Caffeine, 4,6 -diamidino-2-phenylindole dihydrochloride (DAPI), kaempferol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Medium 200, Low Serum Growth Supplement (LSGS), Trypsin-EDTA, 2,7-dichlorodihydrofluorescein diacetate (H₂DCFDA) and Fluo-4/AM were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Caspase-3, Caspase-8 and Caspase-9 Colorimetric Assay kits and caspase-8 inhibitor Z-IETD-FMK were bought from R&D Systems Inc. (Minneapolis, MN, USA). Primary antibodies [Fas/CD95, DR4, DR5, p-ATM (Ser1981), ATM and β-actin], horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse immunoglobulin and p53 siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-caspase-3, -8 and -9 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against p53 and p-p53 (Ser15) were obtained from Abcam (Cambridge, Cambridgeshire, UK).

Human umbilical vein endothelial cells (HUVECs) were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) and cultured in Medium 200 plus LSGS at 37°C in a humidified atmosphere with 5% CO₂. The cells were used between the second to fifth passages.

HUVECs were plated onto 96-well microplates at a density of 5×10³ cells/100 μl per well and then incubated with kaempferol at the concentrations of 0, 50, 100, 150 and 200 μM for 24, 48 and 72-h treatment. Cell viability was determined by MTT assay as previously described (15), and the optical density ratio of the treatment to the control (% of control) was calculated.

HUVECs were treated with 100 μM kaempferol for 24 and 48 h. The cells were examined and photographed using a phase-contrast microscope. Then the cells were collected and fixed in 75% ethanol overnight at −20°C before being stained with 0.1 M phosphate/citric acid buffer (0.2 M NaHPO₄ and 0.1 M citric acid, pH 7.8) and 40 μg/ml PI for 30 min at room temperature in the dark. The cells were determined with BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) as previously described (6).

HUVECs were treated with 100 μM kaempferol for 48 h. The cells were fixed and incubated with 1 μg/ml DAPI following a previously reported method (6,16). After being harvested, cells were combined with molten low-melting agarose (Sigma-Aldrich) at a density of 1×10⁵ cells/ml. The agarose-cell mixture (50 μl) was immediately pipetted onto comet slides. The slides were then immersed in pre-chilled lysis solution for 30 min at 4°C as previously described (10). After lysis, horizontal electrophoresis was performed for 30 min at 300 mA. The slides were fixed by 70% ethanol for 5 min before being stained with 50 μl nuclear counterstain DAPI solution (final concentration: 1 μg/ml) and viewed under a fluorescence microscope.

HUVECs (5×10⁴ cells/well) on 4-well chamber slides were treated with 100 μM kaempferol for 24 h. Cells were fixed in 3% formaldehyde (Sigma-Aldrich) for 15 min, permeabilized with 0.1% Triton-X 100 in PBS for 1 h with blocking of non-specific binding sites using 2% bovine serum albumin (BSA) as previously described (6,17). These fixed cells were stained with cleaved caspase-3 antibody (1:100 dilution, Cell Signaling Technology) overnight before being detected using a goat anti-mouse IgG secondary antibody conjugated fluorescein isothiocyanate (FITC) (1:500 dilution, green fluorescence) (Merck Millipore, Billerica, MA, USA), followed by nuclei counterstaining using and PI (red fluorescence). Images were collected with a Leica TCS SP2 Confocal Spectral Microscope (Leica Microsystems, Heidelberg, Mannheim, Germany)

HUVECs (5×10⁶ cells) were pretreated with or without 10 μM Z-IETD-FMK (a specific caspase-8 inhibitor) for 1 h and incubated in 75-T flasks and treated with kaempferol for 24 and 48 h. After treatment, cells were harvested and lysed, and cell lysates (50 μg proteins) were incubated to check relative caspase activity using Caspase-3, Caspase-8 and Caspase-9 Colorimetric Assay Kits (R&D Systems Inc.) following the manufacturer's instructions.

