Kaempferol induces ATM/p53-mediated death receptor and mitochondrial apoptosis in human umbilical vein endothelial cells (HUVECs)
CHIU-FANG LEE1*, JAI-SING YANG2*, FUU-JEN TSAI3,4 , NI-NA CHIANG1, CHI-CHENG LU5, YU-SYUAN HUANG6, CHUN CHEN6, FU-AN CHEN6
1Kaohsiung Veterans General Hospital Pingtung Branch, Pingtung; 2Department of Medical Research, China Medical University Hospital, China Medical University, Taichung; 3Human Genetic Center, China Medical University Hospital, Taichung, Taiwan; 4School of Post-Baccalaureate Chinese, Medicine, China Medical University,
Taichung; 5School of Nutrition and Health Sciences, Taipei Medical University, Taipei; 6Department of Pharmacy and Master Program, Tajen University, Pingtung,
Taiwan, ROC.
Running title: Anti-angiogenic and apoptotic effects and HUVEC apoptosis induced by kaempferol
Correspondence to: Professor Fu-An Chen, Department of Pharmacy and Graduate
Institute of Pharmaceutical Technology, Tajen University, 20 Weixin Road, Yanpu, Pingtung 90741, Taiwan, R.O.C.
E-mail: fachen.tajen@yahoo.com.tw
Kaempferol is a member of the flavonoid compounds found in vegetables and fruits and exhibited biological impacts and anticancer activity, but no report showed the angiogenic effect of kaempferol and induction of cell apoptosis in vitro. In this study, we investigated the role of kaempferol on anti-angiogenic property and the apoptotic mechanism of human umbilical vein endothelial cells (HUVECs). Our results demonstrated that kaempferol decreased HUVEC viability in a time- and concentration-dependent manner. Kaempferol also induced morphology change and sub-G1 phase cell population (apoptotic cells). Kaempferol triggered apoptosis of HUVECs by detecting DNA fragmentation, comet assay and immunofluorescent staining for activated caspase-3. The caspase signals, including caspase-8, caspase-9 and caspase-3, were time-dependently activated in HUVECs after kaempferol exposure. Furthermore, pre-treatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced activities of caspase-8, caspase-9 and caspase-3, indicating that extrinsic pathway is a major signaling pathway in kaempferol-treated HUVECs. Importantly, kaempferol promoted reactive oxygen species (ROS) using flow cytometric assay in HUVECs. We further investigated the upstream of extrinsic pathway and showed that kaempferol stimulated death receptor signals [Fas/CD95, death receptor 4 (DR4) and DR5] through increasing the levels of phosphorylated p53 and phosphorylated ATM pathway in HUVECs, which can be individually confirmed by N-acetylcysteine (NAC), ATM specific inhibitor (cafffeine) and p53 siRNA. Based on these results, kaempferol-induced HUVECs apoptosis was involved in an ROS-mediated p53/ATM/death receptor signaling. Kaempferol might process a therapeutic effect on cancer treatment as an anti-vascular targeting.
p53/ATM/death receptor signaling. Introduction
Angiogenesis is an important physiology process during promoting tumor growth or metastatic tumors (1). Suppression of endothelial cell proliferation or induction of cell apoptosis is a good strategy for blocking tumor angiogenesis (2). It has been known that human umbilical vein endothelial cells (HUVECs) are the most generally demoralized endothelial cell model, which can be examined through many processes for antiangiogenic actions (3). Moreover, the induction of endothelial cell apoptosis is one of the central antiangiogenic mechanisms (3, 4). Two major important pathways contribute to the apoptotic processes, including the intrinsic mitochondria-mediated pathway and the extrinsic death receptor signaling (5). Mitochondrial permeability can be regulated to release various apoptotic factors such as cytochrome c, Apaf-1 and pro-caspase-9 to cytosol to form apoptosome and to activate the downstream of caspase-9 (6, 7). The membrane death receptors (extrinsic apoptotic pathway) located in the membrane include Fas/CD95, death receptor 4 (DR4) and DR5 that can influence the distal executioner caspases (6). In addition, reactive oxygen species (ROS) production and DNA damage caused by anticancer drugs lead to an increase of phosphorylation of ataxia-telangiectasia-mutated kinase (ATM) and p53 to trigger human cancer cell apoptosis (8). p53 phosphorylation on the residue of Ser15 has been linked to apoptosis and shown to be a transcription factor to modulate apoptotic target genes such as Fas and DR5 (9). p53 gene expression has been shown to upregulate both of extrinsic and intrinsic apoptotic signaling pathways (6, 10).
