Critical roles of Rad54 in tolerance to apigenin‑induced Top1‑mediated DNA damage

  • Authors:
    • Zilu Zhao
    • Xiaohua Wu
    • Fang He
    • Cuifang Xiang
    • Xiaoyu Feng
    • Xin Bai
    • Xin Liu
    • Jingxia Zhao
    • Shunichi Takeda
    • Yong Qing
  • View Affiliations

  • Published online on: March 18, 2021     https://doi.org/10.3892/etm.2021.9936
  • Article Number: 505
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Abstract

Apigenin (APG), a flavone sub‑class of flavonoids, possesses a diverse range of biological activities, including anti‑cancer and anti‑inflammatory effects. Previous studies identified the genotoxicity of APG in certain cancer cells, which may be associated with its anticancer effect. However, the DNA damage repair mechanism induced by APG has remained elusive. In order to clarify the molecular mechanisms, the present study determined the toxicity of APG to the wild‑type (WT) DT40 chicken B‑lymphocyte cell line, as well as to DT40 cells with deletions in various DNA repair genes, and their sensitivities were compared. It was demonstrated that cells deficient of Rad54, a critical homologous recombination gene, were particularly sensitive to APG. Cell‑cycle analysis demonstrated that APG caused an increase in the G2/M‑phase population of Rad54‑/‑ cells that was greater than that in WT cells. Furthermore, it was demonstrated by immunofluorescence assay that Rad54‑/‑ cells exhibited significantly increased numbers of γ‑phosphorylated H2AX variant histone foci and chromosomal aberrations compared to the WT cells in response to APG. Of note, the in vitro complex of enzyme assay indicated that APG induced increased topoisomerase I (Top1) covalent protein DNA complex in Rad54‑/‑ cells compared to WT cells. Finally, these results were verified using the TK6 human lymphoblastoid cell line and it was demonstrated that, as for DT40 cells, Rad54 deficiency sensitized TK6 cells to APG. The present study demonstrated that Rad54 was involved in the repair of APG‑induced DNA damage, which was associated with Top1 inhibition.

Introduction

Apigenin (APG), a flavonoid widely distributed in a variety of fruits, vegetables and herbs, including oranges, onions and parsley (1), has been demonstrated to be potent against cancer and effective for chemoprophylaxis (2). Its anticancer effects have been examined in multiple cancer types, including breast (3), prostate (4), colon (5) and cervical cancer (6). The anticancer mechanisms of APG are complex. Previous studies demonstrated that APG inhibited the growth of numerous types of cancer cell via inhibition of epidermal growth factor receptor (7-9). APG is also able to induce cell-cycle arrest in G2/M phase in multiple cell lines (8,10) and trigger apoptosis in leukemia cells (6). APG in combination with tumor necrosis factor-α was able to reduce the cell viability and proliferation of hepatocellular carcinoma cells (11). APG was indicated to target inhibitor proteins of apoptosis in ovarian cancer to induce apoptotic cell death (12). In addition, APG was demonstrated to induce reactive oxygen species and p38, which are essential substances in the process of cell death (7,13,14). Furthermore, it was observed that APG induced DNA lesions involving DNA breaks (15), micronuclei, chromosomal aberrations (CAs) and chromatid exchanges (16-18). APG was further demonstrated to have significant topoisomerase I (Top1) inhibitor activities (19). A previous in vitro study demonstrated that APG inhibited the DNA binding or the DNA re-ligation step of Top1(20).