HUVECs were treated with 100 μM kaempferol for 6, 12 and 24 h. Cells were then harvested and labeled with 2 μM Fluo-4/AM (a specific intracellular Ca²⁺ fluorescence probe) and 500 nM DiOC₆(3), respectively, at 37°C for 30 min. Consequently, intracellular Ca²⁺ and ΔΨm were individually analyzed for fluorescence intensity by flow cytometry as previously described (17).

HUVECs (5×10⁶ cells) were incubated in 100 μM kaempferol for 0, 12 or 24 h. After being harvested and lysed, the 10% SDS-polyacrylamide electrophoresis (SDS-PAGE) gels were used to separate equal amount of protein extract from cell lysate as detailed by Yang et al (18). The appropriate the primary antibodies were hybridized to observe the specific protein signals. Then the HRP-conjugated secondary antibodies were applied before using Immobilon Western HRP substrate kit (Merck Millipore). The densito-metric quantification of each band was performed utilizing NIH ImageJ 1.47 software.

HUVECs were treated with 100 μM kaempferol for 6, 12 and 48 h. Cells were then harvested and labeled with 20 μM H₂DCFDA (a specific ROS fluorescent probe) at 37°C for 30 min. Consequently, ROS was analyzed for fluorescence intensity by flow cytometry as previously described (19). Cells were incubated with 100 μM kaempferol for 48 h before individual pretreatment with or without the 10 mM N-acetylcysteine (NAC, an antioxidant) or 1 mM caffeine (an ATM kinase inhibitor) for 1 h. After that, cells were determined for cell viability by MTT assay as described above.

HUVEC cells were grown to 70% confluence in 6-well culture plates, and control siRNA (100 nM) or p53 siRNA (100 nM) was transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. After transfection, cells were seeded and thereafter exposed to 100 μM kaempferol for 48 h before analyses using western blot and MTT assay, respectively.

The data represent the mean ± standard deviation (SD) from at least three separate experiments. Statistical analysis was carried out using Student's t-test, and P<0.05 was considered statistically significant.

At first, our study focused on the growth inhibition effects of kaempferol on HUVECs. Cells were treated with 0, 50, 100, 150 and 200 μM of kaempferol, and cell number was counted at 24, 48 and 72 h. Our results showed that kaempferol decreased viable HUVECs in a concentration- and time-dependent manner (Fig. 1). The IC₅₀ of kaempferol was 103.25±4.15 μM after 24-h treatment.

To understand whether apoptotic mechanisms are involved in kaempferol-treated HUVECs, the morphological changes and DNA content using flow cytometric analysis were investigated. Our results showed that HUVECs were detached from the surface of the plate and showed shrinkage in kaempferol-treated cells (Fig. 2A, right), and the control group showed normal morphology (Fig. 2A, left). The results from the DNA content demonstrated that kaempferol induced an increase of hypodiploid apoptotic cell population (sub-G1 phase) at 24 and 48 h treatments (Fig. 2B). These effects are time-dependent. Our results indicated that kaempferol provoked apoptotic cell death in HUVECs.

To confirm the apoptotic evidence in kaempferol-treated HUVECs, DAPI stain for DNA condensation and comet assay for DNA damage were monitored. Our results showed that kaempferol induced DNA condensation (Fig. 3A) and DNA damage (Fig. 3B) in HUVECs cells. It is well known that caspase-3 is a key mediator of cell apoptosis (13,14). Next, we used caspase-3 immunofluorescence staining and confocal laser scanning microscopy to observe the caspase-3 protein expression. The caspase-3 protein expression (green color) was observed in the cytosol of kaempferol-treated HUVECs (Fig. 3C). Our results demonstrated that kaempferol provoked apoptotic cell death through DNA damage and caspase-3 activation in HUVECs.