Kaempferol is a dietary flavonoid and is found in fruits and vegetables and in traditional Chinese medicines (11, 12). The pharmacological activities of kaempferol were reported to exhibit anti-inflammatory, antioxidant, cardio-protective and
antitumor activities (12). Our previous study has demonstrated that kaempferol-induced apoptosis in human osteosarcoma cells is mediated through endoplasmic reticulum stress and mitochondria-dependent signaling (13). Kaempferol also induces autophagy by AMPK and AKT signaling and causes G2/M phase arrest via downregulation of CDK1/cyclin B in human hepatocarcinoma cells (14). However, there is no report addressing the possible anti-angiogenetic mechanism of kaempferol. The objective of the current study was to explore apoptotic evidence and its molecular mechanism underlying induced by kaempferol in HUVECs. Therefore, kaempferol might induce both of extrinsic and intrinsic apoptotic pathways in HUVECs cells through an ROS-mediated p53/ATM/death receptor signaling.
Materials and methods
Chemicals and reagents. 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, caspase-8 and
anti-caspase-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).
Cell culture. 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.
Cell viability. 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 methods (15), and the optical density ratio of the treatment to the control (% of control) was calculated.
Cell morphological detection and DNA content analysis by flow cytometry. HUVECs
were treated with 100 μM kaempferol for 24 and 48 h. The cells were examined and photographed using a phase-contrast microscope. Following the cells were collected then 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 darkness. The cells were determined with BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) as described previously (6).
DAPI staining and comet assay. HUVECs were treated with 100 μM kaempferol for
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 described previously (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.
Immunofluorescence staining. 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 described previously (6, 17). These fixed cells were stained with leaved 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)
Determinations of caspase-3/-8/-9 activities and effects of their specific inhibitors.
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 instruction.
Measurements of intracellular Ca2+ levels and mitochondrial membrane potential (ΔΨm). 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 described previously (17).
Western blot analysis. 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. Following the HRP-conjugated secondary antibodies were applied before using Immobilon Western HRP substrate kit (Merck Millipore). The densitometric quantification of each band was performed utilizing NIH ImageJ 1.47 software.
Measurements of ROS production after N-acetylcysteine and caffeine pre-treatment for cell viability. 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 described previously (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.
Small interference RNA transfection. 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.
Statistical analysis. The data represent as the mean ± standard deviation (SD) from at
least three separate experiments. Statistical data was carried out using Student’s t-test, and *P < 0.05 was considered as a statistical significant.
Results
Kaempferol induces growth inhibition in HUVECs. 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 cell of HUVECs in a concentration- and time-dependent manner (Figure 1). The IC50 of kaempferol was 103.25 ± 4.15 μM after 24-h treatment.
Kaempferol triggers morphology change and apoptosis in HUVECs. To understand
whether apoptotic mechanisms are involved in kaempferol-treated HUVECs, the morphology change and DNA content using flow cytometric analysis were
investigated. Our results showed that HUVECs were detached from the surface of plate and showed the morphology shrinkage in kaempferol-treated cells (Figure 2A, right), and the control group well spread with normal morphology (Figure 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 (Figure 2B). These effects are time-dependently. Our results indicated that kaempferol provoked apoptotic cell death in HUVECs.
Kaempferol induces DNA condensation, DNA damage and caspase-3 protein expression 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 (Figure 3A) and DNA damage (Figure 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 shown in cytosol of kaempferol-treated HUVECs (Figure 3C). Our results demonstrated that kaempferol provoked apoptotic cell death through DNA damage and caspase-3 activation in HUVECs.
Kaempferol stimulates intracellular Ca2+ levels and loss of ΔΨm 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 (Figure 4A) and depletion of ΔΨm (Figure 4B) in HUVECs.
