Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells

  • Authors:
    • Min Young Kim
    • Seong-Joon Park
    • Jae Woong Shim
    • Kwangmo Yang
    • Ho Sung Kang
    • Kyu Heo
  • View Affiliations

  • Published online on: January 28, 2015     https://doi.org/10.3892/ijo.2015.2857
  • Pages: 1659-1666
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Abstract

Naphthazarin (Naph, DHNQ, 5,8-dihydroxy-l,4-naphthoquinone) is one of the naturally available 1,4-naphthoquinone derivatives that are well-known for their anti-inflammatory, antioxidant, antibacterial and antitumor cytotoxic effects in cancer cells. Herein, we investigated whether Naph has effects on cell cycle arrest and apoptosis in MCF-7 human breast cancer cells exposed to ionizing radiation (IR). Naph reduced the MCF-7 cell viability in a dose-dependent manner. We also found that Naph and/or IR increased the p53-dependent p21 (CIP/WAF1) promoter activity. Noteworthy, our ChIP assay results showed that Naph and IR combined treatment activated the p21 promoter via inhibition of binding of multi-domain proteins, DNMT1, UHRF1 and HDAC1. Apoptosis and cell cycle analyses demonstrated that Naph and IR combined treatment induced cell cycle arrest and apoptosis in MCF-7 cells. Herein, we showed that Naph treatment enhances IR-induced cell cycle arrest and death in MCF-7 human breast cancer cells through the p53-dependent p21 activation mechanism. These results suggest that Naph might sensitize breast cancer cells to radiotherapy by enhancing the p53-p21 mechanism activity.

Introduction

Breast cancer is a major health problem in women worldwide and the main cause of cancer death among women. Many breast cancer patients fail conventional treatment strategies of chemotherapy, radiation and anti-estrogen therapy (1,2). Therefore, the search for new and effective drugs to treat breast cancer is required. Research into the molecular pathway and biomarkers associated with the development of breast cancer is needed to find successful therapeutic approaches.

Plant-derived drugs play an increasingly important role in cancer therapy due to their low toxicity and high efficacy. Natural products derived from plants, such as camptothecin, vincristine, taxol, etoposide and paclitaxel, have received extensive attention as potential anticancer drugs. These bioactive phytochemicals are known to exert anticancer activity through different mechanisms, including immune activation, suppression of cell cycle progression and induction of apoptosis (311). Naphthazarin (Naph, DHNQ, 5,8-dihydroxy-l,4-naphthoqui-none) is a naturally occurring 1,4-naphthoquinone derivative; a lipophilic red pigment like alkannin and shikonin. Naph derivatives are well-known for their anti-inflammatory, antioxidant, antibacterial antifungal and wound healing effects. They are also known for antitumor cytotoxic effects in cancer cells (1216). The mechanism of antitumor cytotoxicity and the specific molecular target of Naph in cancer cells are yet to be established, but they probably involve the induction of differential expression of cell cycle regulators. Cell cycle progression is governed by cyclin-dependent kinases (CDKs) that are activated by cyclin binding and inhibited by CDK inhibitors (17,18). P21 is a CDK inhibitor (CKI) that plays a crucial role in arresting cellular growth, differentiation and apoptosis. P21 gene expression is induced by the p53 gene, thereby directly mediating p53-induced cell cycle arrest (1924).

UHRF1 (Ubiquitin-like containing PHD and ring finger domains 1), also known as ICBP90 or Np95, is a multi-domain protein associated with cellular proliferation and epigenetic regulation. UHRF1 binds to methylated CpG dinucleotides and recruits the transcriptional repressors DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1) to regulate gene expression (2530). In addition, UHRF1 forms a complex with DNMT1/HDAC1 and binds to the promoter regions of tumor suppressor genes such as p16INK4A, p14ARF and p21 in cancer cells (3136). In this study, we demonstrated that treatment with Naph and irradiation (IR) downregulates the expressions of DNMT1, UHRF1 and HDAC1, whereas it upregulates the expression of p21 in MCF-7 cells. Moreover, cell cycle arrest and apoptosis were significantly increased by combinatorial treatment of Naph and IR. Collectively, our results suggest that Naph might be an effective radiosensitizer and adjuvant therapy for breast cancer.

Materials and methods

Cell and culture conditions

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.

Drug treatment and IR exposure

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 assay and cell morphology

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.

RNA isolation and quantitative real-time PCR

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′.

Western blot analysis

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.

Chromatin immunoprecipitation (ChIP)

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.

Cell cycle analysis

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).

Apoptosis analysis

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).

Results

Naph and IR inhibit proliferation of human MCF-7 breast cancer cells

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).

Naph and IR upregulate p21 expression and downregulate the expression of DNMT1, UHRF1 and HDAC1

Several studies have shown that the DNMT1/UHRF1/HDAC1 complex negatively regulates the expression of p21, which inhibits cell proliferation (3136). 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.

DNMT1/UHRF1/HDAC1 complex and p53 are reciprocally localized on the p21 promoter in MCF-7 cells under treatment with Naph and IR

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 (3136). 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.

Combination of Naph and IR induces G2/M cell cycle arrest and apoptosis 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.

Discussion

Radiotherapy is used in over 50% of patients during the course of cancer treatment both as a curative modality and for palliation. However, radioresistance and toxic side effects impeding dose escalation are major obstacles to the success of radiation therapy (39,40). Therefore, there is a critical need for the discovery of a novel radiosensitizer that can improve the efficacy of radiotherapy. Several studies have reported that plant-derived natural products have cancer chemopreventive and chemotherapeutic properties. The use of natural products as antitumor agents or radiosensitizers for the management of human cancers is an attractive idea because they are readily available and exhibit little or no toxicity (311).

