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Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma. [ABSTRACT FROM AUTHOR]

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon. [ABSTRACT FROM AUTHOR]

[Display omitted] • P. aeruginosa exoA disrupts hemocyte-mediated immune response in G. mellonella. • ExoA injection caused changes in granulocyte and plasmatocyte quantity. • Fluorescent staining showed apoptotic and necrotic cells after exoA injection. • Flow cytometry revealed apoptotic and necrotic cells as well as active caspase-3. • P. aeruginosa exotoxin A induced apoptosis of G. mellonella hemocytes. The cellular immune response of the greater wax moth Galleria mellonella to Pseudomonas aeruginosa exotoxin A was investigated for the first time. The insects were challenged with a sublethal dose of exoA, and then hemocyte parameters were assessed. The analysis showed a statistically significant decrease in the total hemocyte count (THC), which was associated with significant decreases in the number of granulocytes and plasmatocytes. In turn, no statistically significant changes were observed in the number of spherulocytes and oenocytoides. Fluorescent staining indicated that cells collected from the exoA-challenged larvae exhibited features characteristic for apoptotic and autophagic cell death, e.g. cytoplasm vacuolization and chromatin condensation. The flow cytometry analysis revealed a significant increase in the number of phosphatidylserine- and active caspase 3-positive hemocytes challenged with exoA, which proved apoptosis induction. Our results will help in understanding the role of exotoxin A during P. aeruginosa infections not only in insects but also in mammals, including humans. [ABSTRACT FROM AUTHOR]