benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Hemolysis

Serratia marcescens strains are opportunistic pathogens that are increasingly recognized as a cause of severe nosocomial infections. In this study we observed interactions between nonpigmented strains with human epithelial and macrophage-like cells. The strains revealed hemolytic activity only after the contact of the cells with erythrocytes. The contact of the bacteria with the host cells was also essential to their cytotoxicity. Moreover, all strains revealed adherence ability and were invasive to epithelial cells. Analyses of cellular morphology and DNA fragmentation of the HEp-2 and J774 cells exhibited typical features of cells undergoing apoptosis. We observed morphological changes, including condensation of nuclear chromatin and formation of membrane-bound apoptotic bodies. The lowest apoptotic index in HEp-2 cells did not exceed 25%, whereas the highest reached 59% at 24 h and 72% at 48 h after infection. Most of the strains (60%) induced fragmentation of nuclear DNA. The process depended on the activation of caspases, and was completely blocked by the pan-caspase inhibitor z-VAD-fmk. This study provided new insights into the mechanisms of nonpigmented S. marcescens pathogenesis. The results revealed that the strains produce cell-contact toxins that facilitate bacterial invasion, induce hemolysis, cytotoxicity, and apoptosis of host cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspases; Cell Adhesion; Cell Line; Cross Infection; Cytotoxicity Tests, Immunologic; DNA Fragmentation; Enzyme Activation; Epithelial Cells; Hemolysis; Humans; Macrophages; Mice; Serratia marcescens

Fagonia indica is a small spiny shrub of great ethnopharmacological importance in folk medicine. The aqueous decoction of aerial parts is a popular remedy against various skin lesions, including cancer. We used a biological activity-guided fractionation approach to isolate the most potent fraction of the crude extract on three cancer cell lines: MCF-7 oestrogen-dependent breast cancer, MDA-MB-468 oestrogen-independent breast cancer, and Caco-2 colon cancer cells. A series of chromatographic and spectroscopic procedures were utilised on the EtOAc fraction, which resulted in the isolation of a new steroidal saponin glycoside. The cytotoxic activity of the saponin glycoside was determined in cancer cells using the MTT and neutral red uptake assays. After 24h treatment, the observed IC(50) values of the saponin glycoside were 12.5 μM on MDA-MB-468 and Caco-2 cells, but 100 μM on MCF-7 cells. Several lines of evidence: PARP cleavage, caspase-3 cleavage, DNA ladder assays, and reversal of growth inhibition with the pan-caspase inhibitor Z-VAD-fmk, suggested stimulation of apoptosis in MDA-MB-468 and Caco-2 cells, but not in MCF-7 cells, which do not express caspase-3. The haemolytic activity of the saponin glycoside was confirmed in sheep red blood cells, with cell lysis observed at >100 μM, suggesting that, at this concentration, the saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Using the DNA ladder assay, the saponin glycoside (12.5 μM) was not toxic to HUVEC (human umbilical vein endothelial cells) or U937 cells, indicating some selectivity between malignant and normal cells. We conclude that the steroidal saponin glycoside isolated from F. indica is able to induce apoptosis or necrosis in cancer cells depending on the cell type.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cells, Cultured; Erythrocytes; Hemolysis; Human Umbilical Vein Endothelial Cells; Humans; Necrosis; Plant Components, Aerial; Plant Extracts; Saponins; Sheep; U937 Cells; Zygophyllaceae