Naph is a natural component of the roots of several members of the genus Boraginaceae and is well-known to have anti-tumor cytotoxic effects in cancer cells (1216). However, the biological role and mechanism of cytotoxicity and the specific molecular target of Naph in cancer cells are yet to be established. In this study, we demonstrated the role and mechanism of Naph as an effective radiosensitizer and adjuvant therapy in breast cancer. We first evaluated the effect of Naph and IR on the proliferation of MCF-7 breast cancer cells. The results showed that the growth of MCF-7 cells treated with Naph was decreased in a concentration-dependent manner (Fig. 1A).

To diminish the toxic side effect of Naph, we used a low dose (1 μM) of Naph for further experiments to observe its synergistic effect with radiation. Indeed, we observed that the viability of cells treated with a low concentration (1 μM) of Naph and IR of 10 Gy was decreased more than that of MCF-7 cells treated with single Naph and 10 Gy of IR. We also demonstrated that the cells treated with Naph and IR of 10 Gy showed morphologic changes and less number of the prominent cells than untreated cells (Fig. 1C). Therefore, these results suggest that combinational treatment of Naph and IR more effectively inhibits cancer cell viability and proliferation than a single treatment.

Several studies have reported that DNMT1/UHRF1/HDAC1 complex negatively regulates the expression of p21, which inhibits cell proliferation (3136). To determine the mechanism of antitumor cytotoxicity and the specific molecular target of Naph, we next examined the expression of p21 and its regulatory factors (DNMT1/UHRF1/HDAC1). Our results of Real-time PCR showed that Naph downregulated DNMT1, UHRF1, and HDAC1 mRNA expressions in a dose-dependent manner (Fig. 2A). Moreover, we identified that the expression level of p21 was dramatically upregulated by co-treatment of Naph and IR, whereas the expression levels of DNMT1, UHRF1, and HDAC1 were downregulated (Fig. 2B and C).

It is well documented that p21 is a downstream target of p53, which regulates transcription, DNA repair, cell cycle arrest, differentiation, senescence, genomic instability, apoptosis, and survival as well as glucose metabolism, oxidative stress, and angiogenesis (3134). Therefore, we measured the protein levels of p53 and p21 together with levels of repressive factors, including DNMT1, UHRF1, and HDAC1. Our results showed that the expression levels of p53 and its downstream p21 were enhanced in cells co-treated with 1 μM Naph and 10 Gy IR compared to Naph or IR alone treated cells (Fig. 2C). These results support that Naph increases the sensitivity to IR-induced cell cycle arrest or apoptosis through the p53-p21 pathway in breast cancer. In order to identify the regulatory mechanism of p21 expression, we performed the ChIP analysis. Our ChIP results revealed that the repressive factors, DNMT1/UHRF1/HDAC1 were dissociated from the p21 promoter by treatment with Naph and IR compared with that under untreated conditions. On the contrary, the occupancy of p53 on the p21 promoter was significantly increased after combinational treatment of Naph and IR (Fig. 3). Therefore, these results demonstrate that DNMT1/UHRF1/HDAC1 complex and p53 are reciprocally localized on the p21 promoter under treatment with Naph and IR, thereby regulating the transcription of p21 in MCF-7 cells.

Deregulation of the cell cycle and apoptosis are frequent occurrences in cancer development. In this study, p53 and its target gene, p21, were dependently regulated by Naph and/or IR. We investigated whether Naph and IR induce cell cycle arrest in the MCF-7 cells using flow cytometry. The cells treated with 1 μM Naph or 10 Gy IR alone showed a slight increase in the proportion of cells in G2/M phase compared with the untreated cells. However, the cells treated with 1 μM Naph and 10 Gy IR together showed a significant increase in the proportion of cells in G2/M phase compared with the cells treated with Naph and IR alone (Fig. 4A and B). These results suggest that Naph and IR induce G2/M phase arrest. Moreover, the radiosensitizing effect of Naph was assessed through the apoptosis assay. Our data showed that 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 (Fig. 4C and D).

In conclusion, our findings show that combinational treatment of Naph and IR dramatically inhibits cell proliferation. Furthermore, the 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 inducing cell cycle arrest and apoptosis in MCF-7 cells. These findings lead us to believe that Naph might be a potential radiosensitizer in breast cancer (Fig. 5).

Acknowledgements

This study was supported by the National R&D Program through the Dongnam Institute of Radiological & Medical Sciences (DIRAMS) (50595–2014) and the [Basic Science Research Program] through the National Research Foundation of Korea (NRF) (NRF-2014 M2A2A 7043665) funded by the Ministry of Science, ICT and Future Planning.

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April-2015
Volume 46 Issue 4

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Spandidos Publications style
Kim MY, Park S, Shim JW, Yang K, Kang HS and Heo K: Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells. Int J Oncol 46: 1659-1666, 2015
APA
Kim, M.Y., Park, S., Shim, J.W., Yang, K., Kang, H.S., & Heo, K. (2015). Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells. International Journal of Oncology, 46, 1659-1666. https://doi.org/10.3892/ijo.2015.2857
MLA
Kim, M. Y., Park, S., Shim, J. W., Yang, K., Kang, H. S., Heo, K."Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells". International Journal of Oncology 46.4 (2015): 1659-1666.
Chicago
Kim, M. Y., Park, S., Shim, J. W., Yang, K., Kang, H. S., Heo, K."Naphthazarin enhances ionizing radiation-induced cell cycle arrest and apoptosis in human breast cancer cells". International Journal of Oncology 46, no. 4 (2015): 1659-1666. https://doi.org/10.3892/ijo.2015.2857