Frontiers in Marine Science (Jun 2014)
The marine toxin, Yessotoxin, induces apoptosis and increases mitochondrial activity
Abstract
Abstract: Yessotoxin (YTX) group are phycotoxins produced by different species of dinoflagellates. YTX activates the Phosphodiesterases (PDEs). This group of toxins can be consumed by humans, however no intoxication events are reported. Controversial results by measuring cell viability with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide) assay had been described. In this paper mitochondrial activity and cell death are studied through different viability tests, flow cytometry, western blot and caspase activity kit. The results point to a mitochondrial activity increase after YTX treatment that shows a false proliferation increment. In conclusion, when MTT viability test is used, the cell number is an important parameter to determine, since an increase in mitochondrial activity can disguise cell death signal. Introduction: YTX group are synthesized by the dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulax spinifera (Satake et al., 1997). PDEs are described as the YTX target (Alfonso et al., 2003;Alfonso et al., 2005). Through PDEs, YTX activates different metabolic cascades that lead to cell death (Korsnes and Espenes, 2011;Korsnes, 2012). A common method to study cell viability is the MTT assay (Denizot and Lang, 1986; Chiba et al., 1998). In this assay, the yellow MTT tetrazolium salt is metabolized by mitochondrial enzymes resulting in an insoluble violet-blue formazan (Mosmann, 1983;Stockert et al., 2012). Also other viability assays are described independently of the mitochondria, such as the lactate deshydrogenase assay (LDH) (Lobner, 2000;Leirós et al., 2013;Leiros et al., 2014). In this case the LDH enzyme released by damaged cells is measured in the extracellular medium, and cell viability can be estimated (Lobner, 2000). The inconvenience of MTT assay is the possibility of false increased viabilities by the increment in mitochondrial activity (Ulukaya et al., 2004;Dell'Ovo et al., 2008). YTX induces apoptosis in a wide range of tumoral cells (Korsnes, 2012). In addition, it was described that YTX induces intrinsic or mitochondrial apoptotic cell death (Korsnes and Espenes, 2011). However, death receptor (DR) dependent or extrinsic apoptotic pathway activation by YTX is still unstudied (Korsnes, 2012). Extrinsic apoptosis needs a ligand that binds to DR activating caspase 8 by its cytosolic region and downstream caspase 3 is also activated, finally the apoptotic cascade is triggered (Korsnes and Espenes, 2011). In this context the aim of this work was to study mitochondrial activity and apoptotic cell death induced by YTX. Materials and methods: Cell culture: K-562 cell line was purchased from the National Cancer Institute (NCI's) and maintained in RPMI 1640 medium as was explained before (Tobío et al., 2012). Incubations with 30 nM YTX from CIFGA (Lugo, Spain) were carried out for 24 hours. Cell viability: K-562 cells were centrifuged (1500 r.p.m., 5 minutes, 4ºC) after treatment. The pellets were resuspended in saline solution (in mM: Na+ 142.3; K+ 5.94; Ca2+ 1; Mg2+ 1.2; Cl- 126.2; HCO3- 22.85; HPO4 2- 1.2, SO4 2- 1.2; glucose 1 g/L was added to the medium giving an osmotic pressure of 290±10 mOsm/kg of H2O and pH was adjusted to 7.2 with HCl 0.1 N and CO2) with MTT (250µg/mL) (M2128, Sigma) and incubated and measured as described before (Tobío et al., 2012). The supernatant was used to measure LDH by using the in vitro Toxicology Assay Kit (TOX7, Sigma) following the commercial protocol. The assays were carried on with a cell number population per condition of 1.106 and 5.105 cells. Flow cytometry: 2.106 cells per condition were treated with YTX. Cells were washed with saline solution, incubated with 200 nM MitoTracker® Deep Reed FM (MTDR) (Invitrogen, USA) 30 minutes, 37ºC in dark, washed again and fixed as explained before (Tobío et al., 2011). Cells were washed with PBS from Gibco, Life Technologies (UK) and finally resuspended in 100µL of PBS pH 7.2 and analyzed by flow cytometry. Obtained data were analysed by the IDEAS 4.0 Cell Image Analysis software. Caspase 8 activity: Caspase 8 activity was measured with the Caspase-8/FLICE Colorimetric Protease Assay from Invitrogen (Madrid, Spain) following the manufacturer’s instructions. Subcellular fractionation: 3.106 cells per condition were incubated with YTX. Cells were centrifuged and washed with saline solution, then were resuspended in lysis buffer with the following composition: 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1X Complete Protease Inhibitor and 1X Phosphatase Inhibitor Cocktail. The extract was sonicated and centrifuged (10000 r.p.m., 10 minutes, 4ºC). The supernatant with the cytosolic franction stored at -20ºC until protein quantification. Western blotting analysis: Bradford assay and the Direct Detect Spectrometer from Millipore (Germany) were used to know sample protein concentration and BSA was used as protein standard. The