- Open Access
Phenotype-dependent apoptosis signalling in mesothelioma cells after selenite exposure
© Nilsonne et al; licensee BioMed Central Ltd. 2009
- Received: 16 March 2009
- Accepted: 29 June 2009
- Published: 29 June 2009
Selenite is a promising anticancer agent which has been shown to induce apoptosis in malignant mesothelioma cells in a phenotype-dependent manner, where cells of the chemoresistant sarcomatoid phenotype are more sensitive.
In this paper, we investigate the apoptosis signalling mechanisms in sarcomatoid and epithelioid mesothelioma cells after selenite treatment. Apoptosis was measured with the Annexin-PI assay. The mitochondrial membrane potential, the expression of Bax, Bcl-XL, and the activation of caspase-3 were assayed with flow cytometry and a cytokeratin 18 cleavage assay. Signalling through JNK, p38, p53, and cathepsins B, D, and E was investigated with chemical inhibitors. Furthermore, the expression, nuclear translocation and DNA-binding activity of p53 was investigated using ICC, EMSA and the monitoring of p21 expression as a downstream event. Levels of thioredoxin (Trx) were measured by ELISA.
In both cell lines, 10 μM selenite caused apoptosis and a marked loss of mitochondrial membrane potential. Bax was up-regulated only in the sarcomatoid cell line, while the epithelioid cell line down-regulated Bcl-XL and showed greater caspase-3 activation. Nuclear translocation of p53 was seen in both cell lines, but very little p21 expression was induced. Chemical inhibition of p53 did not protect the cells from apoptosis. p53 lost its DNA binding ability after selenite treatment and was enriched in an inactive form. Levels of thioredoxin decreased after selenite treatment. Chemical inhibition of MAP kinases and cathepsins showed that p38 and cathepsin B had some mediatory effect while JNK had an anti-apoptotic role.
We delineate pathways of apoptosis signalling in response to selenite, showing differences between epithelioid and sarcomatoid mesothelioma cells. These differences may partly explain why sarcomatoid cells are more sensitive to selenite.
- Mitochondrial Membrane Potential
- Electrophoretic Mobility Shift Assay
- Malignant Mesothelioma
- Epithelioid Cell
Selenite is a redox-modulating compound which is increasingly investigated for use as an anticancer agent. We have recently shown that selenite induces apoptosis in malignant mesothelioma cells in a dose-, time- and phenotype-dependent manner, with a more potent effect on sarcomatoid cells [1, 2]. Promising anti-cancer effects have also been shown in in vitro models of lung, prostate, breast, skin, and hematologic cancers [3–12], with a selective effect upon malignant cells compared to normal cells [1, 4, 13]. Several investigators have showed independently that selenite cytotoxicity can be inhibited by antioxidants [1, 14–19]. Redox regulation is likely to influence cellular sensitivity to selenite, and we have reported that selenite decreases the activity of thioredoxin reductase (TrxR) . Together with thioredoxin (Trx) and NADPH, it forms the thioredoxin system, which is highly active in redox signalling and defence against oxidative stress.
Malignant mesothelioma is a tumor of the serosal membranes, most often arising in the pleura after prolonged asbestos exposure. This tumor has a peculiar pattern of differentiation, where the malignant cells may assume either an epithelioid or a sarcomatoid phenotype. These two phenotypes exhibit differences in their biological behavior, as evidenced by gene expression analyses [20–23] and the fact that presence of sarcomatoid cells is associated to poor prognosis and increased therapy resistance [24–26]. The median survival time from diagnosis is around 12 months . Response rates to current pharmacological therapies are low, reaching only 40% at best [28, 29].
This study aimed to investigate apoptosis signalling during selenite treatment in an epithelioid and a sarcomatoid mesothelioma cell line. Both were initially derived from the same tumor , and the latter is more sensitive to selenite. Thus, we anticipated the emergence of differences in apoptosis signalling in response to selenite that might explain the differential sensitivity of the two cell lines.
