Abstract
Background/Aim: Azoxystrobin (AZOX), a methoxyacrylate derivative, has potent antimicrobial and antitumor activities. Here, we report the anticancer effects of AZOX on the p53-negative human myelogenous leukemia cell line HL-60RG and the p53 positive human T-cell leukemia cell line MOLT-4F. Materials and Methods: Using both leukemia cells, the anticancer effect of AZOX treatment was analyzed throughout the cell cycle. Results: AZOX damaged both cell lines dose-dependently, and the cell damage rates were almost the same in both lines. Cell cycle distribution analysis showed that the treated MOLT-4F cells arrested at the S phase, whereas HL-60RG cells increased during the subG1 phase, suggesting that cell death was occurring. AZOX-induced cell death in HL-60RG was inhibited with the addition of uridine, which is used as a substrate for the salvage pathway of pyrimidine nucleotides. Conclusion: AZOX has p53-independent anticancer effects in leukemia cells, but the mechanisms underlying the damage differ between cell lines.
Mitochondria play an important role in the production of adenosine triphosphate (ATP) via oxidative phosphorylation. Because the high energy obtained by ATP hydrolysis is used for cellular proliferation, mitochondria are essential organelles for maintaining cell survival. With the progress of cancer research in recent years, mitochondria have been targeted as a cancer therapy strategy (1-4). This is because mitochondrial functions are essential for tumor initiation, growth, invasion, and metastasis (5-9). For example, treatment of glioblastoma multiforme cells with mahanine, a mitochondrial complex-III inhibitor, induced cell cycle arrest at the G0/G1 phase, resulting in the inhibition of cell proliferation (10). Furthermore, treatment of lymphoma T-cells (Jurkat cells) with pyrvinium, an anthelmintic drug approved by the Food and Drug Administration (FDA), induced apoptosis via the mitochondrial pathway (11). These findings indicate that the induction of mitochondria-mediated apoptosis can be utilized as a target pathway for cancer treatment.
It has been recently reported that azoxystrobin (AZOX) (Figure 1A), which is used as a systemic fungicide in agriculture, functions as a potential chemotherapeutic target drug for the treatment of cancer. This compound is a methoxyacrylate derived from the naturally occurring strobilurins (12). Upon treatment of fungal infections with AZOX, the ubiquinol oxidation (Qo) center of mitochondrial electron transport chain (mETC) complex III was inhibited through the cytochrome pathway (13). However, AZOX has been shown to have not only antifungal effects but also anticancer effects in cancer cells. For example, AZOX induced apoptosis of esophageal cancer cells via the mitochondrial pathway (14). Furthermore, the development of oral cancer was inhibited following AZOX treatment via the specific inhibition of mETC complex III activity and subsequently the induction of mitochondria-mediated apoptosis (15). Therefore, AZOX may be utilized as a novel therapy for human cancer cells.
Azoxystrobin (AZOX) induced damage in MOLT-4F and HL-60RG cells. (A) Chemical structure of AZOX. (B) Both cells were incubated with the indicated doses of AZOX for 24 h. Cell damage was estimated by the MTT assay. The data are presented as a comparison with the untreated control. Each bar denotes the standard deviation (SD; n=3).
Here, we report a novel anticancer effect of AZOX both in the human myelogenous leukemia cell line HL-60RG that does not express p53 and in the human T-cell leukemia cell line MOLT-4F that expresses p53. The anticancer effects of AZOX on leukemia cells with or without p53 were examined through the analysis of the distribution of cells in the various phases of the cell cycle.
Materials and Methods
Cells. The human T-cell leukemia cell line MOLT-4F was provided by the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). The human myelogenous leukemia cell line HL-60RG was provided by the Human Science Research Resources Bank (Osaka, Japan).
Chemicals and reagents. AZOX and uridine were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). All other reagents were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA), Nacalai Tesque (Kyoto, Japan), and FUJIFILM Wako Pure Chemical.
Medium and cell cultures. The cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Nichirei Corporation, Tokyo, Japan) and 75 mg/l kanamycin sulfate (FUJIFILM Wako Pure Chemical) and maintained at 37°C in a humidified chamber under an atmosphere of 95% air and 5% CO2.
