Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Kaempferol Activates G2-Checkpoint of the Cell Cycle Resulting in G2-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells

  • Kim, Ki Yun (Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Jang, Won Young (Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Lee, Ji Young (Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University) ;
  • Jun, Do Youn (Institute of Life Science and Biotechnology, Kyungpook National University) ;
  • Ko, Jee Youn (Nation Institute of Crop Science, Rural Development Administration) ;
  • Yun, Young Ho (Nation Institute of Crop Science, Rural Development Administration) ;
  • Kim, Young Ho (Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University)
  • Received : 2015.11.20
  • Accepted : 2015.12.03
  • Published : 2016.02.28
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Abstract

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2-arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondria-dependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.

Keywords

Introduction

Three major checkpoints are involved in cell cycle control; the first is the G1 checkpoint, which acts in the G1 phase and confirms that the environment is favorable for committing to the S phase; the second is the G2 checkpoint, which acts in the G2 phase and prevents entry into mitosis until damaged DNA is repaired and DNA replication is completed; and the third is the mitotic spindle assembly checkpoint, which acts during mitosis and ensures correct attachment of the replicated individual chromosomes to the mitotic spindles [24]. During cell cycle arrest resulting from checkpoint activation, cellular mechanisms are triggered to repair the damage and to recover cellular integrity. However, if the damage is beyond repair, the cells generate a signal to undergo apoptosis [1,13].

Apoptosis induction by chemotherapy in tumor cells leads to their own destruction into apoptotic bodies, which are cleared by neighboring cells [12]. This apoptotic process is known to be an efficient mechanism by which malignant tumor cells can be removed during treatment with chemotherapeutic drugs. The intrinsic mitochondria-dependent apoptotic pathway is frequently implicated in tumor cell death caused by chemotherapeutic drugs that are DNA-damaging and microtubule-damaging agents [9].

Recently, we started to examine various edible plants to isolate an apoptogenic substance that is pharmacologically safe, as edible plant-derived cytotoxic components against tumors may be less toxic against normal cells. Isolation of an antitumor compound from barnyard millet (Echinochloa crus-galli var. frumentacea) grains, by a serial solvent extraction method followed by HPLC analysis of phenolic compounds in the individual solvent fractions, has led to identification of the flavonoid compound kaempferol (3,4,5,7-tetrahydroxyflavone), which is frequently found in plant-derived foods. Several epidemiological studies have raised the possibility that the consumption of foods containing kaempferol can reduce the risk of developing tumors [6,10,27]. Kaempferol has been reported to induce cell cycle arrest mainly at the G2/M phase and/or induce apoptosis in several tumors [5,8,21,23,26,31,34]. However, the correlation between G2/M arrest and apoptosis induced in tumor cells after kaempferol treatment requires further investigation in order to clarify whether the G2/M arrest was the upstream and causal event of kaempferol-induced apoptosis.

From in silico docking analysis, kaempferol has been identified as having a broad inhibitory effect on human acetyl deacetylases (HDACs), a mechanism by which it exerts a cytotoxic effect on tumor cells [2]. Since HDACs are known to play a role in the cellular DNA-damage response [25], it is reasonable to assume that kaempferol-induced cell cycle arrest and apoptosis may be due to DNA damage and subsequent activation of the G2-checkpoint of the cell cycle.

To study the potential link between kaempferol-induced G2-checkpoint activation and kaempferol-induced apoptosis, we utilized the overexpression of Bcl-xL, which blocks mitochondrial apoptosis [14,17]. In this study, kaempferol-induced cell cycle arrest and the kaempferol-induced apoptotic signaling pathway were investigated using a Jurkat T cell clone stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL).

