Abstract
Safrole, a component of Piper betle inflorescence, is a carcinogen which has been demonstrated to induce apoptosis on human oral cancer HSC-3 cells in vitro and to inhibit HSC-3 cells in xenograft tumor cells in vivo. In our previous study, safrole promoted phagocytosis by macrophages and natural killer cell cytotoxicity in normal BALB/c mice. The cytotoxic effects of safrole on HL-60 cells were investigated by using flow cytometric analysis, comet assay, 4’,6-diamidino-2-phenylindole (DAPI) staining, western blotting and confocal laser microscopy. The obtained results indicate that safrole induced a cytotoxic response through reducing the percentage of viable cells and induction of apoptosis in HL-60 cells in a dose-dependent manner. DAPI staining and comet assay also showed that safrole induced apoptosis (chromatin condensation) and DNA damage in HL-60 cells. The flow cytometric assay showed that safrole increased the production of reactive oxygen species (ROS) and Ca2+ and reduced the mitochondrial membrane potential in HL-60 cells. Safrole enhanced the levels of the pro-apoptotic protein BAX, inhibited those of the anti-apoptotic protein BCL-2 and promoted the levels of apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in HL-60 cells. Furthermore, safrole promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and of activating transcription factor 6α (ATF-6α). Based on these findings, we suggest that safrole-induced apoptosis in HL-60 cells is mediated through the ER stress and intrinsic signaling pathways.
Chewing betel quid containing Piper betle inflorescence reportedly generates a high concentration of safrole (420 μM) in the saliva (1). Safrole can bind to DNA and generate safrole-DNA adducts (2-4), causes marked increase in intracellular Ca2+ and reduces the cell viability of human osteosarcoma cells (5). Additionally, safrole increased the levels of Ca2+ release from the endoplasmic reticulum (ER) in Madin-Darby canine kidney (MDCK) cells (6). Recently, in our laboratory, we have found that safrole induced apoptosis in human oral cancer HSC-3 cells in vitro through a mitochondria-dependent pathway and reduced the HSC-3 cells in a xenograft mouse model in vivo (7). Although safrole is a group 2B carcinogen and a documented rodent carcinogen (8), there is no adequate evidence to show that exposure to safrole can induce human cancer cell apoptosis. We investigated whether or not safrole could induce apoptosis in human leukemia HL-60 cells, because there is no available information regarding its cytotoxic effects on human leukemia cells.
Materials and Methods
Materials and chemicals. Safrole, dimethyl sulfoxide (DMSO), propidium iodide (PI), trypan blue and Triton X-100 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Culture medium and supplements were obtained from Gibco Life Technologies (Carlsbad, CA, USA). All primary and secondary antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The fluorescent probes 4’,6-diamidino-2-phenylindole (DAPI), 2’,7’-dichlorofluorescin diacetate (H2DCF-DA), 1H-indole-6-carboxylic acid, 2-[4-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl] amino]-3-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]-, (acetyloxy)methyl ester (Indo-1/AM) and 3,3’-dihexyloxacarbocyanine iodide (DiOC6) were purchased from Molecular Probes Life Technologies.
Cell line. The human leukemia cell line (HL-60) was purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan, ROC), and was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT, USA), 2 mM L-glutamine and 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in an incubator with a 5% CO2 and 95% air (9, 10).
Morphological changes and viable cell examinations. Cells were seeded on 24-well plates at the density of 2×105 viable cells/well, and were treated with 0, 25, 50, 75 and 100 μM safrole, or with a vehicle (DMSO, 0.1% in culture media), and all cells were incubated for 24, 48 and 72 h. For the examination of morphological changes, cells in the wells were photographed under a phase-contrast microscope. For the determination of the percentage of viable cells, a PI exclusion method and a FACSCalibur instrument (BD Biosciences, San Jose, CA, USA) equipped with BD CellQuest Pro software were used, as previously described (11-13).
Determination of cell cycle distribution and apoptosis. Cells (2×105/well) seeded on 24-well plates, were treated with or without 0, 25, 50, 75 and 100 μM safrole, or with vehicle (DMSO, 0.1% in culture media) for 24 h. Cells from each treatment were harvested and gently fixed with 70% ethanol at −20°C overnight. Then cells were washed twice with phosphate buffered saline (PBS) and were then incubated with 20 μg/ml PI, 100 μg/ml RNase and 0.1% Triton X-100 in PBS for 30 min in the dark. Finally, the PI-stained cells were analyzed for cell cycle distribution and sub-G1 phase population (apoptosis) by using flow cytometry, as described elsewhere (11, 14).
DAPI staining and comet assay for apoptosis and DNA damage. Cells placed in 24-well plates at a density of 2×105 viable cells/well were treated with or without 0, 25, 50, 75 and 100 μM safrole, or with vehicle (DMSO, 0.1% in culture media) and all cells were incubated for 24 h. Cells in each treatment were individually fixed with 4% formaldehyde (Sigma-Aldrich Corp.) for 15 min and were stained with DAPI (Molecular Probes Life Technologies) for nucleic acid condensation (apoptosis), as described elsewhere (15, 16). The examined cells were prepared as previously described (16, 17) for the comet assay, and the DNA was stained by PI in order to determine the DNA tail in safrole-treated HL-60 cells. All samples were photographed on a fluorescence microscope (9, 10).