This data indicated that kaempferol-provoked apoptotic death in HUVECs might be mediated through Ca2+ signal and mitochondrial pathway.
Kaempferol induces caspase-8, caspase-9 and caspase-3 activities in HUVECs. To
determine the major caspase pathway of apoptotic death induced by kaempferol, we further detected the activities of caspase-8, caspase-9 and caspase-3 at 24 and 48 h, respectively. Our data inducted that the activities of 8 9 and caspase-3 were significantly increased in kaempferol-treated HUVECs in a time-dependent manner (Figure 5A). Those results suggested that both of intrinsic mitochondria-mediated pathway and extrinsic death receptor signaling are involved in kaempferol-induced apoptosis in HUVECs. To confirm our suggestion, we used the western blotting to detect the cleavage form of caspase-8 caspase-9 and caspase-3. Our results showed that the cleaved caspase-8, caspase-9 and caspase-3 protein level were significantly increased in HUVECs prior to kaempferol challenge at 48 h (Figure 5B). Strikingly, caspase-8 activity was significantly increased at 24-h treatment in treated HUVECs. This data indicated that extrinsic death receptor pathway is a key signal in kaempferol-induced apoptosis of HUVECs.
Z-IETD-FMK blocks caspase-8, caspase-9 and caspase-3 activities in kaempferol-treated 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, caspase-3, and caspase-9 activities. Our results demonstrated that pre-incubation with the specific inhibitor of Z-IETD-FMK strongly decreased the activities of caspase-8 (Figure 6A), caspase-3 (Figure 6B), and caspase-9 activities (Figure 6C) compared with
kaempferol treatment alone. Overall, these data demonstrated that extrinsic death receptor pathway plays a crucial element in kaempferol-triggered apoptosis of HUVECs.
Kaempferol increases ROS generation and N-acetylcysteine reduced kaempferol -induced growth inhibition effect in 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) (Figure 7A). Cells showed a significantly inhibitory effect on kaempferol-induced growth inhibition after pre-treatment with NAC (Figure 7B). These data indicated that ROS production is important in kaempferol-triggered apoptosis of HUVECs.
ATM-p53-mediated death receptor pathway is involved in kaempferol-induced apoptosis. It was report that ROS can modulate death receptor pathway in cancer cells
(8, 10). Our hypothesis showed that extrinsic death receptor pathway plays 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 (Figure 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 the levels of Fas, DR4 and DR5 based on the exposure time (Figure 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 recheck 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 (Figure 9A). On the other hand, p53 siRNA also had a similar effect in kaempferol-treatment group (Figure 9B). The results from our experimental approaches conclude that kaempferol-induced apoptosis of HUVECs is mediated through ATM-p53-mediated pathway.
Discussion
Kaempferol is one of flavonol which is present in fruits and vegetables, including onions, kale, broccoli, apples, cherries, berries, tea and red wine (11, 12). Kaempferol has many biological properties, including anti-cancer effects, antioxidant activity, anti-inflammatory (12, 21). Kaempferol induces apoptosis and cell cycle arrest in various cancer cell lines, including colon cancer (22), liver cancer (23), gastric cancer (24), bladder cancer (25) cells. Kim et al. (26) demonstrated that kaempferol can modulate angiogenesis and immune-endothelial cell adhesion. Zhao
et al. (27) showed that the kaempferol from Pu-erh tea has cancer and
angiogenesis effects. Currently, the mechanism involved in kaempferol of anti-angiogenesis effects is unknown. In this study, we are the first to report that kaempferol induced growth inhibition (Figure1) and apoptosis (Figure 2 and 3) in HUVEC cells. Our results also showed that kaempferol induced caspase-8/-9 and -3 activities (Figure 4) and Fas/CD95, DR4 and DR5 protein levels (Figure 8) on HUVEC cells. Moreover, major cell signaling involved in kaempferol-treated HUVECs were investigated, we focused on an ROS-ATM-p53 signaling. Our results demonstrated that kaempferol induced ROS production (Figure 7), ATM, p53,
phosphorylation of ATM and phosphorylation of p53 protein levels (Figure 9). We used the different specific inhibitors that include Z-IETD-FMK (a specific caspase-8 inhibitor), N-acetylcysteine (NAC, an antioxidant), caffeine (an ATM kinase inhibitor) and p53 siRNA to confirm this pathway. We found that kaempferol triggered HUVEC apoptosis through the ROS-mediated ATM/p53 signaling.
