Pre-treatment of pterostilbene enhances H2O2-induced cell apoptosis through caspase-dependent pathway in human keratinocyte cells

Abstract

Background/Aim: Hydrogen peroxide (H2O2) is one of the reactive oxygen species (ROS), which can induce apoptotic cell death in numerous cancer cells. Pterostilbene (PTE), a natural polyphenolic compound, induces cell apoptosis in many human cancer cells. Materials and Methods: We investigated whether PTE could enhance H2O2-induced cell apoptosis in human keratinocyte HaCaT cells in vitro. The morphological change of HaCaT cells was observed and photographed under a contrast-phase microscope. The percentage of cell viability was measured by propidium iodide exclusion assay. Cell apoptosis was performed by Annexin V/PI double staining and assayed by flow cytometer. DNA condensation was measured by DAPI staining. The protein expression was determined by western blotting. ROS production-associated proteins were also assayed by confocal laser scanning microscopy. Results: PTE pre-treatment enhanced H2O2 (600 μM)-induced cell morphological changes and reduced the total cell number (cell viability). The decreased cell viability in HaCaT cells was through induction of apoptotic cell death, which was confirmed by Annexin V/PI double staining and DAPI staining. Western blotting studies indicated that HaCaT cells which were pre-treated with PTE (100 μM) and then co-treated with H2O2 (600 μM) for 12 h showed significantly increased levels of SOD (Cu/Zn), SOD (Mn), Bax, caspase-3, caspase-8, caspase-9, PARP, p53, p-p53, and p-H2A.X but decreased levels Bcl-2 and catalase. Results also showed that HaCaT cells pre-treated with PTE and then co-treated with H2O2 had increased expression of SOD (Cu/Zn) and glutathione but decreased catalase. Conclusion: These observations suggest that PTE pre-treatment can enhance the H2O2-induced apoptotic cell death in keratinocyte cells and may be an effective candidate for the treatment of proliferative keratinocytes.