Cell damage assay. The cells were incubated in 96-well plates at 37 °C with or without the test agents for 23 h. Then, 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Dojindo, Kumamoto, Japan) was added to each well, and the plates were incubated at 37 °C for 1 h. The media were discarded, and 100 μl of dimethyl sulfoxide (DMSO; FUJIFILM Wako Pure Chemical) was added to dissolve MTT formazan. The absorbance of each well was measured using a microplate reader (Awareness Technology, Palm City, FL, USA) at 570 nm. The absorbance of culture wells without the test agents was set as 100%.
Assessment of DNA content. Cells (1×106) were washed with phosphate-buffered saline (PBS) and suspended in permeabilizing buffer (0.1% Triton-X 100 in PBS). Then, 0.5 mg/ml RNase A and 2 μg/ml propidium iodide (PI; FUJIFILM Wako Pure Chemical) were added and flow cytometric analysis was conducted (BD FACS Calibur; Becton Dickinson, Mountain View, CA, USA). Data were analyzed using Cell Quest (Becton Dickinson).
Apoptosis DNA ladder assay. Cells (1×106) were washed with PBS and suspended in permeabilizing buffer A (10 mM Tris–HCl pH 7.8, 3 mM MgCl2, and 2 mM 2-mercaptoethanol). After centrifugation at 350 × g for 5 min, the pellet was incubated in lysis buffer [50 mM Tris–HCl, 10 mM EDTA-2Na, and 0.5% (w/v) sodium lauroyl sarcosinate (SLS), pH 7.8] supplemented with 1 mg/ml proteinase K at 50°C for 30 min. Then, the lysate was supplemented with 20 μg/ml RNase A, and incubated at 50°C for 15 min. Each lysate was mixed with TAE buffer containing 0.025% (w/v) bromophenol blue. The products were analyzed by electrophoresis at 100 V in 2% (w/v) agarose gel and stained with ethidium bromide.
Statistical analysis. All statistical analyses were performed using Student’s t-test. Significance was established at the p<0.05 level.
Results
The cytotoxic effect of AZOX on MOLT-4F and HL-60RG cells. The concentration-dependent effects of AZOX on MOLT-4F and HL-60RG cells were analyzed by MTT assay. The results show the cytotoxicity of AZOX after treatment of cells for 24 h. As shown in Figure 1B, the 50% inhibitory concentration (IC50) values were 33.4 μM for MOLT-4F and 31.5 μM for HL-60RG. Therefore, the cytotoxic effect of AZOX on HL-60RG cells was almost identical to that on MOLT-4F cells.
AZOX induced apoptosis in HL-60RG cells. The effects of AZOX in the distribution of HL-60RG cells in the different phases of the cell cycle were then analyzed by using flow cytometry. HL-60RG cells were treated with 0, 30, and 60 μM AZOX, and flow cytometry analysis was performed 24 h later. As shown in Figure 2, the number of HL-60RG cells in the subG1 phase increased as the AZOX concentration increased, suggesting the induction of cell death. To confirm this, apoptosis-induced DNA fragmentation was analyzed by DNA ladder assay (Figure 3). Fragmentation of genomic DNA was increased as AZOX concentration increased. Thus, apoptosis was induced in AZOX-treated HL-60RG cells.
Azoxystrobin (AZOX) inhibited HL-60RG cell proliferation by increasing the proportion of cells in the sub- G1 phase. (A) HL-60RG cells were incubated with 30 μM or 60 μM of AZOX for 24 h. The cells were assayed by using flow cytometry as described in Materials and Methods. Data are representative of three independent experiments. (B) Analyzed graph of (A). Each bar denotes the standard deviation (SD; n=3).
Azoxystrobin (AZOX) induced apoptosis in HL-60RG cells. HL-60RG cells were incubated with 30 μM or 60 μM of AZOX for 24 h. The cells were lysed and genomic DNA was extracted. DNA fragmentation was analyzed by agarose gel electrophoresis.
AZOX induced cell cycle arrest at the S phase in MOLT-4F cells. The effect of AZOX on the cell cycle in MOLT-4F cells was analyzed by using flow cytometry. The cells were treated with 0, 30, and 60 μM AZOX, and flow cytometry analysis was performed 24 h later. As shown in Figure 4, the number of cells in the subG1 phase did not increase significantly as the AZOX concentration increased. In contrast, the number of cells in the S phase increased with AZOX, suggesting the induction of cell cycle arrest through p53.