p53, which is the tumor suppressor protein, is an essential regulator in controlling cell growth and cell death (6, 20). In response to intracellular and extracellular stress, p53 is activated and serves as a transcription factor that orchestrates various targets, which in turn modulating multitude of cellular processes such as DNA repair, cell cycle arrest and apoptosis (28, 29). It is reported that p53-inducible pro-apoptotic genes trigger apoptosis through both of extrinsic and intrinsic apoptotic molecular pathways (30). Our results showed that the kaempferol significantly increased ROS production (Figure 7A) and the protein levels of Fas/CD95, DR4, DR5, ATM, p-ATMSer1981, p53, and p53 (Ser15) in HUVECs (Figure 8). In addition, knockdown of p53 expression by p53 siRNA significantly inhibited the cell growth inhibitory effects (Figure 9A) after treatment with kaempferol in HUVECs. Based on our results, we suggest that p53 might be involved in kaempferol-upregulated death receptor signaling.
In addition to death receptor pathway, our results suggest that kaempferol induced apoptosis through mitochondria-dependent pathway. The elevation of DiOC6(3) fluorescence indicated the loss of ΔΨm in kaempferol-treated HUVECs (Figure 4B). The dissipation of ΔΨm is attributed to the opening of mitochondrial permeability transition (MTP) pore. Hence, we suggest that kaempferol led to the persistent opening of MTP pore, which resulted in mitochondrial swelling and the rupture of mitochondrial outer membrane, ultimately the release of intermembrane
proteins such as cytochrome c, Apaf-1, pro-caspas-9, AIF and Endo G that trigger cell apoptosis (6, 19).
Oxidative stress is closely related to cancer and often associated with cancer prevention and cancer therapy agents (15, 31). It was report that reactive oxygen species (ROS) not only function as a regulator of subcellular events but are also able to induce cell apoptosis (8). Yang et al. (32) demonstrated that kaempferol reduced the glutamate-induced oxidative stress in mouse-derived hippocampal neuronal HT22 cells. Ondricek et al. (33) showed that kaempferol rescued RGC-5 cells from iodoacetic acid-induced cell death, as well as reduced caspase activation and ROS generation. However, Jeong et al. (34) demonstrated that kaempferol caused an increase in reactive oxygen species (ROS) generation and induced cell death in human glioma cells. Kim et al. (35) also showed that kaempferol induced the generation of fluorescent DCF in the MCF-7 cells, and treatment with N-acetylcysteine suppressed kaempferol-induced PARP cleavage. Sharma et al. (36) also showed kaempferol in glioblastoma cells induced apoptosis through oxidative stress. In this study, kaempferol was found to be less cytotoxic towards HUVECs after pre-treatment with
N-acetylcysteine, suggesting that kaempferol induced oxidative stress in HUVECs
(Figure 7B). Based on the result from DCFH-DA assay, surprisingly kaempferol was found to stimulate the ROS formation in HUVECs.
In conclusion, the molecular signaling pathway in HUVECs caused by kaempferol is summarized in Figure 10. Our study discovered that kaempferol reduced HUVEC viability and induced DNA danage and DNA fragmentation through activated the levels of caspase-3, caspase-8, and caspase-9 signaling, which were upregulated by ROS-mediated p53/ATM molecules following stimulations of p53
downstream protein levels of Fas/CD95, DR4, and DR5. Our results suggest that kaempferol warrants further development as an anti-angiogenetic agent in the future.
Acknowledgments
This study was financially supported by research grants from Kaohsiung Veterans General Hospital Pingtung Branch, Pingtung, Taiwan.
References
1. Chung AS, Lee J and Ferrara N: Targeting the tumour vasculature: insights from physiological angiogenesis. Nat Rev Cancer 10: 505-514, 2010.