different subcellular fractions were blotted to PVDF membrane by reduced SDS-PAGE. Precision Plus ProteinTM Standards KaleidoscopeTM was used as molecular weight marker in the electrophoretic runs. After blockage with 0.5% BSA the membranes were incubated 10 minutes with anti-caspase 3, washed with PBS and 0.1% Tween®20 and incubated for 10 minutes with secondary anti-mouse IgG horseradish peroxidase-linked species-specific whole antibody. After three washes, chemiluminescence was visualized with SuperSignal® West Pico (low intensity) or with SuperSignal® West Femto (high intensity) both from Pierce (ThermoScientific, USA). The chemiluminiscent signal was detected with the Diversity GeneSnap software and analized by the Diversity 4 gel documentation and analysis system. Relative protein expression was calculated respect to β-actin expression in each experiment. Experiments were carried out at least three times by duplicate. Histone 1 was used as negative control of nuclear fraction and β-tubulin as cytosolic positive control. Results: YTX is highly related to apoptosis activation in different cell lines (Leira et al., 2002;Malaguti et al., 2002;Korsnes and Espenes, 2011). In tumoral K-562 cell line, after 24 hours incubation with YTX the viability is decreased (Tobío et al., 2012). In these conditions the percentage of K-562 cell viability was studied by using different methods, MTT and LDH viability assays after YTX treatment (figure 1). By MTT, an increase of about 30% of cell viability after YTX treatment was observed when the sample size was 1.106 cells per condition. However, when the number of cell was reduced to 5.105 cells per condition, the percentage of cell viability in the presence of toxin was significantly decreased (20%). On the other hand, when the viability was checked with the LDH test, a significantly decreased, 20% and 45%, was observed depending on the sample size, 1.106 cells and 5.105 cells respectively. The false increased in proliferation detected by the MTT assay in the sample size of 1.106 cells per condition after YTX incubation, points to an increase in mitochondrial activity. Therefore, in figure 2, mitochondrial activity was measured by using the MitoTracker® Deep Reed FM (MTDR) dye. The mitochondrial activity was stained after oxidation and fixation of the compound by the mitochondrial metabolism. In figure 2A and 2B the representative histogram and cell images in brightfield (channel 05) and MTDR intensity (channel 06) are shown. The diagram, figure 2A, represents in X axis the MTDR intensity. A displacement of MTDR intensity to the right was observed after YTX treatment. The results from three experiments are collected in figure 2C, where the mitochondrial activity was significantly increased, around 30%, in K-562 incubated with YTX. Finally, apoptotic hallmarks were studied in these conditions. Figure 3 represents caspase 8 activity and caspase 3 expression levels after YTX exposure. As figure 3A shows, a significant increase of caspase 8 activity, 0.16 units respect to untreated cells, and caspase 3 expression levels, 0.21 units, were observed. Therefore, YTX induces K-562 cell death by activating apoptosis and increases mitochondrial activity. Discussion: Colorimetric MTT assay is widely used as a viability measurement method (McHale and L., 1988;Chiba et al., 1998). But after YTX treatment, MTT assay had shown problems to detect a cell viability decrease. In this sense, in primary cardiac cell cultures, a false increment of the proliferation rate opposite to Sulforhodamine B assay (SRB) results was reported after YTX treatment (Dell'Ovo et al., 2008). Also the same effect was obtained in different cancer cell lines after assaying anticancer therapies (Ulukaya et al., 2004). In our study, an increase in cell viability using MTT was observed when the number of cells was high, while by using the LDH assay a significant viability decrease was measured. In these conditions, YTX is activating extrinsic apoptosis cell death by increasing caspase 8 activity and caspase 3 levels. The explanation for this increase was found when the mitochondrial activity was quantified cell by cell in a cytometer. In these conditions a significant increment of mitochondrial activity was detected. Since the cell population is too high, the increase in mitochondrial activity that detects the MTT test disguised the decrease of signal due to the cell death and point to a false proliferation increase. In this sense, a mitochondrial activity decrease was observed after 48 hours YTX treatment in BE(2)-M17 neuroblastoma cell line (Leira et al., 2002). However, this study was done in a microplate reader with a small number of cells (Leira et al., 2002). Therefore, to measure the viability by MTT assay is very important to take into account the number of cells per condition when the experiment is designed. Alternative assays, such as LDH test, independently of the direct mitochondrial activity, can be used.
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