Azoxystrobin (AZOX) inhibited MOLT-4F cell proliferation by inducing S-phase arrest. (A) MOLT-4F cells were incubated with 30 μM or 60 μM of AZOX for 24 h. The cells were assayed by using flow cytometry as described in Materials and Methods. Data are representative of three independent experiments. (B) Analyzed graph of (A). Each bar denotes the standard deviation (SD; n=3).
Uridine inhibited AZOX-induced cell death. Human dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme catalyzing the fourth step in the de novo pyrimidine synthesis pathway (16, 17). DHODH converts dihydroorotate to orotate, generating electrons, which are transferred via redox cycling of ubiquinone to mETC complex III. Furthermore, AZOX has been previously reported to inhibit mETC complex III (18). Thus, AZOX-induced cell death in HL-60RG cells may be caused by the inhibition of the biosynthesis pathway of pyrimidine nucleotides. Here, we evaluated whether the lack of pyrimidine nucleotides induces cell death in AZOX-treated HL-60RG cells. HL-60RG cells treated with 0, 30, and 60 μM AZOX were cultured with or without the addition of uridine, and flow cytometry analysis was performed 24 h later. As shown in Figure 5, without AZOX, addition of uridine did not affect the number of cells in the subG1 phase. However, after treatment with 60 μM AZOX, the addition of uridine of the number of cells in the subG1 phase was approximately 5-fold lower than that without the addition of uridine. Thus, the addition of uridine significantly inhibited AZOX-induced cell death in HL-60RG cells.
Uridine inhibited azoxystrobin (AZOX)-induced DNA damage in HL-60RG cells. HL-60RG cells dosed with 30 μM or 60 μM of AZOX were incubated with 50 μg/ml of uridine for 24 h. The cells in the subG1 phase were assayed by flow cytometry as described in Materials and Methods. The data are presented as a comparison with the untreated control. Each bar denotes the standard deviation (SD; n=3). *p<0.05; ns: Non-significant.
Discussion
In this report, we analyzed the mechanism through which AZOX, a novel β-methoxyacrylate derivative, induced cell death in leukemia cells expressing or not expressing p53 (MOLT-4F and HL-60RG). AZOX treatment reduced the viability of MOLT-4F cells with an IC50 value almost identical to that of HL-60RG cells. Thus, AZOX inhibited the proliferation of leukemia cells regardless of the presence or absence of p53 expression. However, in the cell cycle distribution analysis, the treatment of HL-60RG cells with AZOX arrested cells at the subG1 phase, whereas MOLT-4F cells arrested at the S phase. Cell cycle arrest of HL-60RG cells at the subG1 phase was associated with the population of dead cells, suggesting that cell death progressed rapidly in the absence of p53. However, the cell cycle arrest at the S phase in the MOLT-4F cells is thought to result from the actions of both p53 and the cyclin-dependent kinase inhibitor (CDKi) p21, which are critical for p53-mediated G1/S cell cycle arrest (19, 20). Previous reports have shown that AZOX induced cell death by inhibiting the mECT complex III activity of the mitochondrial electron transport system in human cancer cells (14, 15, 18). Inhibition of mECT complex III inhibits the de novo biosynthesis of pyrimidine nucleotides, reducing the levels of these nucleotides, which are used as substrates for DNA replication, transcription, and other functions (21-25). Thus, the depletion of pyrimidine nucleotides, which are essential for cell survival, particularly inhibits cell survival in human cancer cells (16, 25). In support of this finding, we demonstrated that the induction of cell death in AZOX-treated HL-60RG cells following addition of uridine was 5-fold lower than that without the addition of uridine. Cellular uridine is synthesized intracellularly from UTP and CTP via the nucleotide salvage pathway and is then used as a substrate for transcription and other functions (22, 26-28). Our results suggest that the addition of uridine inhibited AZOX-induced death of HL-60RG cells. However, it has been reported that the inhibition of pyrimidine biosynthesis activates p53 (29). Thus, the induction of cell cycle arrest in AZOX-treated MOLT-4F cells may be induced by the activation of p53.
Acknowledgements
The Authors thank Asuka Higashiura, Yuki Uchiyama, and Yuta Semba for technical assistance.
Footnotes
Authors’ Contributions
ST, TS, and YN contributed to the design and implementation of the research, to the analysis of the results and to the writing of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received January 12, 2022.
- Revision received February 3, 2022.
- Accepted February 4, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.