Induction of DNA lesions is an important mechanism of action of numerous clinical anticancer agents. DNA double-strand breaks (DSBs), the most severe type of DNA lesion, induces cell death if not repaired (21). DSBs were induced by numerous exogenous factors, including ionizing radiation (IR) (22) and endogenous factors, including topoisomerase (23,24). Camptothecin (CPT), a DNA Top1 inhibitor, has been widely used in the clinic as an anticancer drug. Regarding the mechanism of action, CPT has been demonstrated to selectively bind to and stabilize the 3'phospho-tyrosyl bond formed between Top1 and DNA [Top1 covalent protein DNA complex (Top1cc)] to trap Top1 in Top1cc, which inhibits DNA and RNA synthesis when colliding with the replication fork or transcription machinery to cause DSBs and cell death (25-28). To avoid CPT-induced cell death, two major DNA repair pathways are in place: One is the removal of covalent 3'-DNA adducts by tyrosyl-DNA phosphodiesterase I (TDP1), which hydrolyzes the drug-stabilized 3'phospho-tyrosyl bond of Top1cc (29,30); the other is DSB repair by homologous recombination (HR) (31). Cells deficient of either TDP1 or HR factors, including Rad54, were demonstrated to enhance DNA damage and cell death (32,33).

The DT40 cell line is derived from bursal B-lymphocyte cells (34). It has a stable karyotype and exhibits extraordinarily high gene-targeting efficiency; thus, it has been widely used in genetic studies, including comprehensive research on the mechanisms of DNA damage and repair (35-38). In the present study, the sensitivity to APG, induction of DNA damage and formation of Top1cc were investigated using wild-type (WT) DT40 cells, as well as DT40 cells with deletions in several DNA repair genes. The TK6 cell line, a human lymphoblastoid cell line, has numerous advantages, including stable spontaneous mutation frequencies, ability to grow in suspension and simplicity of culture. As a human-derived cell line, TK6 has been widely used for genotoxicity testing (39).

In the present study, it was identified that Rad54, an HR factor, has a critical role in the tolerance to APG-induced DNA damage in DT40 cells. The Rad54-/- TK6 cell line was also used to verify these results and it was demonstrated that human Rad54 has the same function in response to APG as that in the chicken DT40 cell line.

Materials and methods

Chemicals

APG and CPT were purchased from MedChemExpress. APG (50 mM) and CPT (100 µM) were prepared and stored at -20˚C. The chemicals were dissolved with DMSO to produce stock solutions.

Cell culture

DT40 cells and human lymphoblast TK6 cells were provided by Dr Shunichi Takeda (Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Table I). DT40 cells were maintained in RPMI-1640 medium (Wisent, Inc.) containing 10% heat-inactivated FBS (Wisent, Inc.), 1% chicken serum (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin-streptomycin (Wisent, Inc.) and 50 µM β-mercaptoethanol (Gibco; Thermo Fisher Scientific, Inc.). The medium for TK6 cells was RPMI-1640, supplemented with 10% heat-inactivated horse serum (HyClone; Cytiva) and 1% penicillin-streptomycin. All cell lines were maintained at 37˚C in a humidified atmosphere with 5% CO2.

Table I

DNA repair genes mutated in the DT40 and TK6 clones analyzed.

Table I

DNA repair genes mutated in the DT40 and TK6 clones analyzed.

GeneName of cell lineFunctionReference
Fen1Chicken DT40Base excision repair, processing 5'flap in long-patch and lagging strand DNA(48)
Rad18Chicken DT40TLS(49)
Parp1Chicken DT40Poly(ADP) ribosylation, related to single-strand break and BER(50)
Rad54Chicken DT40DSB repair by HR(51)
Rad54Human TK6DSB repair by HR(53)
TDP1Human TK6Tyrosyl-DNA phosphodiesterase I(54)

[i] TLS, translesion DNA synthesis; HR, homologous recombination; BER, base excision repair; DSB, double-strand break; TDP1, tyrosyl-DNA phosphodiesterase 1; PARP1, poly(ADP-ribose) polymerase; FEN1, flap structure-specific endonuclease 1; ADP, adenosine diphosphate.

Cell viability assay

The cells were exposed to APG (0, 10, 20 and 30 µM) and CPT (0, 10, 20, 30 and 40 nM) for 72 h, and subsequently, 20 µl Cell Counting Kit-8 (CCK-8; MedChemExpress) reagent was added to each well. The absorbance of each well was measured at 450 nm using a microplate reader (SynergyMx; BioTek Instruments, Inc.). Cell survival curves were constructed from the CCK-8 assay as previously described (40). SPSS software version 20.0 (IBM Corp.) was used to calculate the survival percentage compared with the control. Relative IC50 value = (The IC50 of deletion cells/The IC50 of WT cells) x100%.