2. Wang J, Li G, Wang Y, et al: Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1alpha/VEGF secretion axis. Oncotarget 2015.
3. He MF, Gao XP, Li SC, et al: Anti-angiogenic effect of auranofin on HUVECs in vitro and zebrafish in vivo. Eur J Pharmacol 740: 240-247, 2014.
4. Beedie SL, Peer CJ, Pisle S, et al: Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues. Mol Cancer Ther 14: 2228-2237, 2015. 5. Liang CH, Wang GH, Chou TH, et al: 5-epi-Sinuleptolide induces cell cycle
arrest and apoptosis through tumor necrosis factor/mitochondria-mediated caspase signaling pathway in human skin cancer cells. Biochim Biophys Acta 1820: 1149-1157, 2012.
6. Chiang JH, Yang JS, Lu CC, et al: Newly synthesized quinazolinone HMJ-38 suppresses angiogenetic responses and triggers human umbilical vein endothelial cell apoptosis through p53-modulated Fas/death receptor signaling. Toxicol Appl Pharmacol 269: 150-162, 2013.
7. Ray PD, Huang BW and Tsuji Y: Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 24: 981-990, 2012.
8. Indran IR, Tufo G, Pervaiz S and Brenner C: Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Biochim Biophys Acta 1807: 735-745, 2011.
9. Tsai AC, Pan SL, Sun HL, et al: CHM-1, a new vascular targeting agent, induces apoptosis of human umbilical vein endothelial cells via p53-mediated death receptor 5 up-regulation. J Biol Chem 285: 5497-5506, 2010.
human osteogenic sarcoma cell apoptosis through induction of oxidative stress and up-regulation of ATM/p53 signaling pathway. J Orthop Res 29: 1448-1456, 2011.
11. Huang WW, Tsai SC, Peng SF, et al: Kaempferol induces autophagy through AMPK and AKT signaling molecules and causes G2/M arrest via downregulation of CDK1/cyclin B in SK-HEP-1 human hepatic cancer cells. Int J Oncol 42: 2069-2077, 2013.
12. Chen AY and Chen YC: A review of the dietary flavonoid, kaempferol on human health and cancer chemoprevention. Food Chem 138: 2099-2107, 2013.
13. Huang WW, Chiu YJ, Fan MJ, et al: Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells. Mol Nutr Food Res 54: 1585-1595, 2010.
14. Chen HJ, Lin CM, Lee CY, et al: Kaempferol suppresses cell metastasis via inhibition of the ERK-p38-JNK and AP-1 signaling pathways in U-2 OS human osteosarcoma cells. Oncol Rep 30: 925-932, 2013.
15. Lu CC, Chen HP, Chiang JH, et al: Quinazoline analog HMJ-30 inhibits angiogenesis: involvement of endothelial cell apoptosis through ROS-JNK-mediated death receptor 5 signaling. Oncol Rep 32: 597-606, 2014.
16. Lu CC, Yang SH, Hsia SM, Wu CH and Yen GC: Inhibitory effects of Phyllanthus emblica L. on hepatic steatosis and liver fibrosis in vitro. J Funct Foods 20: 20-30, 2016.
17. Lu CC, Yang JS, Chiang JH, et al: Cell death caused by quinazolinone HMJ-38 challenge in oral carcinoma CAL 27 cells: dissections of endoplasmic reticulum stress, mitochondrial dysfunction and tumor xenografts. Biochim Biophys Acta 1840: 2310-2320, 2014.
18. Yang JS, Hour MJ, Huang WW, Lin KL, Kuo SC and Chung JG: MJ-29 inhibits tubulin polymerization, induces mitotic arrest, and triggers apoptosis via cyclin-dependent kinase 1-mediated Bcl-2 phosphorylation in human leukemia U937 cells. J Pharmacol Exp Ther 334: 477-488, 2010.
19. Lu CC, Huang BR, Liao PJ and Yen GC: Ursolic acid triggers nonprogrammed death (necrosis) in human glioblastoma multiforme DBTRG-05MG cells through MPT pore opening and ATP decline. Mol Nutr Food Res 58: 2146-2156, 2014. 20. Haupt S, Berger M, Goldberg Z and Haupt Y: Apoptosis - the p53 network. J Cell
Sci 116: 4077-4085, 2003.
