Pro-apoptotic action of protein-carbohydrate fraction isolated from coelomic fluid of the earthworm Dendrobaena veneta against human colon adenocarcinoma cells.

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF. In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays. Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.

Influence of short peptides with aromatic amino acid residues on aggregation properties of serum amyloid A and its fragments.

Serum amyloid A variant 1.1 (SAA1.1) is an acute phase protein. In response to injury, inflammation or infection its production increases highly, which may lead to aggregation of the protein and accumulation of its deposits in various organs. Due to the cellular toxicity of the aggregates, as well as the fact that accumulated deposits are a burden that obstructs proper functioning of the affected tissues, it is vital to find a way to suppress the process of pathological aggregates formation. To make this possible, it is necessary to investigate thoroughly the oligomerization process and recognize factors that may influence its course. Some previous studies showed that aromatic interactions are important to the potential of an inhibitor to suppress the aggregation process. In our research we had proved that a five-residue peptide RSFFS (saa1-5) is an efficient inhibitor of aggregation of the most amyloidogenic fragment of SAA1.1, SAA1-12. In the present work the oligomerization and aggregation propensity of SAA1-12 was compared to that of SAA1-27, in order to determine the contribution of the sequence which extends beyond the most amyloidogenic region but encompasses residues reportedly involved in the stabilization of the SAA native conformation. Thioflavin T fluorescence assay, quantitative chromatographic analysis of the insoluble fraction and transmission electron microscopy allowed for a deeper insight into the SAA aggregation process and the morphology of aggregates. Substitutions of Phe3 and/or Phe4 residues in saa1-5 sequence with tryptophan, tyrosine, homophenylalanine, naphthylalanine and ß,ß-diphenylalanine allowed to study the influence of different aromatic systems on the aggregation of SAA1-12 and SAA1-27, and evaluate these results in relation to hSAA1.1 protein. Our results indicate that compounds with aromatic moieties can affect the course of the aggregation process and change the ratio between the soluble and insoluble aggregates.