To determine the roles of intracellular Ca²⁺ levels and ΔΨm levels of apoptotic death induced by kaempferol, we detected the intracellular Ca²⁺ by Fluo-4/AM dye and ΔΨm by DiOC₆(3) dye at 6, 12 and 24 h, respectively. Kaempferol increased intracellular Ca²⁺ levels (Fig. 4A) and depletion of ΔΨm (Fig. 4B) in HUVECs. The data indicated that kaempferol-provoked apoptotic death in HUVECs might be mediated through Ca²⁺ signal and mitochondrial pathway.

To determine the major caspase pathway of apoptotic death induced by kaempferol, we further detected the activity of caspase-8, -9 and -3 at 24 and 48 h, respectively. Our data inducted that the activities of caspase-8, -9 and -3 were significantly increased in kaempferol-treated HUVECs in a time-dependent manner (Fig. 5A). These results suggested that both the intrinsic mitochondria-mediated pathway and the extrinsic death receptor signaling are involved in kaempferol-induced apoptosis in HUVECs. To confirm our suggestion, we used western blotting to detect the cleaved form of caspase-8, -9 and -3. Our results showed that the cleaved caspase-8, -9 and -3 protein level were significantly increased in HUVECs prior to kaempferol challenge at 48 h (Fig. 5B). Strikingly, caspase-8 activity was significantly increased at 24-h treatment in treated HUVECs. The data indicated that extrinsic death receptor pathway is a key signal in kaempferol-induced apoptosis of HUVECs.

Our hypothesis that kaempferol-provoked apoptosis is mediated mainly through extrinsic death receptor pathway. To prove our hypothesis, Z-IETD-FMK (a specific caspase-8 inhibitor) was used to block caspase-8, -3, and -9 activities. Our results demonstrated that pre-incubation with the specific inhibitor of Z-IETD-FMK strongly decreased the activity of caspase-8 (Fig. 6A), caspase-3 (Fig. 6B), and caspase-9 (Fig. 6C) compared with kaempferol treatment alone. Overall, these data demonstrated that extrinsic death receptor pathway is a crucial element in kaempferol-triggered apoptosis of HUVECs.

Our findings showed that kaempferol increased intracellular ROS production at 6, 12 and 24 h in HUVECs by using flow cytometry and H₂DCFDA (a specific fluorescent probe) (Fig. 7A). Cells showed a significant inhibitory effect on kaempferol-induced growth inhibition after pre-treatment with NAC (Fig. 7B). These data indicated that ROS production is important in kaempferol-triggered apoptosis of HUVECs.

It was reported that ROS can modulate death receptor pathway in cancer cells (8,10). Our hypothesis showed that extrinsic death receptor pathway is a central component in kaempferol-triggered apoptosis of HUVECs. The results revealed that kaempferol stimulated the death receptor-associated protein levels, including Fas/CD95, DR4 and DR5 in HUVECs (Fig. 8A). It is well documented that p53 gene and its phosphorylation at the Ser15 interact Fas/CD95 activation during cell apoptosis (6,20). To elucidate the possible signaling pathway in kaempferol-provoked apoptosis, the levels of associated proteins were evaluated. Kaempferol increased the protein level of ATM, p53, phosphorylation of ATM and p53, followed by increasing levels of Fas, DR4 and DR5 based on the exposure time (Fig. 8B). Our results indicated that kaempferol increased the protein level of Fas/CD95, DR4 and DR5 through the ATM-p53-dependent regulation of transcription levels. We also re-checked the kaempferol-caused ATM-p53-dependent signal in HUVECs, and caffeine (an ATM kinase inhibitor) and p53 siRNA were used to block ATM and p53 function. Pre-treatment with caffeine reversed the inhibition of cell viability in treated group (Fig. 9A). Moreover, p53 siRNA also had a similar effect in kaempferol-treatment group (Fig. 9B). The results from our experimental approaches indicate that kaempferol-induced apoptosis of HUVECs is mediated through ATM-p53-mediated pathway.