 

Materials and Methods

Chemicals, Reagents, Antibodies, Cells, and Culture Medium

Ethanol (99.9%) was purchased from Duksan (Seoul, Korea). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethythaizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,3'dihexyloxacarbocyanine iodide (DiOC6) were obtained from Sigma-Aldrich (St. Louis, MO, USA). An ECL western blotting kit was purchased from Amersham (Arlington Heights, IL, USA), and Immobilon-P membrane was obtained from Millipore (Bedford, MA, USA). Anti-caspase-3, anti-poly (ADP-ribose) polymerase (PARP), anti-Bak, anti-Bcl-xL, anti-Bcl-2, anti-PUMA, anti-p-Cdc25C (Ser-216), anti-p53, anticyclin B1, anti-p-histone H3 (Ser-10), and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-ATM (Thr-389), anti-ATM, anti-p-ATR (Ser-428), anti-p-Chk1 (Ser-317), anti-Chk1, anti-p-Chk2 (Ser-19), anti-Chk2, anti-p-Cdc25C (Thr-48), anti-Cdc25C, anti-p-Cdk1 (Thr-161), antip-Cdk1 (Tyr-15), anti-p-p53 (Ser-15), anti-caspase-8, anti-caspase-9, and anti-Bid antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-Bak (Ab-1) antibody was obtained from Calbiochem (San Diego, CA, USA). Rabbit antiserum raised against the C-terminal 7 amino acids of human Cdk1 protein was prepared as previously described [16]. A stable transfectant of human acute leukemia Jurkat T cells with the vector (clone J/Neo) and a stable transfectant of Jurkat T cells with the antiapoptotic protein Bcl-xL gene (clone J/Bcl-xL) were provided by Dr. Dennis Taub (Gerontology Research Center, NIA/NIH, Baltimore, MD, USA). Jurkat T cells were maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD, USA) containing 10% FBS, 20 mM HEPES (pH 7.0), 5 × 10-5 M β-mercaptoethanol, and 100 μg/ml gentamicin. For both JT/Neo cells and JT/Bcl-2 cells, 200 μg/ml G418 was added to RPMI 1640 medium.

Extraction and HPLC Analysis of Phenolic Compounds from Barnyard Millet Grains

Barnyard millet grains harvested in 2011 in Korea were provided by the National Institute of Crop Science (Rural Development Administration, Gyeongnam, Korea), and a voucher specimen has been deposited in the Laboratory of Immunobiology, College of Natural Sciences, Kyungpook National University (Daegu, Korea). The dried grains (250 g) were milled on a Blender 7012 (Dynamics Corporation, USA) for 10 min, and then extracted three times with 80% ethanol for 3 h at 80℃The ethanol extract and the organic solvent fraction were concentrated using a rotary vacuum evaporator (Heidolph LR 4000, Germany). The dry weights of the 80% ethanol extract and its organic solvent fractionations are described in Supplement 1 [15].

The contents of phenolic compounds in the 80% ethanol extract of barnyard millet grains were analyzed as previously described [29]. Samples were analyzed by HPLC (Agilent 1200; Agilent Technologies, Waldbronn, Germany). The analytical column was a ZORBAX ODS (4.6 × 250 mm; Agilent Technologies) with a guard column (Phenomenex, Torrance, CA, USA). The detection wavelength was set at 280 nm and the solvent flow rate was held constant at 1.0 ml/min. The mobile phase used for the separation consisted of solvent A (0.1% acetic acid in distilled water) and solvent B (0.1% acetic acid in acetonitrile). A gradient elution procedure was used as 0 min 92% A, 2-27 min 90% A, 27-50 min 70% A, 50-51 min 10% A, 51-60 min 0% A, and 60-62 min 92% A. The injection volume used for analysis was 20 μl. The standard phenolic compounds used for HPLC analysis were biochanin A, caffeic acid, (±)-catechin hydrate, chlorogenic acid, trans-cinnamic acid, formononetin, gallic acid, hesperidin, homogentisic acid, isoorientin, kaempferol, naringin, orientin, protocatechuic acid, pyrogallol, quercetin, resveratrol, rutin hydrate, syringic acid, vanillic acid, vanillin, and veratric acid (Sigma-Aldrich), and all samples were analyzed in triplicates. The column temperature was set at 60℃ and the effluent was monitored at 279 nm. As shown in Supplement 2, among 22 phenolic compounds detected in the 80% ethanol extract, isoorientin, kaempferol, protocatechuic acid, quercetin, and biochanin A were the major phenolic ingredients, and their contents were 3.56, 2.69, 2.02, 1.60, and 1.18 μg/mg of the 80% ethanol extract, respectively.

