The olive leaf extract oleuropein exerts protective effects against oxidant-induced cell death, concurrently displaying pro-oxidant activity in human hepatocarcinoma cells

Abstract

Objectives: Oleuropein (OP), the predominant natural constituent of leaves of the olive tree, exerts anti-inflammatory and antioxidant effects. The purpose of this study was to assess the protective effects of OP under the conditions of paraquat (PQ)-induced oxidative stress in vitro, using the human hepatocarcinoma cell line, HepG2.

Methods: Cell viability and death were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and 4′,6-diamidino-2-phenylindole-propidium iodide staining, respectively. Superoxide anion and lipid peroxidation levels were evaluated using nitroblue tetrazolium and thiobarbituric acid-reactive substances assays, respectively. Apoptosis was assessed by measuring poly(ADP-ribose) polymerase (PARP) and caspase-3 (Casp-3) cleavage via immunoblotting and immunofluorescence analyses.

Results: PQ induced a decrease in cellular viability by promoting necrosis through a mechanism involving superoxide generation and nuclear translocation of cleaved Casp-3. Co-treatment with OP afforded significant protection against the suppressive effects of PQ, as evident from increased cell viability, reduction of Casp-3 immunofluorescence, and normalization of β-tubulin expression levels. Unexpectedly, these OP-mediated protective effects were associated with increased superoxide and malondialdehyde generation and PARP cleavage.

Discussion: OP protects HepG2 cells against PQ-induced necrosis by suppressing Casp-3 cleavage while concomitantly acting as a pro-oxidant agent. This paradoxical mechanism of action of OP requires further investigation.

Keywords:

• Oleuropein
• Paraquat
• Oxidative stress
• Superoxide
• Hydroxyl radical
• Lipid peroxidation
• Apoptosis
• Necrosis

Acknowledgements

The author thanks E. Tsilibary and K. Machera for providing experimental materials and A. Kaminari, E. Tsalavoutas, K. Kyriakopoulou, and E. Katsanou for their support. Oleuropein was isolated and kindly provided by Dr N. Fokialakis. The language of the manuscript has been edited by the Charlesworth Group Author Services.

Disclaimer statements

Contributors None.

Funding Part of this work was carried out at Benaki Phytopathological Institute (BPI), Athens, Greece, and was partly funded by BPI Plant Heal – FP7-REGPOT-2008-1, No. 230010.

Conflicts of interest There are no conflicts of interest.

Ethics approval No ethical approval was required. The study is entirely based in vitro.