Alexander Tzintzarov

Sangre de drago, the sap of Croton lechleri Müll. Arg. tree, has been used for centuries in traditional medicine owing to its diverse biological activities. Extracts derived from different parts of the species contain a multitude of phytochemicals with varied applications. Twigs, however, are among the least studied parts of the plant. This study unveils new biological activities of Croton lechleri twig extracts recovered by applying Soxhlet and advanced green techniques. For all extracts, total phenolic content and antioxidant activity were determined. Subsequently, four were selected, and their cytotoxic effects were assessed on both normal (HaCat) and malignant melanoma (A375) skin cell lines using the MTT assay and trypan blue exclusion assay. All showed dose-dependent cytotoxicity, with the Soxhlet ethanol extract demonstrating the highest selectivity towards A375 cells over HaCat cells. The extracts induced apoptosis and necrosis, as confirmed by Annexin V/PI dual-labeling and flow cytometry, highlighting their ability to trigger programmed cell death in cancer cells. The selective inhibition of cell cycle progression in A375 compared to HaCat observed both for Soxhlet ethanol and pressurized ethanol extracts induces cell cycle arrest at multiple points, primarily in the G1 and G2/M phases, and significantly reduces DNA synthesis as evidenced by the decrease in the S-phase population, confirmed by the EdU assay. Consequently, the Soxhlet extract composition was analyzed using LC-MS, which revealed their richness in polyphenolic compounds, particularly flavonoids from the flavonol subclass.

This study investigated the anti-proliferative effects of biologically active substances (BAS) derived from the mucus of the garden snail Helix aspersa on breast cancer cell lines (MCF-7 and MDA-MB-231) and a non-cancerous cell line (MCF-10A). Two mucus fractions with different molecular weights (Mw) were examined, and their interactions with the chemotherapy drugs cisplatin and tamoxifen were evaluated. The results showed that the mucus fractions exhibited a slight anti-proliferative effect on breast cancer cells, with the fraction having Mw above 50 kDa demonstrating stronger activity compared to the fraction with Mw above 20 kDa. Furthermore, the combination of the mucus fractions with cisplatin or tamoxifen resulted in significant reductions in the half effective dose, indicating possible synergistic effects which were more pronounced with the fraction having Mw above 20 kDa. Dose-response curves and microscopic images supported the presence of such effects, revealing lower IC50 values and significant cell death when the cancer cells were treated with the combination of the mucus fractions and chemotherapy drugs. However, the analysis using the (highest single agent) HSA model indicated mostly additive interactions between the mucus fractions and chemotherapy drugs. These findings suggest the potential of biologically active substances derived from Helix aspersa mucus in enhancing the anti-proliferative effects of chemotherapy drugs in breast cancer treatment. The identification of additive interactions provides insights into the development of combination therapies with reduced drug dosage. Overall, this study contributes to the understanding of the anti-proliferative effects of Helix aspersa mucus fractions and their interactions with chemotherapy drugs, highlighting their potential as novel therapeutic approaches in breast cancer treatment.

The fungus Amanita muscaria is universally recognizable for its iconic appearance; it is also widely regarded as poisonous, inedible, and even deadly. In spite of that, there have been documented cases of use of A. muscaria-containing preparations against various diseases, including cancer, to no apparent ill effect. The search for compounds that can be used to treat cancer among various plants and fungi has been intensifying in recent years. In light of this, we describe an HPLC HILIC analytical method for the evaluation of the content of the anticancer compound ergosterol (ERG) and the neuroactive alkaloids ibotenic acid (IBO) and muscimol (MUS) that contribute significantly to the unpleasant physiological syndrome associated with A. muscaria consumption. A ‘homemade’ A. muscaria tincture made using 80-proof rye vodka as the solvent, an A. muscaria extract made with a standardized water–ethanol solution as the solvent, and fractions obtained from the second extract via liquid–liquid extraction with nonpolar solvents were analyzed. The study also presents the results of capillary zone electrophoresis with contactless conductivity detection and UHPLC-MS/MS analyses of the IBO and MUS content of the two native A. muscaria extracts and an evaluation of the standardized extract’s cytotoxic effect against a small panel of lung cell cultures in vitro. Our results show that the standardized extract has a significant cytotoxic effect and does not contain the compounds of interest in any significant quantity.

This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa—a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines—six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50–100 kDa (HRv 50–100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50–100 with cis-platin for all tested cell lines. The combination of HRv 50–100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50–100 as an antitumor agent used alone or as a booster in combination with different chemotherapies.