Role of yessotoxin in calcium and cAMP‐crosstalks in primary and K‐562 human lymphocytes: The effect is mediated by Anchor kinase a mitochondrial proteins

Abstract

Yessotoxin (YTX) is a marine polyether toxin previously described as a phosphodiesterase (PDE) activator in fresh human lymphocytes. This toxin induces a decrease of adenosine 3',5'-cyclic monophosphate (cAMP) levels in fresh human lymphocytes in a medium with calcium (Ca(2+) ), whereas the contrary effect has been observed in a Ca(2+) -free medium. In the present article, the effect of YTX in K-562 lymphocytes cell line has been analysed. Surprisingly, results obtained in K-562 cell line are completely opposite than in fresh human lymphocytes, since in K-562 cells YTX induces an increase of cAMP levels. YTX cytotoxicity was also studied in both K-562 cell line and fresh human lymphocytes. Results demonstrate that YTX does not modify fresh human lymphocytes viability, whereas in K-562 cells, YTX has a highly cytotoxic effect. It has been described in a previous study that YTX induces a small cytosolic Ca(2+) increase in fresh human lymphocytes but no effect was observed on Ca(2+) pools depletion in these cells. However, our results show that, in K-562 cells, YTX has no effect on cytosolic Ca(2+) levels in a medium with Ca(2+) and induces an increase on Ca(2+) pools depletion followed by a Ca(2+) influx. As far as Ca(2+) modulation is concerned these results demonstrate that YTX has a clear opposite effect in tumoural and fresh human lymphocytes. In addition, intracellular Ca(2+) reservoirs affected by YTX are different than thapsigargin-sensible pools. Furthermore, YTX-dependent Ca(2+) pools depletion was abolished by cAMP analogue (dibutyryl cAMP), phosphodiesterase-4 (PDE4) inhibitor (rolipram), protein kinase A inhibitor (H89) and oxidative phosphorylation uncoupler carbonyl cyanide p-(trifluoromethoxy) (FCCP) treatments. This evidences the crosstalks between Ca(2+) , YTX and cAMP pathways. Also, results obtain demonstrate that YTX-dependent Ca(2+) influx was only abolished by FCCP pre-treatment, which indicates a link between YTX and mitochondria in K-562 cell line. Cytosolic expression of A-kinase anchor proteins (AKAPs), the proteins which integrates phosphodiesterases (PDEs) and PKA to the mitochondria, was determined in both cell models. On the one hand, in human fresh lymphocytes, YTX increases AKAP149 cytosolic expression. This fact is accompanied with a decrease in cAMP levels, and therefore PDEs activation, which finally leads to cell survival. On the other hand, in tumoural lymphocytes, YTX has an opposite effect since decreases AKAP149 cytosolic expression and increase cAMP levels which leads to cell death. This is the first time that YTX and mitochondrial AKAPs proteins relationship is characterised.

Key role of phosphodiesterase 4A (PDE4A) in autophagy triggered by yessotoxin

“…Cells treated with YTX for 24 and 48 h decreased their viability (Tobío et al, 2012). After 24 h of YTX treatment, the apoptotic programmed cell death was activated, while after 48 h apoptotic hallmarks were not detected.…”

Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

“…The pellets were first washed with saline solution and centrifuged (1100 × g, 5 min, 4°C), and then re-suspended in saline solution with MTT (250 μg/mL) (M2128, Sigma) and incubated for cell viability (Tobío et al, 2012). The supernatants were utilized to measure LDH release by using the in vitro Toxicology Assay Kit (TOX7, Sigma) following the commercial protocol.…”

“…Since YTX modulates PDEs, the complex was studied after toxin treatment in the tumor K-562 cell line. In this sense, a close relation between the complex expression and cell death activated by the toxin was discovered (Tobío et al, 2012; Fernandez-Araujo et al, 2014). This was supported by the fact that silencing the expression of PDE4A, the effect of YTX on K-562 cell viability is avoided and changes in the cytosolic expression of the rest of the proteins of the complex is observed (Fernandez-Araujo et al, 2014).…”

“…YTXs are modulators of phosphodiesterases (PDEs) and consequently affect the levels of cyclic adenosine 3′,5′-cyclic monophosphate (cAMP) (Alfonso et al, 2003, 2004, 2005; Pazos et al, 2006). The final effect is different depending on the cellular model studied, human fresh lymphocytes or human leukemic K-562 cell line (Alfonso et al, 2003; Tobío et al, 2012). Moreover, YTX has been described as a mitochondrial apoptosis inducer (Korsnes and Espenes, 2011; Korsnes, 2012).…”

“…As mentioned, large differences, in terms of YTX toxicity, cAMP levels and AKAP149 expression, were found depending on the cellular model studied. In this sense, while no effect on cell viability was observed in human fresh lymphocytes, high cell death was detected in leukemic K-562 cells after YTX treatment (Tobío et al, 2012). Later on, the effect in the K-562 line was studied in depth and YTX was described as apoptotic and autophagy inductor in these cells (Fernandez-Araujo et al, 2014).…”