Cells and culture
This study was carried out using a well-established model system for mesothelioma differentiation, consisting of the two cell sub-lines STAV-AB and STAV-FCS. Cells were derived from a single tumor, and subsequently induced to differentiate into the epithelioid (STAV-AB) and the sarcomatoid phenotype (STAV-FCS), respectively, by altering the serum composition . Hence, STAV-AB cells were grown in Gibco RPMI 1640 medium (Invitrogen) and 10% human AB serum, whereas STAV-FCS cells were grown in the same medium and 10% fetal calf serum. The specific differentiation of these cells has been evidenced by immunoprofiling showing that STAV-AB cells express more cytokeratin, whereas STAV-FCS cells have stronger reactivity to vimentin antibodies  as well as by morphometry. The elongated sarcomatoid cell morphology of the STAV-FCS cells and the more round epithelioid morphology of the STAV-AB cells have been confirmed by average length:width ratios of 3.42 in the STAV-FCS cells and 1.58 in the STAV-AB cells . Jurkat cells were obtained from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium and 20% fetal calf serum. All cells were grown at 37°C with 5% CO2 and passaged approximately twice per week.
Treatment of cell cultures and inhibition of signalling enzymes
Chemical inhibitors against apoptosis signalling enzymes
Cathepsin D, E
SERVA Electrophoresis GmbH
Cell viability assays
Viability assays were performed in conjunction with flow cytometry experiments to obtain internal controls. Aliquots of cell suspensions prepared for flow cytometry were plated in triplicates in 96-well plates, with a density of approx. 5000 cells per well. They were then analysed using the WST-1 assay (Roche), whereby a tetrazolium salt is cleaved by mitochondrial enzymes to yield a coloured product, to measure viability. The plates were read in a Spectramax spectrophotometer at 450 nm with subtraction of background absorbance at 600 nm.
Flow cytometric analyses
Flow cytometric assays for detection of apoptosis were carried out using the Annexin V kit (Caltag Laboratories) according to the manufacturer's protocol. Briefly, trypsinized cells were resuspended in Binding Buffer with Annexin V-FITC and Propidium Iodide (PI), and incubated for 15 minutes in the dark.
For analysis of Bax expression, cells were fixed in 0.25% paraformaldehyde, and permeabilised with 100 μg/ml Digitonin. Aliquots were then incubated for 30 minutes with phycoerythrin-conjugated mouse anti-human Bax antibody (Santa Cruz, sc20067) or mouse IgG as a control, both in final dilution 1:10. Morphological controls were established using cytospins. Slides were fixed in 4% buffered formaline, washed 2 × 5 min in PBS, and air-dried. Staining was performed with the same antibody concentration and incubation time, and the staining was evaluated by confocal microscopy.
For analysis of Bcl-XL expression, cells were fixed in 1% formaldehyde, and permeabilised with 0.1% Saponin. Aliquots were then incubated for 15 minutes with phycoerythrin-conjugated rabbit anti-human Bcl-XL antibody (GeneTex, GTX46035), final dilution 1:800, or rabbit IgG as a control. The secondary antibody Alexa 488 goat anti-rabbit IgG was diluted 1:1600, and incubation was performed for 15 minutes in the dark.
The mitochondrial membrane potential was measured using two independent methods. 1) The Mitochondria Staining Kit (Sigma) was used according to the manufacturer's instructions. Briefly, cells were trypsinised and then resuspended in a solution of 45% medium, 5% serum and 50% staining solution containing the JC-1 probe. They were incubated for 20 min in 37°C, and then washed with staining buffer. Cells treated with Valinomycin were used as a positive control. 2) With the fluorescent probe DiOC6(3) (3,3-dihexyloxacarbocyanine iodide; Molecular Probes), cells were incubated for 15 minutes with concentrations ranging from 1 to 100 nM DiOC6(3). After staining, an aliquot of cells was prepared for confocal microscopy to verify that the staining was localized to the mitochondria.