Cytotoxicity Assay

Cytotoxic activity of kaempferol against Jurkat T cells was determined by the MTT assay as previously described [14].

Flow Cytometric Analysis

Flow cytometric analysis of the cell cycle profile of Jurkat T cells treated with kaempferol was carried out as previously described [16]. The extent of apoptosis and necrosis was determined with an Annexin V-FITC apoptosis kit (Clontech, Takara Bio Inc., Shiga, Japan) as previously described [14]. The alteration in mitochondrial membrane potential (Δψm) following kaempferol treatment was analyzed after staining with DiOC6 [35]. Intracellular Bak activation following kaempferol treatment was measured by flow cytometry as previously described [11].

Protein Extraction and Western Blot Analysis

Total cell lysates were prepared by suspending Jurkat T cells (5 × 106 cells) in 300 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl2, 25 mM MOPS, 1 mM PMSF, 5.0 μg/ml proteinase inhibitor E-64, and 0.1% Triton X-100, pH 7.2). The cells were disrupted by sonication and extracted at 4℃ for 30 min prior to recovery of total cell lysate by centrifugation at 16,000 ×g for 20 min. An equivalent amount of protein lysate (20 μg) was electrophoresed on 4–12% NuPAGE gradient gels (Invitrogen/Novex, Carlsbad, CA, USA) with MOPS buffer or MES buffer, and then electrotransferred to Immobilon-P membranes. An ECL western blotting kit was used to detect each protein as described elsewhere [14].

Statistical Analysis

Unless otherwise indicated, results in this paper represent at least three separate experiments. Statistical analysis was performed using Student’s t-test to evaluate the significance of differences between two groups and one-way ANOVA between three or more groups. In all graphs, * indicates p < 0.05 between the untreated and treated cells. All data are expressed as the mean ± standard deviation (SD, for each group n ≥ 3). One-way ANOVA followed by Dunnett’s multiple-comparison test was also used for statistical analysis using IBM SPSS Statistics ver. 19.

 

Results and Discussion

Apoptogenic Effect of Kaempferol on Human Jurkat T Cells

To understand the mechanism of kaempferol cytotoxicity, its effect on Jurkat T cell viability and cell cycle distribution was investigated in stably transfected J/Neo and J/Bcl-xL cells. In the MTT assay, the viability of J/Neo cells after exposure to 25, 50, and 75 μM kaempferol for 36 h was 81.2%, 55.8%, and 36.5%, respectively, whereas the viability of J/Bcl-xL cells was 92.3%, 79.2%, and 76.5%, respectively. These results demonstrated that kaempferol induced cytotoxicity in a dose-dependent manner, and cytotoxicity was reduced by Bcl-xL overexpression (Fig. 1A).

Fig. 1.Effect of kaempferol on cell viability (A) and cell cycle distribution (B) in a Jurkat T cell clone transfected with an empty vector (J/Neo) or a Bcl-xL-expression vector (J/Bcl-xL). Continuously growing Jurkat T cells (5 × 104/well) were incubated with the indicated concentrations of keampferol in a 96-well plate for 40 h and in the final 4 h were incubated with MTT to assess cell viability as described in Materials and Methods. Each value is expressed as the mean ± standard deviation (n = 3; three replicates per independent experiment). Where indicated by *, p < 0.05 as compared with the control. Cell cycle distribution of Jurkat T cells exposed to kaempferol for 36 h was determined flow cytometric analysis as described in Materials and Methods.