Assays for mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and cytosolic Ca2+ release. Cells were seeded on 24-well plates at a density of 2×105 cells/well and treated with or without 75 μM safrole, or with vehicle (DMSO, 0.1% in culture media) and all cells were incubated for 0, 0.5, 1, 3, 6, 12 or 24 h to determine the level of ΔΨm, as well as the production of ROS and cytosolic Ca2+. All cells in each treatment were harvested, washed twice by PBS, then re-suspended in 500 μl of DiOC6 (1 μmol/l) for ΔΨm, in 500 μl of H2DCF-DA (10 μM) for ROS and in 500 μl of Indo-1/AM (3 μg/ml) for cytosolic Ca2+ production at 37°C in a dark room for 30 min. Then all samples were analyzed immediately by flow cytometry, as previously described (18-20).
Western blotting analysis for the changes of apoptosis-associated protein levels. Cells at a density of 1×106 cells/well were exposed to 75 μM safrole, or vehicle (DMSO, 0.1% in culture media) and all cells were incubated for 0, 6, 12, 24, 48 and 72 h. Then cells were harvested and washed twice with PBS for the determination of protein levels [cyclin D and E, cyclin-dependent kinase 6 (CDK6), p16, p27, p57, checkpoint kinase 2 (CHK2), B-cell lymphoma 2 (BCL-2), BCL-2-associated X protein (BAX), apoptotic protease activating factor-1 (APAF-1), cytochrome c, apoptosis-inducing factor (AIF), X-linked inhibitor of apoptosis protein (XIAP), caspase-9, fatty acid synthase (FAS), caspase-3, caspase-8, poly (ADP-ribose) polymerase (PARP), glucose-regulated protein 78 (GRP78), growth arrest- and DNA damage-inducible gene 153 (GADD153) and activating transcription factor-4 (ATF-4) and ATF6-α] associated with G0/G1 arrest and of apoptosis, which were determined by western blotting. Cells from each treatment were lysed by using the PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea), as described previously (13, 21, 22). Samples containing equal amounts of protein (40 μg) from the lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk in TBST buffer (20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH 8.0) for 1 h at 25°C, prior to incubation with the specific antibodies overnight at 4°C. Then they were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies that were detected by the enhanced chemiluminescence (ECL) reagent kit (Immobilon Western HRP substrate, Millipore) and then with autoradiography using X-ray film (GE Healthcare, Piscataway, NJ, USA) (14, 20, 23). For ensuring equal protein amounts, each membrane was re-probed with anti-β-Actin antibody.
Confocal laser scanning microscopy for protein translocation. Cells were seeded in 4-well chamber slides at a density of 2×104 cells and then treated or not with 75 μM safrole for 24 h. Cells in each chamber were fixed in 4% formaldehyde in PBS for 15 min, and where then permeabilized with 0.3% Triton-X 100 in PBS for 1 h. The fixed cells were stained with antibodies against AIF and GADD153 (1:100 dilution) overnight before being washed twice with PBS, and were then stained with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG secondary antibody (green fluorescence) at 1:100 dilution, followed by DNA staining with PI (red fluorescence), as previously described (14, 16). All samples were examined and photomicrographed by the use of a Leica TCS SP2 Confocal Spectral Microscope (19, 24).
Statistical analysis. The quantitative data are presented as the mean±SD. The statistical differences between the safrole-treated and control samples were calculated by the Student's t-test. A p-value of less than 0.05 was considered significant.
Results
Safrole induced morphological changes and reduced the percentage of viable HL-60 cells. Cells were treated with or without various doses of safrole for 24, 48 and 72 h, and the induced morphological changes were examined and photographed under a phase-contrast microscopy. The results shown in Figure 1A indicate that safrole induced morphological changes such as membrane blebbing and cell shrinkage in a dose-dependent manner. Cells were harvested for determining the percentage of viable cells from each treatment by the PI exclusion method. Results are presented in Figure 1B, indicating that safrole decreased the percentage of viable HL-60 cells.
Safrole induced cell cycle arrest and apoptosis of HL-60 cells. Cells were treated with different concentrations of safrole for 24 h and were then isolated for determining the cell cycle distribution. The results shown in Figure 2A indicate that the number of cells in each stage of the cell cycle was expressed as a percentage that of the total number of cells. The results indicate that safrole induced G0/G1 phase arrest in HL-60 cells (Figure 2A).