For analysis of procaspase-3 expression, cells were fixed in 1% paraformaldehyde, and permeabilised with 10 μg/ml Digitonin. Aliquots were then incubated for 30 minutes with a rabbit monoclonal antibody to procaspase-3 (Abcam, ab32150), final dilution 1:150, or rabbit IgG as a control. The secondary antibody Goat anti-Rabbit IgG-FITC (Abcam, ab6717) was diluted 1:300, and incubation was performed for 30 minutes in the dark.
Detection of the active form of caspase-3 was performed with a FITC-conjugated antibody (BD Biosciences, 559341). Cells were fixed in 1% paraformaldehyde, and resuspended in 100 μg/ml Digitonin solution with antibody in final dilution 3:20, and incubated for 30 minutes at 4°C. Cells treated with 2 μM doxorubicin for 24 h were used as positive controls.
Flow cytometry was always performed immediately after the staining was completed. All analyses were performed on a Becton Dickinson flow cytometer and the data were processed in the Cell Quest program.
Cytokeratin 18 cleavage assay
The M-30 Apoptosense assay (Peviva, Stockholm) measures cytokeratin 18 fragments specifically produced by cleavage by caspases 3, 6, 7, and 9 [32, 33]. Cells were seeded in 96-well microtiter plates with or without 10 μM selenite and 0.2 μg/ml doxorubicin. After 24 h, cells were lysed by the addition of 10 μl 10% Tergitol-type NP-40 (Sigma-Aldrich) to each well. The ELISA analysis was carried out according to the manufacturer's instructions. Briefly, 25 μl samples were incubated together with 75 μl horseradish peroxidase-conjugate solution on the ELISA microplate for 4 h on a shaker. 200 μl of tetramethylbenzidine substrate solution were added and the plate was incubated for a further 20 min. The reaction was stopped by the addition of 50 μl 1.0 M H2SO4, and the absorbance at 450 nm was determined on a Spectramax spectrophotometer.
Immunocytochemistry and confocal microscopy
For analysis of nuclear translocation of p53 and p21, cytospins were prepared. For p53 analysis, the slides were fixed in ice-cold dry acetone. Prior to staining, they were heated to 100°C for 5 min in citrate buffer, pH 6.0. Staining was performed using the p53 Refine kit (Novacastra). For p21 analysis, the slides were fixed in 4% buffered formaline, and air-dried. Staining was performed with a mouse monoclonal antibody (Calbiochem, OP64), diluted 1:200, for 30 minutes.
For analysis with monodansyl cadaverine (MDC), cells were grown on sterilised Superfrost Plus slides (Menzel GmbH &Co). The slides were stained for 10 minutes with 10 μM MDC (BioChemica), and immediately analysed by confocal microscopy.
DNA binding assay for p53
Nuclear extracts were prepared as described previously . Electrophoretic Mobility Shift Assay (EMSA) was conducted using the LightShift Chemiluminescent EMSA Kit (Pierce). 20 μg of nuclear protein was used for each sample. The double-stranded oligonucleotide probes for the p53 binding site (sense 5'-TACAGAACATGTCTAAGCATGCTGGGG-3') were annealed and labeled with biotin. To label DNA probes, the Biotin 3' End DNA Labeling Kit (Pierce) was used according to the manufacturer's protocol.
Measurement of Thioredoxin
ELISA was used to quantify the amounts of thioredoxin (Trx) in the cells. The assay was adapted from Pekkari et al . Wells were coated with a primary monoclonal antibody (2G11, kindly provided by dr. Anders Rosén of the University of Linköping) overnight at 4°C, 5 μg/ml diluted in carbonate buffer, pH 9.6. Secondary biotinylated antibody (IMCO Corporation) was added in a concentration of 5 μg/ml. Absorbance at 405 nm was measured using a SpectraMax 250 spectrophotometer (Molecular Devices). Data were analyzed using the SOFTmax Pro software, v. 2.6.
All experiments were performed at least three times. When one representative experiment is shown, it was chosen on the basis of being closest to the average of all the experiments performed. Student's t-test, two-way ANOVA with Dunnett's post test or Bonferroni's multiple comparison test, and χ2-tests were used to determine statistical significance. The choice of statistical method for each experiment is indicated in the respective legend text.
Selenite is a strong prooxidant when used in cytotoxic doses, and may induce apoptosis. Many independent researchers have confirmed that the cytotoxicity of selenite is mediated by oxidative stress, in cell types so various as malignant mesothelioma , hepatoma [14, 15, 36], cancers of the breast , prostate [4, 17, 19, 37], and uterine cervix , glioma , lymphoma , and Jurkat T-cells . Cell death with apoptotic characteristics has also been observed in erythrocytes following selenite treatment . Selenite-induced oxidation may target many cellular components, and the resulting damage and cell signalling is therefore likely to be dependent on the constitution of the cell in question, and may vary between cell types, and indeed between mesothelioma cells of different phenotypes. This study is the first to our knowledge to investigate whether such differentiation-dependent variation accounts for differences in sensitivity between cell phenotypes.
Selenite induced apoptosis and sarcomatoid cells were more sensitive
Selenite-induced loss of mitochondrial membrane potential and effects of inhibition of apoptosis signalling enzymes
Loss of δΦ m after selenite treatment a
Statistical significance vs. no selenite b
Statistical significance vs. selenite only c
Loss of δΦ m after selenite treatment a
Statistical significance vs. no selenite b
Statistical significance vs. selenite only c
2.89 (± 0.68)
1.28 (± 0.18)
3.41 (± 0.57)
p < 0.01
3.30 (± 0.24)
p < 0.001
0,94 (± 0.06)
1.05 (± 0.05)
JNK + selenite
3,96 (± 0.58)
p < 0.001
3.74 (± 0.25)
p < 0.001
0.99 (± 0.04)
0.88 (± 0.03)
p38 + selenite
4.06 (± 0.63)
p < 0.001
4.15 (± 0.52)
p < 0.001
0.74 (± 0.05)
0.92 (± 0.03)
p53 + selenite
2.62 (± 0.57)
p < 0.05
3.59 (± 0.52)
p < 0.001
1.27 (± 0.12)
1.46 (± 0.10)
Cathepsin B + selenite
5.68 (± 0.70)
p < 0.001
6.27 (± 0.75)
p < 0.001
p < 0.01
Cathepsin D, E
0.93 (± 0.06)
0.90 (± 0.03)
Cathepsin D, E + selenite
3.95 (± 0.77)
p < 0.001
3.45 (± 0.37)
p < 0.001
To further delineate the role of signalling molecules among the MAP kinases and cathepsins, chemical inhibitors against these enzymes were used (Table 1). In the untreated epithelioid cells, the inhibitors decreased the baseline apoptotic fraction by 20–50% [see Additional file 1]. This demonstrates the efficacy of the inhibitors at the concentrations in which they were used. None of the enzyme inhibitors affected the proportion of viable cells during Annexin-PI apoptosis assays, although the WST-1 viability assays indicated a modest growth inhibitory effect of CA 074-Me and SB 203580 (data not shown).
Further controls to verify the efficacy of the chemical inhibitors were obtained by testing them on Jurkat cells over a 25 h time course following apoptosis induction with 0,2 μM staurosporine. The inhibitors of JNK, p53 and cathepsin D and E successfully decreased the apoptosis induction, whereas the cathepsin B inhibitor increased it [see Additional file 2].
p38 inhibition reduced apoptosis frequency slightly in sarcomatoid cells
In the sarcomatoid cells, the p38 inhibitor SB203580 caused a small decrease in the apoptotic response to selenite (Figure 1D). In the epithelioid cells, p38 inhibition had no effect on the ability of selenite to induce apoptosis. However, selenite caused an even more marked drop of the δΦm after p38 inhibition in both cell types (Table 2). This indicates that p38 was involved in apoptotic signalling particularly in the more sensitive sarcomatoid cells. The effect of inhibition was small however, and it cannot be regarded a key pathway. Activation of p38 after selenite exposure has previously been shown in cervix , leukemia  and prostate cancer cells .
Inhibition of JNK increased the apoptotic response of epithelioid cells
Inhibition of JNK increased the proportion of selenite-induced early apoptotic cells by more than two thirds in the epithelioid cells (Figure 1C). In the sarcomatoid cells the effect was comparable to that without the inhibitor (Figure 1D). Scant effect on the loss of δΦm was observed (Table 2). JNK apparently played no role in apoptosis signalling in the sarcomatoid cells. In the epithelioid cells, JNK even had a small antiapoptotic effect. The lack of proapoptotic activity is concordant with earlier findings in cervix cancer cells  but different from findings in prostate cancer cells .
Selenite caused nuclear accumulation but inactivation of p53
The expression of p21 (also known as Cip1 and WAF1) in response to genotoxic stress is tightly regulated by p53 (reviewed in ), and we therefore measured it as an additional indicator of p53 activity. The fraction of p21-positive cells was approximately doubled by selenite treatment (Figure 2F–J). Although these changes are statistically significant, the positive fraction was very small even after selenite treatment. As a positive control, epithelioid cells were treated with 2 μM doxorubicin and showed a 22% positive fraction (not shown).
Cells of either phenotype treated with the p53 inhibitor Pifithrin did not show a decreased apoptosis frequency as judged by Annexin-PI (Figure 1), nor a smaller loss of δΦm (Table 2). This is particularly interesting since p53 inhibition decreased the baseline apoptosis in untreated cells (Figure 1, Additional file 1). Consequently, p53 was active in the control cells but was inactivated by selenite. Apoptosis was still induced by selenite, implicating p53-independent pathways in this process.
To find the mechanism of inhibition, we considered the complex regulation of p53 activity. The central DNA-binding core domain of p53 contains one zinc atom. Zinc chelators have been shown to cause accumulation of wild-type p53 in a structurally aberrant form with inhibited DNA-binding activity . Selenium is a known chelator of zinc and when applied in vivo as selenite or its reduced form selenide, it forms nanocrystals of zinc-selenium with free or loosely bound zinc . Another possibility is that selenite as an oxidizing agent may act directly upon regulatory cysteines on the p53 molecule, leading to an accumulation of oxidized p53 incapable of DNA-binding . Also, secondary mediated redox regulation needs to be considered. The multifunctional protein Redox Effector Factor 1 (Ref-1) is involved in the redox regulation of stress inducible transcription factors such as Activating Protein-1, Nuclear Factor-κB, Hypoxia Inducible Factor-1 and p53, and may play an important role in this system. Ref-1 depends on thioredoxin (Trx) to maintain its active reduced state [49–51]. In a yeast experimental system, it has been shown that deletion of thioredoxin reductase (TrxR) downregulates p53 activity by keeping it in its oxidized form [52, 53]. Trx overexpression on the other hand has been shown to increase p53 transactivation of reporter genes in human cell lines .
Protein levels of Trx were reduced by selenite treatment in sarcomatoid cells, from 175 ng/mg to 100 ng/mg. The epithelioid cells had a baseline expression of 57 ng/mg, decreasing slightly to 52 ng/mg after selenite treatment (Figure 3). It has been demonstrated previously that levels of TrxR decrease significantly in both cell lines with selenite treatment . Consequently, redox inactivation of p53 is a plausible explanation for the lack of activity that was seen despite nuclear accumulation following selenite exposure.
Selenite induced Bax up-regulation and Bcl-XL down-regulation
In mesothelioma, the antiapoptotic Bcl-2 family member Bcl-XL is frequently overexpressed , and this has been shown to be an important mechanism by which mesothelioma cells gain apoptosis resistance . In the epithelioid cells, the Bcl-XL expression decreased markedly after selenite treatment, whereas only a subpopulation of the sarcomatoid cells showed lower expression after treatment (Figure 4).
