Tetrandrine Enhances H2O2-Induced Apoptotic Cell Death Through Caspase-dependent Pathway in Human Keratinocytes

Abstract

Background: Tetrandrine, a bis-benzylisoquinoline alkaloid, induces apoptosis of many types of human cancer cell. Hydrogen peroxide (H₂O₂) is a reactive oxygen species inducer; however, there are no reports to show whether pre-treatment of tetrandrine with H₂O₂ induces more cell apoptosis than H₂O₂ alone. Thus, the present study investigated the effects of tetrandrine on H₂O₂-induced cell apoptosis of human keratinocytes, HaCaT, in vitro.

Materials and methods: HaCaT cells were pre-treated with and without tetrandrine for 1 h, and then treated with H₂O₂ for examining cell morphological changes and cell viability using contrast-phase microscopy and propidium iodide (PI) exclusion assay, respectively. Cells were measured apoptotic cell death by using annexin V/PI double staining and further analyzed by flow cytometer. Cells were further assessed for DNA condensation using 2-(4-amidinophenyl)-6-indolecarbamidine staining. Western blotting was used to measure expression of apoptosis-associated proteins and confocal laser microscopy was used to measure the protein expression and nuclear translocation from the cytoplasm to nuclei.

Results: Pre-treatment of tetrandrine for 1 h and treatment with H₂O₂ enhanced H₂O₂-induced cell morphological changes and reduced cell viability, whilst increasing apoptotic cell death and DNA condensation. Furthermore, tetrandrine significantly increased expression of reactive oxygen species-associated proteins such as superoxide dismutase (Cu/Zn) and superoxide dismutase (Mn) but significantly reduced the level of catalase, which was also confirmed by confocal laser microscopy. It also increased expression of DNA repair-associated proteins ataxia telangiectasia mutated, ataxia-telangectasia and Rad3-related, phospho-P53, P53 and phosphorylated histone H2AX, and of pro-apoptotic proteins BCL2 apoptosis regulator-associated X-protein, caspase-3, caspase-8, caspase-9 and poly ADP ribose polymerase in HaCaT cells.

Conclusion: These are the first and novel findings showing tetrandrine enhances H₂O₂-induced apoptotic cell death of HaCaT cells and may provide a potent approach for the treatment of proliferated malignant keratinocytes.

Keywords: HaCaT cells; Tetrandrine; caspase; cell apoptosis; hydrogen peroxide.