreatic cancer cells. (a–d) Whole lysate from cells treated as indicated in the figure for 48 h were analyzed by Western blot employing antibodies directed against the indicated proteins or their phosphorylated form, belonging to the PI3K/AKT, MAPK and NFκB signal transduction pathways, respectively. Analysis of the expression and phosphorylation status of Cdc37, an endogenous substrate of CK2, and expression levels of the CK2 subunits is also shown. In all experiments, β-actin was detected as control for equal loading. Numbers below the blots refer to quantitation of band signal intensity with ImageJ software. Experiments were performed at least three times obtaining similar results.

Fig. 7

Cell incubation with PCP influences negatively the TNFα-induced NFκB/p65 translocation into the nucleus. Cells were left untreated or incubated with 100 μM PCP for 48 h. Where indicated, cells were added 20 ng/ml TNFα in the last 15 min of incubation time. After fixation, cells were subjected to immunofluorescence analysis. Nuclei were visualized by DAPI staining. Negative control (NC) refers to cells immunostained with only the biotinylated secondary antibody and FITC-conjugated streptavidin. Original magnification: 400×. At least 50 cells were analyzed per treatment condition.