A New Edible Plant-Derived Toxin as Potential Candidate for Immunotherapy: Evaluation of Cytotoxic Profile on Intestinal Cells

Introduction: Ribosome-inactivating proteins (RIPs) are toxic plant enzymes. Several RIPs have been conjugated to specific carriers to obtain immunotoxins, which gave promising results in pre-clinical and clinical studies. However, some adverse effects were reported, mainly immunogenicity. Recently, a new RIP from the edible plant Salsola soda, named sodin-5, has been identified and characterized. The aim of this work was to evaluate the sodin-5 cytotoxic profile on intestinal cell lines, comparing it with saporin, one of the best known and most studied RIPs for the construction of immunotoxins.

Methods: Different endpoints were considered to evaluate RIP cytotoxicity on colon adenocarcinoma Caco-2 and HT29 cell lines: cell viability experiments through tetrazolium salt reduction and cell death through cytofluorimetric analysis of AnnexinV/PI positivity. The effect of the RIPs on the membrane integrity was monitored by Trans-Epithelial- Electrical Resistance (TEER) measurements on Caco-2 monoculture and/or Caco-2/HT29 co-culture. RIP immunological cross-reactivity was evaluated through ELISA.

Results: Our results showed that sodin-5 had high cytotoxicity, very similar to that of saporin. Both RIPs reduced cell viability after 72 h, with EC₅₀s in the nM range. Apoptosis was the main cell death pathway triggered by RIPs, and no necrosis involvement was observed. The lack of necrosis is very important as apoptosis does not cause inflammation. Sodin-5 and saporin significantly reduced TEER values both in Caco-2 cell mono- and Caco-2/HT29 co-culture in comparison to untreated controls. Finally, no immunological cross-reactivity was observed with saporin anti-serum.

Conclusions: Since sodin-5 and saporin had similar cytotoxic activities, sodin-5 could be proposed for further study as a potential toxic payload in immunotoxin construction to overcome some adverse effects of saporin and other RIPs. Theoretically, RIPs from edible sources should be less immunogenic and better tolerated by humans; therefore, it could represent a better candidate for immunotoxin-based experimental therapy.