How different serols contribute to cytotoxic action of sea cucumber saponins

Saponins form a diverse class of secondary metabolites found in both plants and some marine invertebrates. Holothuroids, or sea cucumbers, produce these molecules as a chemical defense against predators and parasites, but interestingly, tolerate the cytotoxic nature of these chemicals. This tolerance is poorly understood. The aim of this study was therefore to elucidate the mechanisms behind the tolerance of holothuroid cells to the cytotoxic saponins (e.g. Frondoside A) they produce. This investigation was conducted using a suite of complementary biophysical tools, firstly using in vitro techniques such as Isothermal Titration Calorimetry (ITC) and calcein leakage experiments, for measuring interactions between lipid models of various compositions and the saponin Frondoside A, then using in silico approaches such as docking methods and insertion into implicit lipid bilayers, to provide a molecular point of view on our observations. Interactions between Frondoside A and cholesterol were more favorable than those with the holothuroid Δ7 and Δ9(11) sterols. Liposomes containing cholesterol resulted in exothermic interactions with the holothuroid saponin Frondoside A whereas liposomes containing the Δ7 sterol resulted in endothermic interactions. Lipid phase simulations using settings previously developed for plant saponins revealed that the holothuroid saponin Frondoside A has an agglomerating effect on cholesterol domains, similar to that previously observed for the plant saponin α-Hederin. However, when interacting with the Δ7 sterols, the sterol domains were fragmented into small clusters. A significantly lower leakage was observed with liposomes containing the Δ7 holothuroid sterol than that with liposomes containing cholesterol. Our results suggest that the structural peculiarities of holothuroid sterols provide the organisms with a mechanism to mitigate the sterol-agglomerating effect of saponins on the cell membranes, and therefore to protect sea cucumber cells from the cytotoxicity of the saponins they produce.