21. Calderon-Montano JM, Burgos-Moron E, Perez-Guerrero C and Lopez-Lazaro M: A review on the dietary flavonoid kaempferol. Mini Rev Med Chem 11: 298-344, 2011.
Colon Cancer Cells. J Cancer Prev 18: 257-263, 2013.
23. Zhang Q, Cheng G, Qiu H, et al: The p53-inducible gene 3 involved in flavonoid-induced cytotoxicity through the reactive oxygen species-mediated mitochondrial apoptotic pathway in human hepatoma cells. Food Funct 6: 1518-1525, 2015. 24. Song H, Bao J, Wei Y, et al: Kaempferol inhibits gastric cancer tumor growth:
An in vitro and in vivo study. Oncol Rep 33: 868-874, 2015.
25. Dang Q, Song W, Xu D, et al: Kaempferol suppresses bladder cancer tumor growth by inhibiting cell proliferation and inducing apoptosis. Mol Carcinog 54: 831-840, 2015.
26. Kim JD, Liu L, Guo W and Meydani M: Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. J Nutr Biochem 17: 165-176, 2006.
27. Zhao X, Song JL, Kim JD, Lee JS and Park KY: Fermented Pu-erh tea increases in vitro anticancer activities in HT-29 cells and has antiangiogenetic effects on HUVECs. J Environ Pathol Toxicol Oncol 32: 275-288, 2013.
28. Janicke RU, Sohn D and Schulze-Osthoff K: The dark side of a tumor suppressor: anti-apoptotic p53. Cell Death Differ 15: 959-976, 2008.
29. Tor YS, Yazan LS, Foo JB, et al: Induction of Apoptosis in MCF-7 Cells via Oxidative Stress Generation, Mitochondria-Dependent and Caspase-Independent Pathway by Ethyl Acetate Extract of Dillenia suffruticosa and Its Chemical Profile. PLoS One 10: e0127441, 2015.
30. Kuribayashi K, Finnberg N, Jeffers JR, Zambetti GP and El-Deiry WS: The relative contribution of pro-apoptotic p53-target genes in the triggering of apoptosis following DNA damage in vitro and in vivo. Cell Cycle 10: 2380-2389, 2011.
31. Reuter S, Gupta SC, Chaturvedi MM and Aggarwal BB: Oxidative stress, inflammation, and cancer: how are they linked? Free Radic Biol Med 49: 1603-1616, 2010.
32. Yang EJ, Kim GS, Jun M and Song KS: Kaempferol attenuates the glutamate-induced oxidative stress in mouse-derived hippocampal neuronal HT22 cells. Food Funct 5: 1395-1402, 2014.
33. Ondricek AJ, Kashyap AK, Thamake SI and Vishwanatha JK: A comparative study of phytoestrogen action in mitigating apoptosis induced by oxidative stress. In Vivo 26: 765-775, 2012.
34. Jeong JC, Kim MS, Kim TH and Kim YK: Kaempferol induces cell death through ERK and Akt-dependent down-regulation of XIAP and survivin in human glioma cells. Neurochem Res 34: 991-1001, 2009.
kaempferol-induced apoptosis of breast cancer cells and is more evident under 3-D culture condition. Cancer Biol Ther 7: 1080-1089, 2008.
36. Sharma V, Joseph C, Ghosh S, Agarwal A, Mishra MK and Sen E: Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Mol Cancer Ther 6: 2544-2553, 2007.
Figure 1. Kaempferol reduces cell viability in HUVECs. Cells were exposed to various concentrations of kaempferol (0, 50, 100, 150 or 200 μM) for 24, 48 and 72 h and determined and analyzed the viability using the MTT assay. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus untreated control.
Figure 2. Kaempferol induces morphological changes and hypodiploid DNA contents in HUVECs. (A) Cells in response to 100 μM kaempferol for a 24-h exposure were photographed at 200x magnification and showed apoptotic morphological changes. (B) Cells were treated with 100 μM of kaempferol for 24 and 48 h, Cells with hypodiploid DNA contents (%) represented the fractions undergoing apoptotic DNA degradation by flow cytometry and the Modfit program. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus untreated control.