Cell-cycle assays

Flow cytometry was used to determine cell-cycle arrest (41). In total, 4x105 cells were cultured with 20 µΜ APG or 6 nM CPT for 16 h. Subsequently, the cells were fixed with 70% ethanol for 4˚C overnight prior to staining with propidium iodide (PI) in the presence of RNase A (cat. no. ST579; Beyotime Institute of Biotechnology) (42). The results were determined using a flow cytometer (CytoFLEX; Beckman Coulter, Inc.).

Colony formation assay

The colony formation assay was performed as previously described (39,43). The medium for DT40 cells included various amounts of APG, mixed with DMEM-F12 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 15% heat-inactivated FBS (Wisent, Inc.), 1.5% chicken serum (Wisent, Inc.), 1.5% (w/v) methylcellulose, 200 mΜ L-glutamine (Gibco; Thermo Fisher Scientific, Inc.) and 50 µΜ β-mercaptoethanol (Gibco; Thermo Fisher Scientific, Inc.) using a slowly rotating shaker overnight at 4˚C. The medium for TK6 cells contained various amounts of drugs, which were mixed with 1.5% (w/v) methylcellulose medium containing 10% heat-inactivated horse serum using slowly rotating tubes overnight at 4˚C. Cells (100 cells/well) were seeded into six-well plates containing 3 ml methylcellulose medium per well and after 14 days, visible white colonies were counted in each well.

Immunofluorescence staining and image analysis

Immuno-fluorescence staining was performed as previously described (43). In total, 5x105 cells were treated with 30 µM APG or 100 nM CPT for 3, 6 and 9 h. The cells were then fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were permeabilized with 0.1% Nonidet P-40. The cells were then blocked with PBS containing 3% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 30 min and incubated with a mouse monoclonal γ-phosphorylated H2A.X variant histone (γ-H2AX; Ser139) antibody (1:1,000; cat. no. 05-636; EMD Millipore) at 4˚C overnight. Anti-mouse Alexa Fluor 488-conjugated antibody (1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology) was used as the secondary antibody at 37˚C for 1 h. The γ-H2AX foci were visualized under a fluorescent microscope (AX10 imager A2/AX10 Cam HRC; Zeiss). The nuclei were stained with DAPI for 10 min at room temperature. The foci of 100 nuclei were counted per well as described in previous reports using Photoshop (version 12.0.3; Adobe Systems, Inc.) (39,43). Experiments were performed three times independently.

Detection of CAs

Karyotype analysis was performed according to a previous study (44). In brief, cells were treated with APG or CPT for different durations. At 3 h prior to harvesting, 0.2 µg/ml colchicine solution (Gibco; Thermo Fisher Scientific, Inc.) was added. The suspended cells were incubated in 75 mM KCl solution for 25 min and then fixed in Carnoy's solution. The cell suspension was dripped onto ethanol-soaked glass slides and dried using a flame. The dried slides were stained with 5% Giemsa solution and rinsed with water.

In vitro complex of enzyme (ICE) assay

Top1-DNA adducts were isolated using the ICE Bio-assay as described in a previous study (36). In total, 1.5x107 cells per sample were treated with APG or CPT and then suspended in 2 ml buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton X-100; 1 mM phenylmethylsulfonyl fluoride) and put on ice for 10 min. The precipitate was then resuspended in 2 ml buffer B (2 mM EDTA, 0.3 mM egtazic acid, 0.1% Triton X-100 and protease inhibitor) and incubated for 30 min on ice. The chromatin sediment was washed with buffer C (25 mM Tris-Cl, 300 mM NaCl, 10% glycerol and protease inhibitor) five times. After being resuspended in 2 ml 6 M guanidinium chloride, DNA solutions were loaded onto the top of a cushion of CsCl (densities of CsCl solutions: 1.82, 1.7, 1.5 and 1.45 g/ml) and samples were centrifuged at 100,000 x g for 16 h at room temperature. Subsequently, 1 ml supernatant of each part was collected. For dot blot analysis, 100 µl of each fraction was loaded onto a Bio-Dot apparatus (cat. no. 1706545; Bio-Rad Laboratories, Inc.). After washing with Tris-buffered saline with Tween-20, the membrane was incubated with anti-Top1 antibody at 4˚C for 24 h (1:10,000; cat. no. ab109374; Abcam) for 24 h and subsequently incubated with an anti-rabbit antibody conjugated with horseradish peroxidase at 37˚C for 1 h (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology).

