Seasonal cellular stress phenomena and phenotypic plasticity in land snail Helix lucorum (L.) populations from different altitudes

Temperature, a major abiotic environmental factor, regulates various physiological functions in land snails and therefore determines their biogeographical distribution. Thus, species with different distributions may present different thermal tolerance limits. Additionally, the intense reactivation of snail metabolic rate upon arousal from hibernation or aestivation may provoke stress. Land snails, Helix lucorum, display a wide altitudinal distribution resulting in populations being exposed to different seasonal temperature variations. The aim of the present study was to investigate the expression of heat shock proteins (Hsps), mitogen activated protein kinases (MAPKs) and proteins that are related to apoptosis (Bcl-2, ubiquitin), that have "cytoprotective" roles and are also considered to be reliable indicators of stress because of their crucial role at maintaining cellular homeostasis. These proteins were assessed in H. lucorum individuals from two different populations, one at Axios (sea level, 0m) and the other one at Kokkinopilos (Olympus, 1250 m), as well as after mutual population exchanges. Therefore, this study aimed to answer whether the different responses of these stress-related proteins depend solely on the environmental temperatures. The results of the present study showed seasonally altered levels in all studied proteins in the hepatopancreas and foot of snails, as much among different populations as between the same populations being exposed to varying altitudes. However, the changes between individuals of the same population acclimatized to a different habitat, showed a relatively similar pattern of expression supporting the induction of the specific proteins under the prism of the life history of each species.

Non-native Helix lucorum Linnaeus, 1758 (Gastropoda: Eupulmonata: Helicidae) after twelve years in Prague, Czech Republic

The first occurrence of Helix lucorum Linnaeus in the Czech Republic was reported 12 years ago, at the closed train station Žižkov in Prague. A part of the station is a ruderal habitat while large patches are covered with partly damaged concrete. At the site where it was first recorded, and where the density of H. lucorum is still the highest, this invasive snail has now almost completely replaced the original H. pomatia Linnaeus. However, it has not expanded either inside or outside the station area.

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

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.

Folate-conjugated Helix lucorum hemocyanin – preparation, stability, and cytotoxicity

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance–Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 μg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 μg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.