Assessment of the toxicity and antiproliferative activity of hemocyanins from Helix lucorum, Helix aspersa and Rapana venosa

Abstract

Hemocyanins (Hcs) are respiratory, oxygen-carrying metalloproteins that are freely dissolved in the hemolymph of many molluscs and arthropods. The interest in hemocyanins has grown significantly since it was found that they can be successfully used in immunotherapy of neoplastic diseases as non-specific or active stimulators of the immune system. The present study aims to assess the cytotoxicity, in vivo toxicity and antiproliferative activity of hemocyanins isolated from marine snail Rapana venosa (RvH), garden snails Helix lucorum (HlH) and Helix aspersa (HaH). For in vitro safety testing, 3T3 Neutral Red Uptake (NRU) test was used. The experiments for antiproliferative activity of the hemocyanins were performed by MTT assay on a panel of cell lines - a model of breast cancer. The in vivo toxicological assessment was performed by regular clinical examinations of hemocyanin-treated laboratory mice and histopathological analysis of hematoxylin/eosin stained preparations of parenchymal organs. The evaluation of the in vitro cytotoxicity showed that the tested hemocyanins does not induce toxic effects in nontumorigenic epithelial cell lines. In contrast, significant reduction of the viability of human breast carcinoma cell lines was found after treatment with high concentrations of hemocyanins. The in vivo experiments showed no signs of organ and systemic toxicity in the hemocyanin-treated animals. The presented data indicate that Hcs show a potential for development of novel anticancer therapeutics due to their beneficial properties, biosafety and lack of toxicity or side effects. Key words: hemocyanins (Hcs); cytotoxicity; antitumor activity; in vivo biosafety testing.