Statistical analysis

Statistical significance of differences was examined using Student's t-test or two-way analysis of variance with Bonferroni's test. Statistical analysis was performed with SPSS 25.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

Rad54-/- DT40 cells are sensitized to APG

DT40 chicken B-lymphocyte cells (45) have been widely used in research on DNA lesions and repair (46,47). To examine the molecular mechanisms of APG-induced DNA lesions and their repair, the effects of APG on the viability of WT DT40 cells, as well as DT40 cells deletions in several DNA repair genes, flap structure-specific endonuclease 1 (FEN1)-/- (48), Rad18-/- (49), poly(ADP-ribose) polymerase 1 (PARP1)-/- (50) and Rad54-/- (51), were studied and compared. Those cells were separately defective in several DNA repair pathways (Table I). Cells were treated with different concentrations of APG for 72 h and a CCK-8 assay was used to determine their viability. The 50% inhibitory concentration was calculated using SPSS 20.0 software (IBM Corp.). As presented in Fig. 1A and B, Rad54-/- cells deficient of HR repair demonstrated significant sensitivity to APG, while the cells deficient in other DNA repair pathways, were not sensitive to APG. The sensitivity of Rad54-/- cells to APG was also demonstrated in the colony formation assay, as presented in Figs. 1C and S1A. CPT, a Top1 inhibitor, which has been previously demonstrated to sensitize Rad54-/- DT40 cells (52), was used as the positive control, as presented in Fig. 1D.

Figure 1

Rad54-/- cells are sensitized to APG. (A) Sensitivity of Parp1-/-, Rad18-/-, Fen1-/- and Rad54-/- cells to APG. The x-axis represents the concentration of APG and the y-axis represents the relative percentage of cell survival at 72 h. Two-way analysis of variance with Bonferroni’s test was used to test for differences in the linear dose-response curves between WT and mutant cells. *P<0.01 WT vs. Rad54-/- cells; WT vs. Parp1-/- cells; WT vs. Rad18-/- cells; WT vs. Fen1-/- cells. (B) Relative IC50 values of APG in DT40 cells. The x-axis represents the DT40 cells and the y-axis represents the relative number of surviving cells at 72 h. IC50 is the concentration at which APG reduces cellular survival to 50% relative to cellular survival without APG treatment. The IC50 was calculated from the results of CCK-8 survival assay. Proliferative ability of (C) APG- and (D) CPT-treated DT40 cells determined by clonogenic assays. The x-axis represents the concentration of drugs and the y-axis represents the fractions of surviving colonies on a logarithmic scale. Survival data were log-transformed to approximate normality. Values are expressed as the mean ± standard deviation. The experiments were performed three times independently. Two-way analysis of variance was used to test for differences in the linear dose-response curves between WT cells and deletion cells. *P<0.05, control vs. Rad54-/- cells. APG, apigenin; CPT, camptothecin; WT, wild-type; PARP1, poly(ADP-ribose) polymerase; FEN1, flap structure-specific endonuclease 1; ADP, adenosine diphosphate.

APG treatment leads to G2/M-phase cell-cycle arrest of Rad54-/- DT40 cells

Changes in the cell cycle of Rad54-/- DT40 cells compared with WT cells were determined. After 16 h of APG treatment, flow cytometric analysis of PI-stained cells demonstrated that the WT and Rad54-/- cells were arrested at the G2/M phase of the cell cycle. Furthermore, APG led to a higher degree of accumulation of in the G2/M phase for Rad54-/- cells compared with that in WT cells. CPT also promoted G2/M-phase arrest of Rad54-/- cells (Fig. 2).

