Human breast cancer cell line, MCF-7, was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in DMEM (Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA) and 5% antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Cells were cultured at 37°C in a humidified atmosphere of 5% CO₂.

Stock solutions of 10 mM naphthazarin (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium to the indicated final concentration for cell treatment. Cells were incubated at 37°C overnight and then treated with naphthazarin (Naph). After 2 h, cells were exposed to gamma-rays from a 137Cs γ-ray source (Eckert & Ziegler, Berlin, Germany) at a dose rate of 2.6 Gy/min. Following IR at a 10 Gy dose, the cells were incubated under naphthazarin conditions for the indicated times.

Cell proliferation was assessed using the MTT colorimetric assay. MCF-7 cells (2×10⁵ cells/well) were seeded in 6-well plates and incubated at 37°C overnight (O/N). After 24 h of culture, the medium was removed and replaced with experimental medium. Cells were pretreated with Naph (respective concentration) before 2 h and then exposed to IR at a 10 Gy dose for 24 h. Subsequently, cells were washed twice with PBS and 5 mg/ml MTT in PBS was added to each well for 4 h. After removal of the MTT solution, a solubilization solution (DMSO/EtOH, 1:1 ratio) was added to each well to dissolve the formazan crystals. The absorbance at 570 nm was measured using a Paradigm™ Detection Platform (Beckman Coulter, Inc., Fullerton, CA, USA). For investigation of morphological changes, Naph or/and IR treated cells were examined under an inverted light microscope (Nikon) after 48 h.

Total RNA was isolated from the MCF-7 breast cancer cells using TRI-Solution (Bio Science Technology, Rockaway, NJ, USA) according to the manufacturer’s protocol. The quantity of isolated RNA was measured using NanoDrop (Thermo Scientific, Rockford, IL, USA) and 1 μg of RNA was reverse-transcribed using the iScript™ cDNA synthesis kit (Bio-Rad). The following qPCR primers were used: sense HDAC1 5′-TGGAAATCTATCG CCCTCAC -3′ and antisense HDAC1 5′-TCTCTGCATCTGCT TGCTGT-3′; sense DNMT1 5′-GAGCTACCACGCAGAC ATCA-3′ and antisense DNMT1 5′-CGAGGAAGTAGAA GCGGTTG-3′; sense UHRF1 5′-CTGGGGGATGATTCT CTGAA-3′ and antisense UHRF1 5′-CTCTTCCGTCTCA TGGGGT-3′; sense p21 5′-ATGGAACTTCGACTTTGTC ACC-3′ and antisense p21 5′-AGGCACAAGGGTACAA GACAGT-3′; sense β-actin 5′-AGCGAGCATCCCCCAAA GTT-3′ and antisense β-actin 5′-GGGCACGAAGGCTC ATCATT-3′.

Total cell lysates were loaded onto SDS-PAGE and transferred to PVDF (GE Healthcare Life Sciences, Piscataway, NJ, USA). The membranes were incubated overnight at 4°C with the primary antibodies. The following primary antibodies were used: anti-DNMT1 (Sigma-Aldrich), anti-UHRF1 (BD Bioscience), anti-HDAC1 (Abcam), anti-p53 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-p21 (Abcam) and anti-β-actin (Sigma-Aldrich). On the following day, the membranes were washed for 10 min, 3 times each, and incubated with the secondary antibody: polyclonal anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) or monoclonal anti-mouse antibody (Invitrogen). The immunoreactive proteins were detected using enhanced chemiluminescence (Thermo Scientific). Immunoblots were quantified using the ImageMaster densitometry program.

ChIP assays were performed using the Magna ChIP kit (Millipore) according to the manufacturer’s protocol. The following primers were used: sense p21 5′-TGGACTGGGCACTCTTGTCC -3′ and antisense p21 5′-CA-GAGTAACAGGCTAAGGTT-3′. Anti-DNMT1 (Sigma-Aldrich), anti-UHRF1 (BD Biosciences), anti-HDAC1 (Abcam) and anti-p53 (Santa Cruz Biotechnology Inc.) antibodies were used to immunoprecipitate chromatin fragments.

Cells were treated with 1 μM Naph or/and 10 Gy IR for 24 h. The cells were trypsinized and resuspended in PBS. Then, cells were centrifuged and washed in PBS. Following fixation in cold 70% ethanol for 30 min at 4°C, the cells were stained with PI (40 μg/ml) and RNAse A (50 μg/ml) prior to analysis. The stained cells were subjected to cell cycle analysis using FACSAria (BD Biosciences).

The Annexin V analysis was carried out using the PE Annexin V apoptosis detection kit (BD Biosciences). After treatment of 1 μM Naph or/and 10 Gy, cells were washed in cold PBS and then resuspended in 1X binding buffer and incubated with PE Annexin V (2.5 μg/ml)-conjugated primary antibody and 7-amino-actinomycin (7-AAD, 5 μl) for 15 min on ice. Following incubation, PI (10 μg/ml) was added to the suspension, and the cells were analyzed by FACSAria (BD Biosciences).

Since Naph and/or ionizing radiation (IR) modulate cell proliferation or viability, we first examined the regulatory effect of Naph and IR on cell growth of MCF-7 cells. We treated MCF-7 cells with Naph for 48 h and cell viability was measured by the MTT assay. The viability of MCF-7 cells treated with Naph was decreased in a dose-dependent manner (Fig. 1A). We used the 1 μM of Naph for further experiments to observe its synergistic effect with radiation. To investigate the synergistic effect of Naph and IR, MCF-7 cells were treated for 24, 48 and 72 h with Naph or 10 Gy of IR as a single treatment, or in combination. The results from the MTT assay showed that the combination of Naph and IR was more robust than that of Naph or IR single-treatment (Fig. 1B). MCF-7 cells typically exhibit a cobblestone-like appearance and tight cell-cell junction which is the characteristic of the epithelial phenotype (37,38), but when the cells were treated with Naph and IR, morphological changes and less number of prominent cells were observed (Fig. 1C).

