CYTOTOXIC EFFECTS OF 4-HYDROXYNONENAL IN HUMAN OSTEOSARCOMA CELLS

INTRODUCTION: 4-Hydroxynonenal (HNE) a diffusible product of lipid peroxidation, has been suggested to act as a key mediator in oxidative stress-induced cell death. Exogenously administrated HNE can form protein adducts and cause apoptotic cell death in various cell types. Previously we demonstrated that in a human osteosarcoma cell line, SaOS2, HNE 10#mu#M exhibits an early cytotoxic effect characterized by apoptosis, and cytostatic and differentiating effects characterized by slow growth, increase in alkaline phosphatase and decrease in tumorigenicity[1]. The aim of this study is investigate HNE cytotoxic effects more in depth by analyzing cell distribution in the cell cycle phases, the expression levels of proteins involved in cell growth regulation, the internucleosomal cleavage of DNA, and LDH activity to evaluate necrotic events. MATERIALS AND METHODS SaOS2 cells were seeded at 2x10#A4# cells/cm#A2# and treated with 10#mu#M HNE[1]. Samples stained with PI as DNA probe were analysed by flow cytometry to assess the cell cycle distribution. P21 expression levels were evaluated in Western Blot. The DNA laddering was revealed according to Mc.Conkey [2]. The LDH activity was monitored spectrophotometrically by measuring NADH levels. RESULTS The treatment with HNE 10#mu#M after 24hrs causes an accumulation of osteosarcoma cells in S and in G2/M. Such an effect on the cell cycle may be related to the increased levels of the CDK-inhibitor p21, as shown by western blot analysis. Although specific morphological characteristic of apoptotic cells were reported in literature for SaOS2 treated with HNE, internucleosomal DNA fragmentation, was not found in agarose gel electrophoresis. The measurament of the LDH release in the medium showed the membrane integrity of treated cells. In conclusion HNE-induced cytotoxyc effect in SaOS2 cells is characterized by a growth arrest in the S and G2/M phases, coupled with p21 increased expression. HNE-exerted cell death is an atypical apoptosis because it doesn’t show the classical DNA laddering. On the other hand the absence of LDH activity in the medium excludes that the induced cell death can result in necrosis. References [1] Calonghi N. et al.BBRC 293 (2002) 1502-1507 [2] Mc Conkey DJ. et al. Arch.Biochim.Biophys.Acta 972 (1989) 302-310