APG treatment produces more DSBs in Rad54-/- DT40 cells

To verify the effect of APG to induce DNA damage in DT40 cells, the APG-induced γ-H2AX foci in DT40 cells were quantified. DT40 WT and Rad54-/- cells were treated with 30 µM APG for 0, 3, 6 and 9 h. As presented in Figs. 3A and S2A, the numbers of γ-H2AX foci were significantly increased in the nuclei and peaked at 6 h after APG exposure in both WT and Rad54-/- cells. However, in the Rad54-/- cells, γ-H2AX foci increased more significantly in response to APG (Figs. 3A and S2A) or CPT treatment (Figs. 3B and S2A). The patterns of γ-H2AX foci induced by APG and CPT in Rad54-/- cells were similar. However, compared with the WT, the increase of CPT-induced γ-H2AX foci in Rad54-/- cells was significantly greater than that induced by APG at 9 h.

APG-induced DSBs in Rad54-/- cells were also compared with the WT by measuring CAs in DT40 cells. Cells were incubated with 20 µM APG or 10 nM CPT and CAs were confirmed dynamically at 8, 16 and 24 h. The largest number of CAs was counted after 16 h of treatment with APG (Fig. 4A and B) or CPT (Fig. 4C). As presented in Fig. 4, Rad54-/- cells had significantly increased numbers of CAs than WT cells in response to APG or CPT treatment. These results suggested that Rad54 participated in repairing APG-induced DNA damage.

APG traps Top1cc in Rad54-/- DT40 cells

It was then was further assessed whether APG induced Top1cc in DT40 cells. Cells were lysed and Top1cc was separated from free Top1 in the cellular lysates by subjecting cellular extract to CsCl gradient ultracentrifugation. Free Top1 remained in the top two fractions, while Top1cc moved to lower fractions of the CsCl gradient corresponding to the migration of chromosomal DNA. DT40 cells were cultivated with 100 µM APG or CPT for 2 h and an ICE assay was used to detect Top1cc and free Top1. As presented in Fig. 5, fractions 1-2 were mainly free protein and Top1cc peaked at fraction 3. The results in Fig. 5 demonstrated that both APG and CPT induced more accumulation of Top1cc in Rad54-/- cells than in WT cells (Fig. 5). This observation suggested that APG is able to trap Top1 to remain in a covalent protein-DNA complex in DT40 cells, suggesting that APG is a Top1 inhibitor.

APG induces sensitivity and increases DSBs and Top1cc in Rad54-/- and TDP1-/- TK6 cells

In order to identify whether human Rad54 was also involved in tolerance of DNA lesions induced by APG, the effects of APG in Rad54-/- TK6 cells were studied and compared (53). As presented in Figs. 6A and B, and S1B, Rad54-/- TK6 cells were more sensitive to APG or CPT than WT cells in the colony formation assay. Following treatment with 80 µM APG or 20 nM CPT for 0, 3, 6 and 9 h, the number of γ-H2AX foci in Rad54-/- TK6 cells was significantly increased as compared with that in WT TK6 cells (Figs. 6C and D, and S2B). Furthermore, Top1cc was observed in TK6 cells after treatment with 100 µM APG or 2 nM CPT for 2 h, and the drugs induced increased Top1cc in Rad54-/- compared with WT cells (Fig. 6E). These data were consistent with the results obtained with DT40 cells, suggesting that APG may be a human Top1 inhibitor, which may induce Rad54-dependent tolerance of DNA damage.

