Introduction: Edible mushrooms have many nutritional goods; they are rich in carbohydrates, proteins, minerals, and flavor compounds and are low in fat and calories. Moreover, mushrooms are rich in beneficial bioactive components with pharmacological properties, but they are also known to contain toxic substances. Recently, a new protein synthesis inhibitor enzyme from Boletus edulis, named edulitin 2, has been identified and characterized as a ribotoxin-like protein. The aim of this paper is to evaluate the cytotoxic profile of edulitin 2 on two colon adenocarcinoma cell lines, Caco-2 and HT29.

Methods: Edulitin 2 cytotoxicity was evaluated on Caco-2 and HT29 cells measuring cell viability and apoptotic/necrotic cell death in dose–response and time–response experiments. Furthermore, the effect of edulitin 2 on the integrity of the cultured cell monolayer was assessed using Transepithelial Electrical Resistance (TEER) measurements. TEER was evaluated in both Caco-2 monocultures and Caco-2/HT29 co-cultures, where the latter more closely resembled the intestinal epithelium.

Results: Edulitin 2 seems to require a quite long delay to exert its toxic effects. In fact, in our models, viability was slightly reduced after 24h of incubation with edulitin 2, but it was strongly affected after 48 and 72h. The half-maximal effective concentration (EC₅₀) values, calculated from dose–response curves at 72h, were in the 0.1 µM range. Flow cytometric analysis showed apoptotic cell death (early and/or late), but not necrosis. After 72 h, 1 µM of edulitin 2 strongly reduced TEER values in both models, thus suggesting the damage of cell monolayer integrity.

Conclusions: Our data demonstrate that edulitin 2 exerts a cytotoxic effect on intestinal cells after long time incubations (48-72 h). No necrotic lesions were observed in intoxicated cells at any tested concentration. The discrimination between apoptosis and necrosis is important as apoptosis does not cause inflammation.