Yonsei Med J.  2014 Jul;55(4):886-894. 10.3349/ymj.2014.55.4.886.

A Novel Synthetic Compound 3-Amino-3-(4-Fluoro-Phenyl)-1H-Quinoline-2,4-Dione (KR22332) Exerts a Radioprotective Effect via the Inhibition of Mitochondrial Dysfunction and Generation of Reactive Oxygen Species

• Baek SJ
• Chang JW
• Park KH
• Yang GY
• Hwang HS
• Koh YW
• Jung YS
• Kim CH

• KMID: 2130811
• DOI: http://doi.org/10.3349/ymj.2014.55.4.886

Abstract

PURPOSE
Acute side effects of radiation such as oral mucositis are observed in most patients. Although several potential radioprotective agents have been proposed, no effective agent has yet been identified. In this study, we investigated the effectiveness of synthetic compound 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332) as a radioprotective agent.
MATERIALS AND METHODS
Cell viability, apoptosis, the generation of reactive oxygen species (ROS), mitochondrial membrane potential changes, and changes in apoptosis-related signaling were examined in human keratinocyte (HaCaT).
RESULTS
KR22332 inhibited irradiation-induced apoptosis and intracellular ROS generation, and it markedly attenuated the changes in mitochondrial membrane potential in primary human keratinocytes. Moreover, KR22332 significantly reduced the protein expression levels of ataxia telangiectasia mutated protein, p53, and tumor necrosis factor (TNF)-alpha compared to significant increases observed after radiation treatment.
CONCLUSION
KR22332 significantly inhibited radiation-induced apoptosis in human keratinocytes in vitro, indicating that it might be a safe and effective treatment for the prevention of radiation-induced mucositis.

Keyword

Radiation induced oral mucositis; 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332); apoptosis; radioprotection

MeSH Terms

Apoptosis/drug effects/physiology
Cell Line, Tumor
Cell Survival/drug effects/physiology
Humans
Keratinocytes/metabolism
Membrane Potential, Mitochondrial/drug effects/physiology
Radiation-Protective Agents/chemistry/*pharmacology
Reactive Oxygen Species/metabolism
Radiation-Protective Agents
Reactive Oxygen Species

Figure

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  Fig. 1 Structure of 3-amino-3-(4-fluoro-phenyl)-1H-quinoline-2,4-dione (KR22332).

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  Fig. 2 Effect of KR22332 on HaCaT cell viability after radiation. HaCaT cells were exposed to various doses of radiation (0-20 Gy) or to various concentrations of KR22332 (0-100 µg/mL) with or without radiation (8 Gy). At 72 h after radiation, cell viability was measured by an MTT assay. (A) Radiation decreased cell viability in an intensity-dependent manner. (B) KR22332 alone did not show significant toxic effects on the cells until 10 µg/mL. (C) Cells were pretreated with a 8 Gy single dose of radiation followed by treatment with 1, 10, and 50 µg/mL KR22332 for 72 h. KR22332 significantly protected HaCaT cells from radiation-induced cytotoxicity in a dose-dependent manner, until 10 µg/mL. The data represent mean±SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001.

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  Fig. 3 Effect of KR22332 on the viability of the HNSCC cell line after treatment with radiation. (A) KR22332 did not have protective influence on the viability of HN3 cell. (B) HN3 cell line were pre-treated with 8 Gy single dose of radiation, followed by treatment with 1, 10, and 50 µg/mL KR22332 for 72 h. Then, cell viability was measured by a MTT assay. Radiation significantly decreased cell viability on HN3 cells. KR22332 treatment did not decrease anticancer effect of irradiation on the cell line. The data represent mean±SD of three independent experiments. **p<0.01, ***p<0.001. NS, not significant.

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  Fig. 4 Effect of KR22332 on radiation-induced apoptosis in HaCaT cells. (A) Apoptosis in HaCaT cells was determined by the TUNEL method using an in situ cell detection kit. After monolayers achieved 60-70% confluence, the cells were exposed to radiation (8 Gy), then treated with KR22332 (10 µg/mL). The cells were incubated with 50 µL of TUNEL reaction mixture (TdT and fluorescein-dUTP) and stained with Hoechst 33258 (5 µg/mL). The stained cells (arrow) were then observed under a fluorescence microscope. The TUNEL assay confirmed that radiation induced TUNEL-positive cells (arrow), while KR22332 decreased the number of TUNEL-positive cells. Scale bar=50 µm. (B) To quantify the effects of KR22332 on radiation-induced apoptosis, we used flow cytometry; annexin V-FITC and PI staining were used to analyze the percentage of apoptotic cells in radiation-treated cells (8 Gy) in the absence or presence of KR22332 (10 µg/mL) (upper). The percentage of apoptosis in each fraction is expressed as a graph (lower). The data represent mean±SD of three independent experiments. *p<0.05, ***p<0.001. DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling; V-FITC, V-fluorescein isothiocynate; PI, propidium iodide.

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  Fig. 5 Effect of KR22332 on the MMP in irradiated HaCaT cells. (A) Cells were treated with KR22332 after irradiation (8 Gy) or not, stained with JC-1, and visualized under a fluorescence microscope. KR22332 alone did not affect the MMP of cells. KR22332 stabilized the MMP in radiation-treated cells. (B) The change in the MMP was measured objectively using FACScan. The data represent mean±SD of three independent experiments. Scale bar=50 µm. *p<0.05. NS, not significant; MMP, mitochondrial membrane potential; FACS, fluorescence-activated cell sorting.

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  Fig. 6 Effect of KR22332 on radiation-induced ROS generation in HaCaT cells. Cells were treated with radiation (8 Gy) or KR22332 (5 µg/mL) for 72 h. The level of intracellular ROS was then measured by flow cytometry using the peroxide-sensitive fluorescent probe DCFDA. The results were calculated as a percent of the control group (not exposed to radiation). Radiation significantly increased the generation of intracellular ROS. KR22332 clearly inhibited radiation-induced intracellular ROS generation. To compare the inhibitory effect of a known inhibitor, 10 mM NAC was used to inhibit ROS generation. The data represent mean±SD of three independent experiments. *p<0.05; **p<0.01. NS, not significant; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; DCFDA, 5-(and 6)-carboxyl-20,70-dichlorodihydro fluorescein diacetate.

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  Fig. 7 Effect of KR22332 on radiation-induced apoptosis and the phosphorylation of ATM-p53 and TNF-α in HaCaT cells. The cells were incubated for 72 h after treatment with/without radiation and KR22332. Cell lysates were collected, electrophoresed through an SDS-polyacrylamide gel, and subjected to Western blot analysis with antibodies against cleaved caspase-3, cleaved PARP, ATM, p53, and TNF-α. KR22332 reduced the phosphorylation of ATM, p53 (Ser 15 and 46), and TNF-α that had been augmented by irradiation in HaCaT cells. TNF, tumor necrosis factor; PARP, poly (ADP-ribose) polymerase; ATM, ataxia telangiectasia mutated; HSP, heat shock protein.

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