Okadaic acid toxin at sublethal dose produced cell proliferation in gastric and colon epithelial cell lines

Abstract

The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption.

Figure 1

Effect of Okadaic Acid (OA) treatment on cell proliferation. AGS (A), MKN-45 (B) and Caco 2 (C) were incubated for 24 h or 48 h with different concentrations of OA, and the proliferation was measured by AlamarBlue cell viability assay. In B, D–F, the chart bars show significant differences compared to the control (N = 12). G–I show the effect of treatment with OA for 7 days, in AGS, MKN-45 and Caco 2, respectively, using the Trypan blue method exclusion (N = 8). * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 2

Effect of OA treatment for 4 h on cell viability. The viability was analyzed using the Alamarblue method. The three cell lines show increased resistance to the toxicity of OA with Caco 2 being the most resistant. In B, the chart bar shows the significant differences compared to the control (N = 12). * p < 0.05; ** p < 0.01; *** p < 0.005.

Figure 3

Treatment with OA generates overexpression of Cyclin B. By western blot, an increased expression of Cyclin B after exposure to OA for 4 h or 24 h is shown in all three cell lines. (N = 4 for AGS and MKN-45; N = 3 for Caco 2). * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 4

Treatment with OA generates activation of Akt and Erk pathways. By WB, an increased of p308-Akt1, p-473-Akt1 and p-Erk1/2 is shown relative to basal levels of Akt and Erk as an effect of OA after 4 h (N = 3). * p < 0.05, ** p < 0.01, *** p < 0.005.