To examine if both apoptotic cell death and cell cycle arrest are involved in kaempferol-mediated cytotoxicity, the cells treated with kaempferol were subjected to flow cytometric analysis after staining with propidium iodide (PI). When J/Neo cells were treated with 25, 50, and 75 μM kaempferol for 36 h, the proportion of sub-G1 cells, representing apoptotic cell death, was 9.5%, 21.0%, and 24.8%, respectively (Fig. 1B). However, there was no enhancement in the ratio of sub-G1 cells in J/Bcl-xL cells following kaempferol treatment, and ~50% cells accumulated in the G2/M phase in the presence of 50-75 μM kaempferol, indicating that Bcl-xL could prevent kaempferol-induced apoptosis but not G2/M arrest. These results demonstrated that the cytotoxicity of kaempferol against Jurkat T cells may be primarily due to cell cycle arrest at the G2/M phase and induced apoptotic cell death.

Flow Cytometric Analysis of Kaempferol-Induced Apoptotic Cells by FITC-Conjugated Annexin V Staining

Although several studies have shown that kaempferol can induce apoptosis in tumor cells [5,8,21,23,26,31,34], there have been no reports demonstrating a necrotic effect of kaemperol. To examine if kaempferol induced necrosis as well as apoptosis, both J/Neo and J/Bcl-xL cells treated with kaempferol (25, 50, and 75 μM) for 36 h were analyzed by Annexin V-FITC and PI staining. As shown in Fig. 2A, after treatment of J/Neo cells with kaempferol, early apoptotic cells stained only with Annexin V-FITC as well as late apoptotic cells stained with both Annexin V-FITC and PI were enhanced in a dose-dependent manner. In contrast, the necrotic cells stained only with PI were barely detected. Under these conditions, the levels of neither apoptotic nor necrotic cells were enhanced in J/Bcl-xL cells overexpressing Bcl-xL.

Fig. 2.Effect of kaempferol on Annexin V-FITC and PI staining (A), and forward scatter dot plot (B) of J/Neo and J/Bcl-xL cells. Continuously growing J/Neo cells or J/Bcl-xL cells (2.5 × 105/ml) were incubated with indicated concentrations of kaempferol (0.1% DMSO vehicle, 25, 50, and 75 μM) for 36 h, and harvested to evaluate apoptotic cell death after staining with Annexin V-FITC and PI. The forward scatter properties of individual unstained live, early apoptotic, and late apoptotic cells were measured to analyze changes in cell size during the induced apoptosis. A representative study is shown and two additional experiments yielded similar results.

Previously, it was reported that the Annexin V-FITC-positive/PI-positive category cannot distinguish between late apoptotic cells and necrotic dead cells [18]. Apoptotic cell death progression is characterized by a series of morphological changes that include cell shrinkage and exposure of phosphatidylserine on the cell surface, whereas necrotic cell death progression is characterized by cellular swelling [28]. In order to examine further that the late apoptotic cells stained with both Annexin V-FITC and PI were correlated with apoptosis but not with necrosis, we decided to examine the change in cell size during kaempferol-induced apoptosis. In this context, the forward scatter distributions of the unaffected cells, early apoptotic cells stained only with Annexin V-FITC, and late apoptotic cells stained with both Annexin V-FITC and PI were compared between J/Neo cells and J/Bcl-xL cells after kaempferol treatment. As shown in Fig. 2B, although the light scattering properties of the unstained live cells were not altered following kaempferol treatment, late apoptotic cells and early apoptotic cells exhibited a decrease in forward scatter. This indicated that no cellular swelling but rather a reduction in cell size was provoked during kaempferol-induced apoptosis. These results confirmed that kaempferol-induced apoptosis, which was blocked by Bcl-xL overexpression, was not accompanied by necrosis in Jurkat T cells.