Several studies have shown that the DNMT1/UHRF1/HDAC1 complex negatively regulates the expression of p21, which inhibits cell proliferation (31–36). Since Naph and IR decrease MCF-7 cell proliferation, we first determined the expression levels of p21 and its corepressors, DNMT1, UHRF1 and HDAC1 after treatment with Naph. We treated the MCF-7 cells with Naph at a concentration of 1, 3 and 5 μM for 24 h. The results of Real-time PCR indicated that Naph downregulated DNMT1, UHRF1 and HDAC1 mRNA expressions in a dose-dependent manner (Fig. 2A). In order to determine the combinatorial effect of Naph and IR on the expression of these genes, we treated the MCF-7 cells with Naph and IR under the indicated conditions. The MCF-7 cells were pre-incubated with Naph for 2 h prior to exposure to IR. The results showed that Naph significantly enhanced IR-induced p21 mRNA induction via decrease in DNMT1, UHRF1 and HDAC1 in MCF-7 cells (Fig. 2B). Since p21 is a downstream target of p53 which plays a key role in the regulation of cell cycle and apoptosis, we measured the protein levels of p53 and p21 together with levels of repressive factors including DNMT1, UHRF1 and HDAC1. In MCF-7 cells, the expression levels of p53 and its downstream p21 were increased after 10 Gy IR.

Furthermore, expression levels of p53 and p21 were enhanced in cells treated with a combination of 1 μM Naph and 10 Gy IR. On the contrary, the expression levels of DNMT1, UHRF1 and HDAC1 were decreased after 10 Gy of irradiation. The decreased expression levels of DNMT1, UHRF1 and HDAC1 were further reduced in cells treated with a combination of 1 μM Naph and 10 Gy IR (Fig. 2C). These data suggest that Naph is a potential radiosensitizer that increases the sensitivity to IR-induced cell death in breast cancer.

UHRF1 recruits transcriptional repressors DNMT1 and HDAC1 through its distinct domains. Moreover, it is known that UHRF1 recruits and cooperates with DNMT1 and HDAC1 on the promoter of p21, thereby inhibiting the expression of p21 (31–36). In order to investigate the occupancy of DNMT1/UHRF1/HDAC1 and p53 on the p21 promoter under various conditions such as Naph, IR and NaphsIR treatments, we performed the chromatin immunoprecipitation (ChIP) assay. As expected, binding of DNMT1, UHRF1 and HDAC1 to the p21 promoter after IR and Naph treatment gradually decreased when compared to that under untreated conditions (Fig. 3A–C). In contrast, the occupancy of p53 on the p21 promoter dramatically increased after combinatorial treatment of IR and Naph (Fig. 3D). Taken together, these results demonstrate that dissociation of DNMT1/UHRF1/HDAC1 from the p21 promoter during treatment with Naph and IR enhances the recruitment of p53 to the p21 promoter, thereby activating the transcription of p21 in MCF-7 cells.

Since we identified that mRNA and protein levels of cell cycle related p53 and p21 are regulated by Naph and/or IR, we next investigated whether Naph and IR induce cell cycle arrest in the MCF-7 cells using flow cytometry (FACSAria). In controls, flow cytometry analysis showed 1.5% of cells in G0/G1 phase, 40.3% of cells in S phase, and 17.9% of cells in G2/M phase. In contrast, in cells treated with 1 μM Naph, the proportions of cells in G0/G1, S, and G2/M phases were 0.8, 41.8 and 18.7%, respectively, and the proportions of cells exposed to 10 Gy IR in G0/G1, S, and G2/M phases were 3.9, 35.7 and 25.8%, respectively. When cells were treated with 1 μM Naph and IR, the proportions of cells in G0/G1, S, and G2/M phases were 1.6, 34.0 and 31.2%, respectively. These results suggest that combinatorial treatment of Naph and IR induces G2/M phase arrest compared with single treatment (Fig. 4A and B).

Next, we investigated whether Naph and IR induce MCF-7 cell apoptosis using the Annexin V/7-AAD double staining kit. The combinatorial effect of Naph and IR was evaluated after 48 h of treatment. Cells negative for 7-AAD and positive for PE Annexin V were regarded as early apoptotic cells (Annexin V⁺, 7-AAD⁻); 7-AAD and Annexin V positive cells were defined as late apoptotic cells (Annexin V⁺, 7-AAD⁺); 7-AAD positive and PE Annexin V negative cells were considered as necrotic cells (Annexin V⁻, 7-AAD⁺). The combined treatment of 1 μM Naph and 10 Gy IR in MCF-7 cells significantly increased the apoptotic effect compared with single treatment of IR or Naph. Increased number of late apoptotic cells were observed in the combined treatment group (43.9%) compared with the Naph alone group (15.6%) and IR alone group (38.7%). However, the cells treated with Naph and IR showed a slightly increased necrotic portion (7.3%) and there was a moderate decrease in the number of live cells (47.5%) in comparison with cells treated with Naph or IR treatment alone (Fig. 4C and D). These data suggest that combinatorial treatment of Naph and IR has a potential synergistic effect on the regulation of cell cycle arrest and apoptosis in MCF-7 cells.