Figure 6

Effects of APG on the proliferative ability and DNA damage in Rad54-/- TK6 cells. Proliferative ability of (A) APG- and (B) CPT-treated TK6 cells determined by clonogenic assays. The x-axis represents the concentration of the drugs and the y-axis represents the fractions of surviving colonies on a logarithmic scale. Survival data were log-transformed to approximate normality. Two-way analysis of variance was used to test for differences in the linear dose-response curves between WT and Rad54-/- cells. *P<0.05, Rad54-/- cells compared with WT. (C) Quantification of γ-H2AX foci in WT and Rad54-/- cells after treatment with 80 µM APG. (D) Quantification of γ-H2AX foci in WT and Rad54-/- cells after treatment with 20 nM CPT. TK6 cells were treated for 0, 3, 6 and 9 h, respectively; 100 cells were analyzed for each data-point. (E) APG induced the formation of Top1cc in TK6 cells. TK6 cells were treated with 100 µM APG and 2 µM CPT for 2 h to detect Top1cc. In the dot blot, top to bottom indicates the order in which the fractions were taken out of the sample after ultracentrifugation. Values are expressed as the mean ± standard deviation. Experiments were performed three times independently. *P<0.05, compared with WT after treatment for 3 h; ‡P<0.05, compared with WT after treatment for 6 h; #P<0.05, compared with WT after treatment for 9 h. APG, apigenin; CPT, camptothecin; WT, wild-type; γ-H2AX, γ-phosphorylated H2A.X variant histone; Top1cc, topoisomerase I covalent protein DNA complex.

Based on the function of TDP1 in Top1-associated DNA damage repair by hydrolyzing the 3' phospho-tyrosyl bond of Top1cc (54,55), it was hypothesized that cells deficient in TDP1 should also be sensitive to APG. Therefore, the cytotoxic effects in TDP1-/- TK6 cells were investigated after treating cells with different concentrations of APG (0, 10, 20 and 30 µM) and CPT (0, 2, 4, 6 and 8 nM) for 72 h. The results suggested that TDP1-/- TK6 cells were more sensitive to APG than WT cells with similar effects of CPT in the colony formation assay (Figs. S1B and S3A and B). Subsequently, the effects of APG-induced DNA-damage on TDP1-/- cells were examined. WT and TDP1-/- TK6 cells were treated with 80 µM APG or 20 nM CPT for 0, 3, 6 and 9 h. The results indicated that the numbers of γ-H2AX foci in the nuclei were significantly increased and peaked at 6 h after APG exposure in WT and TDP1-/- TK6 cells. In TDP1-/- TK6 cells, γ-H2AX foci were increased more significantly in response to APG or CPT treatment compared with those in WT cells (Figs. S2B and S3C and D). These results suggested that APG or CPT induced more DNA damage in TDP1-/- TK6 cells. Furthermore, Top1cc was observed in TDP1-/- TK6 cells after treatment with 100 µM APG or 2 nM CPT for 2 h, and the results demonstrated that APG or CPT induced more Top1cc in TDP1-/- TK6 cells than in WT cells (Fig. S3E). These results further suggested that APG is a Top1 inhibitor, which is able to trap Top1 in Top1cc, and increased Top1cc was observed in TDP1-/- vs. WT cells.

Discussion

It has been demonstrated that APG possesses a series of biological effects, including antiviral, antibacterial, antioxidant, anti-inflammatory and anticancer effects (3,56-59). APG was also indicated to cause DNA lesions (60,61). In the present study, DT40 cells with deletions of various DNA damage repair genes were used to examine the possible molecular mechanisms of APG-induced DNA damage repair, and it was identified that Rad54-/- DT40 cells were specifically sensitive to APG. The cell-cycle assay demonstrated that APG also induced more cell-cycle arrest in G2/M phase in Rad54-/- than in WT cells.