Involvement of Bak Activation, Mitochondrial Membrane Potential (Δψm) Loss, and Caspase Cascade Activation in Kaempferol-Induced Apoptosis

To examine the role of the mitochondria-dependent apoptotic pathway, which is known to be frequently involved in chemical-induced apoptosis [9], the change in Δψm of J/Neo cells and J/Bcl-xL cells was measured by flow cytometry using DiOC6 staining following exposure to kaempferol (25, 50, and 75 μM). As shown in Fig. 3A, the percentages of J/Neo cells that displayed decreased fluorescence following treatment with kampferol at concentrations of 25, 50, and 75 μM were 8.4%, 16.2%, and 20.3%, respectively. This indicated that kaempferol (25-75 μM) could induce Δψm loss in J/Neo cells in a dosedependent manner. Under the same conditions, however, kaempferol failed to induce Δψm loss in J/Bcl-xL cells. As Δψm loss is known to be one of the initial intracellular changes of apoptotic cell death [32], these results suggested that Δψm disruption was associated with kaempferol-induced apoptosis in J/Neo cells. These results also indicated that the Δψm loss was mediated by a conserved apoptotic mechanism, which could be inhibited by the antiapoptotic role of Bcl-xL.

Fig. 3.Flow cytometric analysis of Δψm loss (A), western blot analysis of PUMA, Bak, Bid, Bcl-xL, Bcl-2, and activation of caspase-9, -8, and -3 (B), and flow cytometric analysis of Bak activation (C) in J/Neo and J/Bcl-xL cells after treatment with kaempferol. The cells (2.5 × 105/ml) were incubated with vehicle (0.1% DMSO) or the indicated concentrations of kaempferol for 36 h, and harvested. Flow cytometric analysis of Δψm loss and Bak activation, and western blot analyses were performed as described in Materials and Methods. A representative result is presented, and two additional experiments yielded similar results.

Previously, it has been reported that Δψm loss precedes mitochondrial cytochrome c release into the cytosol, followed by caspase-3 activation [19]. Therefore, it is likely that the Δψm loss and subsequent activation of the mitochondria-dependent caspase cascade, which could be blocked by Bcl-xL overexpression, might be prerequisites for kaempferol-induced apoptosis in Jurkat T cells. To test this prediction, western blot analyses were performed for both J/Neo and J/Bcl-xL cells to examine if the induced apoptosis was accompanied by mitochondria-dependent caspase cascade activation. As shown in Fig. 3B, the caspase-9 activation, which proceeded through proteolytic cleavage of the inactive proenzyme (47 kDa) to active forms (37/35 kDa), was detected in J/Neo cells after treatment with kaempferol (25-75 μM) and correlated with the Δψm loss. The cleavage of procaspase-3 (32 kDa) into the active form (17 kDa) as well as the cleavage of procaspase-8 (57 kDa) into the active form (43/41 kDa) was also detected in a dose-dependent manner. In J/Neo cells treated with kaempferol, the PARP cleavage into two fragments, which is known to be mediated by caspase-3 during induction of apoptosis [20], was detected as well as the activation of caspase-3. However, these apoptotic events were completely abrogated in J/Bcl-xL cells.

Numerous studies have reported that the pro-apoptotic multidomain Bcl-2 family member Bak mediates mitochondrial outer membrane permeabilization, leading to mitochondrial cytochrome c release into the cytosol [4,7]. However, antiapoptotic Bcl-2 family members (Bcl-2 and Bcl-xL) block the cytochrome c efflux triggered by Bak via either directly or by inactivating the BH3-only pro-apoptotic Bcl-2 family members (Bad, Bid, Bim, and PUMA) [4,7]. In addition, to examine the cellular events that cause kaempferol-induced Δψm loss, the expression levels of Bcl-2 family proteins, including the pro-apoptotic members (Bak, Bid, and PUMA) and the anti-apoptotic members (Bcl-2 and Bcl-xL), were compared by western blot analysis between J/Neo and J/Bcl-xL cells after kaempferol treatment. As a result, the expression levels of Bak and PUMA were enhanced by 3.0-fold and 4.5-fold, respectively, in J/Neo treated with kaempferol. However, the expression levels of Bcl-2 and Bcl-xL appeared to remain relatively constant in both cell types, regardless of kaempferol treatment. In both J/Neo and J/Bcl-xL cells after kaempferol treatment, the level of Bid protein (22 kDa), which is known to be cleaved by active caspase-8 [22] or by lysosomal cathepsin [3] to generate the truncated Bid (tBid, 15 kDa) causing Δψm loss, was reduced. However, western blot analysis failed to detect the generation of tBid in J/Neo cells treated with kaempferol, presumably attributable to the short half-life of tBid. To confirm that the kaempferol-induced apoptosis is accompanied by Bak activation, the N-terminal conformational change required for Bak activation was analyzed by flow cytometry using the conformation-specific anti-Bak (Ab-1) in J/Neo and J/Bcl-xL cells after kaempferol treatment. As shown in Fig. 3C, the Bak activation was detected in J/Neo cells, but not in J/Bcl-xL cells overexpressing Bcl-xL.

