Apigenin induces DNA damage through the PKCδ-dependent activation of ATM and H2AX causing down-regulation of genes involved in cell cycle control and DNA repair

Abstract

Apigenin, an abundant plant flavonoid, exhibits anti-proliferative and anti-carcinogenic activities through mechanisms yet not fully defined. In the present study, we show that the treatment of leukemia cells with apigenin resulted in the induction of DNA damage preceding the activation of the apoptotic program. Apigenin-induced DNA damage was mediated by p38 and protein kinase C-delta (PKCδ), yet was independent of reactive oxygen species or caspase activity. Treatment of monocytic leukemia cells with apigenin induced the phosphorylation of the ataxia-telangiectasia mutated (ATM) kinase and histone H2AX, two key regulators of the DNA damage response, without affecting the ataxia-telangiectasia mutated and Rad-3-related (ATR) kinase. Silencing and pharmacological inhibition of PKCδ abrogated ATM and H2AX phosphorylation, whereas inhibition of p38 reduced H2AX phosphorylation independently of ATM. We established that apigenin delayed cell cycle progression at G1/S and increased the number of apoptotic cells. In addition, genome-wide mRNA analyses showed that apigenin-induced DNA damage led to down-regulation of genes involved in cell-cycle control and DNA repair. Taken together, the present results show that the PKCδ-dependent activation of ATM and H2AX define the signaling networks responsible for the regulation of DNA damage promoting genome-wide mRNA alterations that result in cell cycle arrest, hence contributing to the anti-carcinogenic activities of this flavonoid.

Fig. 1

Apigenin induces DNA damage in leukemia cells. (A) Comet assays of THP-1 cells treated with indicated concentrations of apigenin or diluent DMSO for 3 h or with 1 mM H₂O₂ for 1 h. (B) % Tail DNA determined by alkaline comet assay in cells treated as described in (A). (C) % Tail DNA in cells treated with 50 μM apigenin for different lengths of time or diluent DMSO for 9 h. (D) Caspase-3 activity was determined in cells treated as described in (C). Data represents mean ± SEM, n = 3. *p < 0.05, **p < 0.01, compared to DMSO control.

Fig. 2

Apigenin induces H2AX phosphorylation. (A) Epigenetic changes were analyzed by LC–MS in THP-1 cells treated with DMSO or 50 μM apigenin for 12 h (upper and lower panel, respectively). Black and red arrows indicate peaks corresponding to non-phosphorylated and phosphorylated H2AX, respectively. (B) Lysates from THP-1 cells treated with 50 μM apigenin for different time periods or diluent DMSO for 6 h were immunoblotted with γH2AX antibodies, membrane were re-blotted with β-tubulin antibodies. (C) THP-1 cells were immunostained with anti-γH2AX antibodies and counterstained with DAPI 3 h after treatment with 50 μM apigenin or DMSO. All results shown are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Apigenin-induced DNA damage is mediated by PKCδ and p38. (A) Comet assays of THP-1 cells pretreated for 1 h with 20 μM EUK-134, 20 μM DEVD-FMK, 10 μM SB203580 or 15 μM rottlerin prior to the addition of 50 μM apigenin or DMSO for 3 h (white and black bars respectively). Data represents the mean ± SEM, n = 4, *p < 0.05 and **p < 0.01 to apigenin treated cells. (B) THP-1 cells pre-treated with 20 μM EUK-134 for 1 h prior the addition of 50 μM apigenin or diluent DMSO for an additional hour were stained with DCFDA (black bars) or DHE (white bars) and visualized under the fluorescence microscope. Fluorescence intensity was determined using the ImageJ software. Data represents mean ± SEM, n = 3. **p < 0.01 compare EUK + Api and Api. (C) Lysates from THP-1 cells pretreated for 1 h with 10 μM SB203580, 15 μM rottlerin or DMSO prior the addition of 50 μM apigenin or diluent DMSO for the times indicated were immunoblotted with γH2AX, p-p38, p38 and β-tubulin antibodies. (D) Lysates from THP-1 cells treated with 50 μM apigenin in the presence or absence of 10 μM SB203580, 15 μM rottlerin or DMSO (indicated as –) were immunoprecipitated with anti-PKCδ antibodies or isogenic IgG control and subsequently subjected to in vitro kinase assays, phosphorylated H2B was visualized by autoradiography. The same membrane was immunoblotted with anti-PKCδ antibodies. Results are representative of three independent experiments.

Fig. 4

Apigenin induces ATM and γH2AX phosphorylation in a PKCδ and p38-dependent pathway. (A) Lysates of THP-1 cells treated with 50 μM apigenin for the indicated times or diluent DMSO for 6 h were immunoblotted with anti-phospho-ATM (p-ATM), anti-ATM, anti-phospho-ATR (p-ATR) and anti-ATR antibodies. (B) THP-1 cells were pretreated for 1 h with 10 μM SB203580 or 15 μM rottlerin prior to the addition of 50 μM apigenin for different time periods or diluent DMSO for 3 h. ATM phosphorylation was analyzed by western blot. (C) Lysates from THP-1 cells transfected with siRNA-control, siRNA-p38 or siRNA-PKCδ and subsequently treated with 50 μM apigenin or diluent DMSO for 3 h were analyzed by western blots with anti-γH2AX, p-ATM, PKCδ, p-p38 or p38 antibodies. In all panels, the same membranes were re-blotted with anti-β-tubulin antibodies to ensure equal loading.

Fig. 5

Apigenin affects cell cycle progression of THP-1 cells by transcriptionally repressing cell cycle and DNA repair genes. (A) Cell cycle distribution was analyzed in THP-1 cells treated for 24 h with various doses of apigenin, DMSO or 200 ng/ml nocodazol for 24 h. All results shown are representative of three independent experiments. (B) Percentage of cells in different stages of the cell cycle as indicated in (A). Data represents the mean ± SEM, n = 3, *p < 0.05 compared to DMSO control as determined by two-way ANOVA followed by Bonferroni's post hoc. (C) Gene expression analysis of THP-1 cells treated with 50 μM apigenin or diluent control for 3 h. Genes significantly changing between groups (p < 0.01) with a fold change greater than 1.2 were analyzed using IPA according to the Gene Ontology Function. Bars correspond to functional categories significantly enriched in the data sets. (D) Heat map representation of cell cycle genes significantly modulated by apigenin (259 genes corresponding to 10.4% of total cell cycle genes). (E) Heat map representation of DNA repair genes significantly modulated by apigenin (140 genes corresponding to 5.5% of total DNA repair genes). (F) mRNA expression of selected genes was analyzed by qRT-PCR. Data represents the mean ± SEM. n = 4, *p < 0.05.

Fig. 6

Working model of apigenin-induced DNA damage. Apigenin induces DSBs, ATM and H2AX phosphorylation in a PKCδ-dependent pathway, while p38 modulates apigenin-induced DNA damage independent of ATM. Apigenin-induced down-regulation of cell cycle control genes and ATM activation led to cell cycle arrest at the G1/S transition. Down-modulation of genes involved in DNA repair by apigenin indicates that cells may be unable to repair apigenin-induced DNA damage, hence triggering apoptosis.