Rad54 is the most highly conserved eukaryotic HR protein, which is involved in various activities that contribute to the progression of HR (62). The Rad54 protein is part of a nucleoprotein filament to confer DNA homology (63,64). It interacts with the Rad51 nucleoprotein filament and induces the activity of DNA pairing in HR (51). HR is an important DNA repair pathway for DSBs. It repairs DSBs in the S phase (62). It was demonstrated that lack of Rad54 in DT40 cells leads to sensitivity to endogenous factors, including topoisomerase (65), and exogenous factors, such as IR (66), which is able to induce DSBs in the S-phase (67,68). In the present study, Rad54-/- DT40 cells were sensitive to APG, demonstrating that APG may induce DSBs that are tolerated in the presence of Rad54. Cell-cycle arrest is an important cell response to DNA lesions that is initiated by activating cell-cycle checkpoint proteins. It was previously demonstrated that APG caused arrest of the cell cycle in G0/G1-phase by reducing the level of cyclin D1 in LNCaP and PC-3 prostate cancer cell lines (1). However, in other studies, APG caused cell-cycle arrest at the G2-phase by decreasing cyclin B in human colon carcinoma and pancreatic cancer cells (10), or regulated other cell-cycle phases by affecting cyclin A and cell division cycle 25A and cell division cycle 25C in different cell types (8,69-71). The results of the cell-cycle assay of the present study suggested that APG arrested Rad54-/- cells in G2/M-phase. To identify whether APG induced DSBs, the γ-H2AX foci were quantified as a marker for DSBs and CAs representing stable DSBs were also evaluated. Analysis of both γ-H2AX foci and CAs identified that APG induced increased DSBs in Rad54-/- cells compared with the WT cells. The results suggested that the enhanced sensitivity and increased amount of cell-cycle arrest of Rad54-/- cells in G2/M-phase were associated with the induction of DSBs by APG.

HR has been identified as the essential Top1-associated repair pathway for DSBs (72). Numerous flavonoids, including APG, quercetin, kaempferol and morin, have been identified as Top1 inhibitors (20,73). Previous studies (20,73) reported that several flavonoids, including APG, were similar to CPT and were able to stabilize Top1cc in vitro and in vivo. Consistent with these previous studies, the present study demonstrated that APG induced significant Top1cc formation in Rad54-/- cells. Furthermore, human Rad54-/- TK6 cells exhibited the same sensitivities to APG and increased DSBs and Top1cc formation as Rad54-/- DT40 cells. These results suggested that APG may be a Top1 inhibitor, which traps Top1 in the form of Top1cc. When Top1cc encounters a replication fork, it leads to cell-cycle arrest and even DSBs, which is mainly repaired by HR. Lack of Rad54 affects the repair of DSBs caused by the sustained existence of Top1cc.

Top1 is an important enzyme for DNA replication, transcription, recombination and chromatin remodeling, which relaxes the supercoil structure of DNA molecules by cutting one strand of duplex DNA and generating Top1cc that causes supercoiled DNA to untwist (74). Under physiological conditions, Top1cc is resolved by TDP1, which catalyzes hydrolysis of the Top1 tyrosine residue covalently linked to the 3'-phosphate of DNA, removes Top1 from the DNA 3'-end so that the broken DNA strand is religated (75). A previous study has demonstrated that deficiency of TDP1 leads to accumulation of Top1cc and DSBs, and a Top1 inhibitor, e.g. CTP, was able to enhance accumulation of Top1cc and DSBs (76). In the present study, it was identified that TK6 cells deficient of TDP1 were also sensitive to APG, which generated increased DSBs and Top1cc. This result further suggested that APG is a Top1 inhibitor.

To the best of our knowledge, the present study was the first to examine the role of Rad54 in the tolerance of APG-induced Top1-mediated DNA damage by using DNA repair-deficient DT40 and TK6 cells. The data suggested that inhibition of Rad54 may enhance the toxicity of APG to tumor cells. The present results also suggested that APG-induced toxicity was enhanced by Rad54 deletion, such that Rad54 may be included in the potential DNA rearrangements caused by APG for the treatment of other diseases. The present study provided insight for developing novel anticancer medicines with higher therapeutic efficacy and less genotoxicity.