Consequently, these results demonstrated that the upregulation of Bak and PUMA levels and the Bcl-xL-sensitive Bak activation were responsible for kaempferol-induced mitochondria-dependent caspase cascade activation in Jurkat T cells.

Contribution of G2-Checkpoint Pathway to Kaempferol-Induced Cell Cycle Arrest

To investigate the involvement of the G2-checkpint machinery in kaempferol-induced G2/M arrest of Jurkat T cells, the activation of ATM/ATR kinases and Chk1/Chk2 kinases, which are G2-checkpoint kinases [1,30], were analyzed by western blot analysis using antibodies recognizing their active phosphorylation forms.

As shown in Fig. 4, the active phosphorylated forms of ATM/ATR kinases and Chk1/Chk2 kinases were detected in Jurkat T cells following kaempferol treatment, irrespective of Bcl-xL overexpression. In accordance with the activation of Chk1/Chk2 kinases, the inhibitory phosphorylation of Cdc25C on Thr-216 as well as the activating phosphorylation of p53 on Ser-15 was enhanced in both cell types; however, the activating phosphorylation of Cdc25C on Thr-48 was not detected in both cell types after kaempferol treatment. Under these conditions, the dephosphorylation of Cdk1 (Tyr-15) by the Cdc25C phosphatase activity, which is a crucial event for the Cdk1 activation during G2/M transition of the cell cycle [33], failed to occur. Recently, kaempferol has been shown to inhibit the HDAC activity that is necessary for DNA-damage repair [25]. Consequently, these results demonstrated that kaempferol-induced cell cycle arrest was due to G2-arrest, which was dictated by DNA-damage-mediated activation of the ATR/ATM-Chk1/Chk2-Cdc25 pathway and the resultant failure to activate Cdk1. These results further suggested that the G2-arrest and the Bcl-xL-sensitive apoptosis were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation.

Fig. 4.Western blot analysis of p-ATM (Thr-389), ATM, p-ATR (Ser-428), p-Chk1 (Ser-317), Chk1, p-Chk2 (Ser-19), Chk2, p-p53 (Ser-15), p53, and β-actin (A), and p-Cdc25C (Ser-216), p-Cdc25C (Thr-48), Cdc25C, pCdk1 (Thr-161), p-Cdk1 (Tyr-15), Cdk1, cyclin B1, p-histone H3 (Ser-10), and β-actin (B) in J/Neo and J/Bcl-xL cells after treatment with kaempferol. The cells (2.5 × 105 cells/ml) were incubated with vehicle (0.1% DMSO) or the indicated concentrations of kaempferol for 36 h, and prepared for the cell lysates. Western blot analyses were performed as described in Materials and Methods. A representative study is shown and two additional experiments yielded similar results.

In conclusion, kaempferol could activate the G2-checkpoint pathway, which caused not only G2-arrest of the cell cycle but also activation of p53 (Ser-15) phosphorylation and several mitochondria-dependent apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, Δψm loss, activation of caspase-9, -3, and -8, and PARP cleavage. These findings will be useful for evaluating the potency of the apoptogenic component kaempferol from barnyard millet (Echinochloa crus-galli var.frumentacea) grains as an antitumor agent.

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