Selenite caused caspase activation particularly in the epithelioid cells
Divergent data have been published regarding the role of caspases in selenite-induced apoptosis. Several studies have shown that selenite causes a caspase-independent apoptotic cell death [6, 18, 40], whereas others have shown caspase-dependence [9, 17, 36, 57]. We report that caspase-3 was activated in a sub-population of epithelioid cells, but little reactivity was seen in sarcomatoid cells. The limited caspase activation in sarcomatoid cells was surprising. A possible explanation could be an upregulation of Inhibitor of Apoptosis (IAP) family members such as survivin and XIAP. Earlier studies have found that overexpression of IAP family members is common in mesothelioma cells [58–61].
Inhibition of cathepsin B but not of cathepsins D and E caused increased loss of δΦm
Cathepsins are a group of proteases that are physiologically present in lysosomes, and may be released upon stimuli such as oxidative stress . Cells that were pretreated with cathepsin B inhibitor CA-074 Me showed slightly less apoptosis after selenite exposure (Figure 1). In the sarcomatoid cells, this was reflected in correspondingly increased viability. In the epithelioid cells, the viable proportion decreased slightly instead. Interestingly, when selenite was combined with the cathepsin B inhibitor, the loss of δΦm was greater than with any other inhibitor (Table 2). Cathepsin D and E inhibitor Pepstatin A did not affect the induction of apoptosis by selenite, nor did it alter the loss of δΦm.
Signs of autophagy were not detected
Autophagy is a form of programmed cell death in which cells do not exhibit apoptotic characteristics. Kim et al have shown that selenite induces autophagy in glioma cells . We wanted to investigate whether some of the cell death that we observe could be due to autophagy. Cells were stained with monodansyl cadaverine and analysed with confocal microscopy for the appearance of granules that might represent autophagic vesicles. No changes in the staining pattern could however be detected between untreated and selenite-treated cells of either phenotype [see Additional file 3].
Our results clearly demonstrate that selenite causes a complex pattern of cell death in malignant mesothelioma cells. Selenite causes both apoptosis and necrosis, but cells exhibiting apoptotic characteristics such as Annexin V externalisation do not necessarily display other classical apoptosis-related changes such as caspase-activation [6, 18, 40]. It appears purposeful to consider selenite-induced cell death to lie on a spectrum between apoptosis and necrosis, where the exact mode of cell death differs depending on phenotype characteristics.
Our results indicate that mesothelioma cells activate p38 and JNK in response to selenite, and that they accumulate p53 in the nucleus, but in a form bereft of DNA-binding activity. We hypothesise that this interesting phenomenon is due to a shift in redox balance towards a prooxidative state with increased levels of reactive oxygen species (ROS) and a loss of thioredoxin system activity.
Sarcomatoid mesothelioma cells, although ordinarily chemoresistant, are more sensitive to selenite than epithelioid cells . The differential activation of apoptosis-signaling proteins on the level of the mitochondrion may partially explain the observed differences in sensitivity.
A better understanding of the proapoptotic mechanisms of selenite as well as of phenotype-dependent response patterns in mesothelioma cells will aid the development of cancer therapies with greater efficacy and which may be better suited to the diverse biology of individual tumors. Malignant mesothelioma is a heterogeneous entity, and further studies on differentiation-related sensitivity to selenite and other cytotoxic drugs are under way in our laboratory using a panel of cell lines of varying epithelioid-sarcomatoid differentiation.
The authors are grateful to Mervi Nurminen, Gunilla Fahlström, and Anette Hofmann for their expert technical assistance, and to Kristin Gustafsson. This study has been supported by the Swedish Foundation for Strategic Research, the Swedish Heart and Lung foundation, the Swedish Cancer Fund, and the Swedish Cancer and Allergy Fund.
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