Supplementary Material

Representative images of colony formation assays in DT40 and TK6 cells. (A) DT40 cells treated with APG. (B) TK6 cells treated with APG. The representative images were taken with a camera. APG, apigenin; WT, wild-type; TDP1, tyrosyl-DNA phosphodiesterase 1.
Representative images of immunostaining in DT40 and TK6 cells. (A) Immunostaining of WT and Rad54-/- DT40 cells using an anti-γ-H2AX antibody and DAPI. Cells were treated with 30 μM APG or 100 nM CPT for 6 h (scale bar, 10 μM; magnification, x1,000). (B) Immunostaining of WT, Rad54-/- and TDP1-/- TK6 cells using an anti-γ-H2AX antibody and DAPI. Cells were treated with 80 μM APG or 20 nM CPT for 6 h (scale bar, 10 μM; magnification, x1,000). TDP1, tyrosyl-DNA phosphodiesterase 1; WT, wild-type; γ-H2AX, γ-phosphorylated H2A.X variant histone; APG, apigenin; CPT, camptothecin.
Effects of APG on proliferative ability and DNA damage in TDP1-/- TK6 cells. Proliferative ability of (A) APG- and (B) CPT-treated TK6 cells determined by colony formation assays. The x-axis represents the concentration of drugs and the y-axis represents the fractions of surviving colonies on a logarithmic scale. Survival data were log-transformed to approximate normality. Two-way analysis of variance was used to test for differences in the linear dose-response curves between WT and TDP1-/- cells after 72 h. *P<0.05 (C) Quantification of γ-H2AX foci in WT and TDP1-/- cells after treatment with 80 μM APG. (D) Quantification of γ-H2AX foci in WT and TDP1-/- cells after treatment with 20 nM CPT. TK6 cells were treated for 0, 3, 6 and 9 h; 100 cells were analyzed for each data-point. (E) APG induced formation of Top1cc in TK6 cells. TK6 cells were treated with 100 μM APG and 2 μM CPT for 2 h to detect Top1cc. Values are expressed as the mean ± standard deviation. The experiments were performed three times independently. *P<0.05, compared with WT after treatment for 3 h; ‡P<0.05, compared with WT after treatment for 6 h; #P<0.05, compared with WT after treatment for 9 h. APG, apigenin; CPT, camptothecin; WT, wild-type; Top1cc, topoisomerase I covalent protein DNA complex; TDP1, tyrosyl-DNA phosphodiesterase 1; γ-H2AX, γ-phosphorylated H2A.X variant histone.

Acknowledgements

The authors would like to thank Dr Hiroyuki Sasanuma (Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Koyot, Japan) for providing the complete protocol of in vitro complex of enzyme analysis.

Availability of data and materials

All data generated or analyzed during the present study are included in this published article.

Authors' contributions

YQ, XW, FH and ZZ conceived and designed the experiments. ZZ and CX performed the experiments. ZZ, YQ, XW, FH, CX, XF and XL analyzed the data. ZZ, YQ, XW, CX, JZ and XB contributed reagents/materials/analysis tools. ZZ, XW and YQ wrote the manuscript. ST was responsible for the generation of gene knock-out cells. XB and JZ developed the method for the ICE assay, verified the results and ensured that the results could be reproduced. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Spandidos Publications style
Zhao Z, Wu X, He F, Xiang C, Feng X, Bai X, Liu X, Zhao J, Takeda S, Qing Y, Qing Y, et al: Critical roles of Rad54 in tolerance to apigenin‑induced Top1‑mediated DNA damage. Exp Ther Med 21: 505, 2021
APA
Zhao, Z., Wu, X., He, F., Xiang, C., Feng, X., Bai, X. ... Qing, Y. (2021). Critical roles of Rad54 in tolerance to apigenin‑induced Top1‑mediated DNA damage. Experimental and Therapeutic Medicine, 21, 505. https://doi.org/10.3892/etm.2021.9936
MLA
Zhao, Z., Wu, X., He, F., Xiang, C., Feng, X., Bai, X., Liu, X., Zhao, J., Takeda, S., Qing, Y."Critical roles of Rad54 in tolerance to apigenin‑induced Top1‑mediated DNA damage". Experimental and Therapeutic Medicine 21.5 (2021): 505.
Chicago
Zhao, Z., Wu, X., He, F., Xiang, C., Feng, X., Bai, X., Liu, X., Zhao, J., Takeda, S., Qing, Y."Critical roles of Rad54 in tolerance to apigenin‑induced Top1‑mediated DNA damage". Experimental and Therapeutic Medicine 21, no. 5 (2021): 505. https://doi.org/10.3892/etm.2021.9936