Figure 3. Kaempferol induces DNA condensation, DNA damage and caspase-3 protein expression in HUVECs. Cells were treated with 100 μM kaempferol for 48 h, (A) DAPI staining to analyze chromatin condensation (a catachrestic of apoptosis).
(B) Comet assay to analyze DNA damage. The images were observed and captured using a fluorescent microscope. (C) Kaempferol stimulated the translocation of caspase-3 trafficking to nuclei in HUVECs by confocal laser scanning microscope as described in Materials and Methods..
Figure 4. Kaempferol stimulates intracellular Ca2+ levels and loss of ΔΨm in HUVECs. (A) Intracellular Ca2+ levels of HUVECs treated with 100 μM kaempferol as determined by Fluo-4/AM fluorescent dye using flow cytometry analysis. (B) The levels of iam in HUVECs after kaempferol treatment as determined by DiOC6(3) fluorescent dye using flow cytometry analysis. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus untreated control.
Figure 5. Kaempferol triggers caspase-8/-9/3 activities and protein levels in HUVECs. Cells were treated with 100 μM kaempferol for 0, 24 and 48 h. (A) Kaempferol-stimulated the activities of caspase-8, caspase-9, and caspase-3 in HUVECs were detected by Colorimetric Assay Kits. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus untreated control. (B) The total proteins were harvested and determined the protein levels of caspase-8, caspase-9 and caspase-3 by Western blotting. β-Actin served as a loading control.
Figure 6. Caspase-8 inhibitor (Z-IETD-FMK) decreases kaempferol-induced caspase-8/-9/3 activities in HUVECs. Cells were pretreated with 10 μM caspase-8 inhibitor (Z-IETD-FMK) for 2 h prior to treatment with 100 μM kaempferol for 48 h. Cells were harvested for measuring the caspase-8 (A), caspase-9 (B) and caspase-3 (C) activities as described in the Materials and methods. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus kaempferol alone sample.
Figure 7. Kaempferol induced ROS generation and N-acetylcysteine reduced kaempferol-induced growth inhibition effect on HUVECs. (A) Treatment with 100 μM kaempferol for 0, 6, 12 and 24 h was subjected to ROS productions by flow cytometry. (B) Pre-treatment with NAC (10 mM, an ROS scavenger) in kaempferol-treated HUVECs restored the cell viability by MTT assay. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus untreated control or kaempferol alone sample.
Figure 8. Kaempferol contributed to p53-correlated ATM/Fas/DR4/DR5 apoptotic signaling in HUVECs. Cells were treated with 100 μM kaempferol for 0, 24 and 48 h, and total protein were prepared and subjected to Western blotting analysis. The membranes were incubated with (A) anti-Fas/CD95, anti-DR4 and anti-DR5 antibodies; (B) p-ATM (Ser1981), ATM, p-p53 (Ser15) and p53 antibodies. The blot was probed with anti-β-Actin antibody to confirm equal loading. Each band was quantified using ImageJ software.
Figure 9. Caffeine and p53 siRNA reduced kaempferol-induced growth inhibition effect in HUVECs. (A) Cells were pretreated with or without 2 mM caffeine (an ATM/ATR inhibitor) for 2 h and further incubated with 100 μM kaempferol. After a 48-h exposure, cell viability was calculated by MTT assay. (B) HUVECs after being transfected with or without control vector or p53 siRNA were exposed to 100 μM kaempferol. Transfected HUVECs following exposure to kaempferol for 48 h was determined for cell viability by MTT assay. The values are presented as the means ± S.D. (n = 3). *P < 0.05 versus kaempferol alone sample.
Figure10. A proposed model for the action and possible signaling pathways of kaempferol on HUVECs. Kaempferol induces apoptosis through both extrinsic and intrinsic apoptotic pathways, resulting from p53-mediated ATM/Fas/DR4/DR5 signaling, which counteracts the induction of apoptotic death in HUVECs.
