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.

Figure 1. Tetrandrine affects cell morphology and cell viability in HaCaT cells. HaCaT cells were treated with H₂O₂ (500 μM) or tetrandrine (20 μM), or treated with tetrandrine (20 μM) for 1 h and then with H₂O₂ (500 μM) for 3, 6 or 12 h. Cells were monitored and photographed under phase-contrast microscopy (A), and total cell viability was calculated by propidium iodide exclusion assay (B) as described in the Materials and Methods. Different letters indicate significant differences (p<0.05) between groups by one-way analysis of variance.

Figure 2. Tetrandrine induced apoptotic cell death of HaCaT cells. HaCaT cells were placed on a 12-well plate overnight and treated with or without tetrandrine for 1 h and then treated with H₂O₂ (500 μM) for 12 h. Cells were harvested for staining with annexin V/propidium iodide (PI) double staining as described in the Materials and Methods. A: Representative profiles of apoptotic cell death. B: Percentage of apoptotic cell death. ***p<0.001 versus the control, ###p<0.001 versus H₂O₂ alone, as analyzed by one-way analysis of variance.

Figure 3. Tetrandrine induced chromatin condensation in HaCaT cells. HaCaT cells (1×105 cells/ml) were seeded on chamber coverslips and treated with or without tetrandrine (20 μM) for 1 h than treated with H₂O₂ (500 μM) for 12 h. The cells were then examined and photographed under fluorescent microscopy at ×100 as described in the Materials and Methods. A: Representative images of cell chromatin condensation. B: 2-(4-Amidinophenyl)-6-indolecarbamidine fluorescence intensity in treated cells. ***p<0.001 versus the control, ###p<0.001 versus the H₂O₂ alone, as analyzed by one-way analysis of variance.

Figure 4. Tetrandrine enhanced expression of reactive oxygen species, DNA damage , and apoptosis-associated proteins in HaCaT cells. HaCaT cells were treated with or without tetrandrine at 20 μM for 1 h then treated with H₂O₂ (500 μM) for 12 h. Cells from each treatment were harvested for determining expression of apoptotic proteins by western blotting as described in the Materials and Methods. A: Superoxide dismutase (SOD) (Cu/Zn), SOD (Mn) and catalase. B: phospho-ataxia telangiectasia mutated (p-ATM), phospho- ataxia-telangectasia and Rad3 related (p-ATR), P53, phospho-P53 (p-P53), and phosphorylated histone H2AX (p-H2A.X). C: BCL2 apoptosis regulator (BCL2)-associated X-protein (BAX), BCL2, caspase-9, caspase-8, caspase-3, and poly ADP ribose polymerase (PARP).

Figure 5. Tetrandrine affected expression and location of reactive oxygen species-associated proteins in HaCaT cells. HaCaT cells (1×105 cells/ml) were maintained on coverslips and pre-treated with tetrandrine at 20 μM for 1 h then treated with or without H₂O₂ (500 μM) for 12 h. Cells were fixed with 4% formaldehyde, permeabilized by using 0.2% Triton-X 100 for 15 min, and were probed with primary antibodies to SOD1 (A), catalase (B), and glutathione (C) and by fluorescein isothiocyanate-conjugated goat anti-mouse IgG (green fluorescence), and the nucleus was stained by propidium iodide (red fluorescence) as described in the Materials and Methods.

Figure 6. The possible molecular mechanism involved in apoptotic cell death by tetrandrine pre-treatment and then H₂O₂ treatment of HaCaT cells. p-ATM: Phosphor-ataxia telangiectasia mutated; p-ATR: phospho-ataxia-telangectasia and Rad3-related; BAX: BCL2 apoptosis regulator-associated X-protein; p-H2A.X: phosphorylated histone H2AX; p-P53: phospho-P53; PARP: poly ADP ribose polymerase; ROS: reactive oxygen species; SOD: superoxide dismutase.