beta-escin and Osteosarcoma

Osteosarcoma is one of the most malignant neoplasms in adolescents, and it generally develops multidrug resistance. Escin, a natural mixture of triterpene saponins isolated from Aesculus hippocastanum (horse chestnut), has demonstrated potent anti-tumour potential in vitro and in vivo. In the present study, we found that escin inhibited osteosarcoma proliferation in a dose- and time-dependent manner. Additionally, escin-induced apoptosis was evidenced by the increased expression of caspase-related proteins and the formation of apoptotic bodies. Escin also induced autophagy, with elevated LC3, ATG5, ATG12 and Beclin expression as well as autophagosome formation. Inhibition of escin-induced autophagy promoted apoptosis. Moreover, p38 mitogen-activated protein kinases (MAPKs) and reactive oxygen species (ROS) were activated by escin. A p38 MAPK inhibitor partially attenuated the autophagy and apoptosis triggered by escin, but a ROS scavenger showed a greater inhibitory effect. Finally, the therapeutic efficacy of escin against osteosarcoma was demonstrated in an orthotopic model. Overall, escin counteracted osteosarcoma by inducing autophagy and apoptosis via the activation of the ROS/p38 MAPK signalling pathway; these findings provide evidence for escin as a novel and potent therapeutic for the treatment of osteosarcoma.

Topics: Antineoplastic Agents; Apoptosis; Autophagosomes; Autophagy; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Escin; Humans; Mitochondria; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.

Topics: Alkaline Phosphatase; Animals; Bacterial Proteins; Calcimycin; Calcium; Cell Membrane; Cholesterol; Cold Temperature; Dose-Response Relationship, Drug; Escin; Glycosylphosphatidylinositols; Osteosarcoma; Phospholipases; Rats; Streptolysins; Tumor Cells, Cultured