Safrole induced chromatin condensation and DNA damage in HL-60 cells. Cells were treated with different doses of safrole for 24 h and were then harvested for determining the apoptosis by DAPI staining and the DNA damage by the comet assay and results are shown in Figure 3A and B, respectively. DAPI staining assay demonstrated that safrole induced chromatin condensation (apoptosis) (Figure 3A). The comet assay demonstrated that the treatment with safrole (25-100 μM) for 24 h induced DNA damage (Figure 3B).
Safrole affected the mitochondria membrane potential (ΔΨm) and levels of ROS and cytosolic Ca2+ in HL-60 cells. Cells were treated with 75 μM safrole for different periods of time, and were collected to measure ΔΨm, the levels of ROS and cytosolic Ca2+ release. The results in Figure 4A, B and C, respectively, show that there was a significant decrease in ΔΨm (Figure 4A) and an increase in intracellular ROS (Figure 4B) and cytosolic Ca2+ levels (Figure 4C) in the safrole-treated cells, as compared to the untreated cells. Safrole significantly decreased the levels of ΔΨm in HL-60 cells after 6-h exposure (Figure 4A). After 3-h treatment with safrole, the levels of cytosolic Ca2+ significantly increased (Figure 4B). In addition, the 3- and 6-h treatment with safrole led to significantly increased ROS levels (Figure 4C).
Safrole affected the levels of G0/G1 phase-, apoptosis- and ER stress-associated proteins levels in HL-60 cells. Cells were treated with 75 μM safrole for 0, 6, 12, 24, 48 and 72 h and were then isolated for determinations for G0/G1 phase, apoptosis- and ER-associated protein levels; the results are shown in Figures 2B and 5. Figure 2B shows that safrole inhibited the expression of cyclins D and E, and CDK6, but it promoted the expression of p16, p27, p57 and CHK2, which may lead to G0/G1 phase arrest. The results also indicated that safrole inhibited the expression of BCL-2, XIAP, and procaspase-9 but promoted the expression of BAX, APAF-1, cytochrome c (Figure 5A) and FAS, caspase-3 and -8 (Figure 5B), which are involved in apoptosis, in HL-60 cells. Furthermore, safrole also promoted the expression of GRP78, GADD153, ATF4 and ATF-6α (Figure 5C), which are involved in ER stress in HL-60 cells.
Safrole promoted translocation of AIF and GADD153 in HL-60 cells. Cells were treated with 75 μM safrole for 24 h and then the location of AIF and GADD153 was determined. Results shown in Figure 6 indicate that safrole induced the translocation of AIF (Figure 6A) and of GADD153 (Figure 6B) to nuclei, leading to cell apoptosis.
Discussion
Our previous study indicated that safrole could induce apoptosis on human oral cancer cells (7). There is no report to show that safrole induced cell cycle arrest and apoptosis in human leukemia cells. In this study, we showed that safrole triggered apoptosis in HL-60 cells through the ER stress-associated signaling and mitochondria-dependent pathways based on these observations: i) safrole induced cell morphological changes and reduced the percentage of viable HL-60 cells; ii) safrole induced G0/G1 phase arrest in HL-60 cells; iii) Safrole induced apoptosis of HL-60 cells dependent on ER stress activation and mitochondria-dependent pathways; iv) safrole promoted the expression of BAX protein and inhibited the expression of BCL-2; v) the biological effects of safrole on HL-60 cells seem to be explained, at least in part, through the ER stress, including promotion of the expression of GRP78, GADD153, ATF-6α and ATF4. Collectively, these findings indicate that the effects of safrole in leukemia may be worthy of further study.
In the present study, our analyses showed that safrole induced a significant increase in BAX (a pro-apoptotic protein) (14) and reduced the levels of BCL-2 (an anti-apoptotic protein) (16) in HL-60 cells, which may have led to the decrease of mitochondrial membrane potential and we suggest that safrole induced apoptosis through the mitochondria-dependent pathway. It is well known that anticancer agents can induce apoptosis through mitochondria-dependent pathways (25). It has also been reported that dysfunction of mitochondria can be induced (reduction of mitochondrial membrane potential) mediated by BAX/BCL2 (14). Our results clearly indicate that safrole increased the BAX/BCL-2 ratio, which may have led to the induction of apoptosis by safrole in HL-60 cells.
Our results also showed that safrole-induced apoptosis may occur through the ER stress pathway based on the production of ROS and the increase of hallmarkers of ER stress such as GRP78 and GADD153 in HL-60 cells (Figure 5C). Therefore, we present the first findings to show that safrole induced apoptosis in human leukemia HL-60 cells through the ER stress signaling pathway.
In conclusion, the present study demonstrated that safrole can induce cytotoxic effects in human leukemia HL-60 cells in vitro. Safrole triggered apoptosis via ER stress and mitochondria-dependent signaling pathways. These possible signaling pathways are summarized in Figure 7.
Acknowledgements
This study was supported by the grant CMU100-TC-10 from China Medical University, Taichung, Taiwan.
Footnotes
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↵* These Authors contributed equally to this study.
- Received February 29, 2012.
- Revision received April 3, 2012.
- Accepted April 4, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved