Cancer Res Treat.  2020 Oct;52(4):1019-1030. 10.4143/crt.2020.012.

Protective Effects of N-Acetylcysteine against Radiation-Induced Oral Mucositis In Vitro and In Vivo

• Kim HJ
• Kang SU
• Lee YS
• Jang JY
• Kang H
• Kim CH

• KMID: 2507929
• DOI: http://doi.org/10.4143/crt.2020.012

Abstract

Purpose
Radiation-induced oral mucositis limits delivery of high-dose radiation to targeted cancers. Therefore, it is necessary to develop a treatment strategy to alleviate radiation-induced oral mucositis during radiation therapy. We previously reported that inhibiting reactive oxygen species (ROS) generation suppresses autophagy. Irradiation induces autophagy, suggesting that antioxidant treatment may be used to inhibit radiation-induced oral mucositis.
Materials and Methods
We determined whether treatment with N-acetyl cysteine (NAC) could attenuate radiation-induced buccal mucosa damage in vitro and in vivo. The protective effects of NAC against oral mucositis were confirmed by transmission electron microscopy and immunocytochemistry. mRNA and protein levels of DNA damage and autophagy-related genes were measured by quantitative real-time polymerase chain reaction and western blot analysis, respectively.
Results
Rats manifesting radiation-induced oral mucositis showed decreased oral intake, loss of body weight, and low survival rate. NAC intake slightly increased oral intake, body weight, and the survival rate without statistical significance. However, histopathologic characteristics were markedly restored in NAC-treated irradiated rats. LC3B staining of rat buccal mucosa revealed that NAC treatment significantly decreased the number of radiation-induced autophagic cells. Further, NAC inhibited radiation-induced ROS generation and autophagy signaling. In vitro, NAC treatment significantly reduced the expression of NRF2, LC3B, p62, and Beclin-1 in keratinocytes compared with that after radiation treatment.
Conclusion
NAC treatment significantly inhibited radiation-induced autophagy in keratinocytes and rat buccal mucosa and may be a potentially safe and effective option for the prevention of radiation-induced buccal mucosa damage.

Keyword

Radiation; Oral mucositis; N-acetylcysteine (NAC); Autophagy; Nuclear factor erythroid 2-related factor 2 (NRF2)

Figure

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  Fig. 1. Effect of N-acetylcysteine (NAC) on DNA damage in the HaCaT cells after radiation treatment. (A) Western blot analysis of signals mediating cell cycle checkpoint and DNA damage. Cell lysates were collected 24 hours after irradiation and NAC treatment, followed by gel electrophoresis, and the levels of p-ATM (Ser1981), total ATM (t-ATM), p21, p-p53 (Ser15), total p53 (t-p53) cyclin B1, γH2AX, and alpha-tubulin were measured. A representative of three experiments is shown in triplicate. (B) Cell cycle analysis by flow cytometry. The distribution of each HaCaT cell line in various stages of the cell cycle was analyzed by propidium iodide staining after radiation and NAC treatment. (C) Cyclin A/cyclin B1 mRNA level was measured using real-time polymerase chain reaction. Asterisks indicate statistically significant differences. *p < 0.05. (D) Immunofluorescence of γH2AX (green spot). Cells were exposed to a single dose of radiation (20 Gy), NAC (10 mM) or radiation+NAC. After 24-hour incubation, immunocytochemistry was performed with an antibody targeting γH2AX, indicative of the cellular response to DNA damage. This experiment was independently repeated at least three times. Scale bars=75 μm. ***p < 0.001.

• 

  Fig. 2. Effect of N-acetyl cysteine (NAC) on radiation-induced intracellular reactive oxygen species (ROS) generation in HaCaT cells. (A) Intracellular ROS generation was measured in HaCaT cells treated with 20 Gy of radiation in the presence or absence of NAC (10 mM). The level of intracellular ROS was measured by flow cytometry using the peroxide-sensitive fluorescent probe, dihydroethidium (DHE). (B) Intracellular ROS generation was evaluated by DHE fluorescence staining for 30 minutes at 37°C. Values are presented as the mean±SD of three experiments in triplicate and was calculated as a percentage of the control. ***p < 0.001. (C) NAC downregulates radiation-induced nuclear factor erythroid 2-related factor 2 (NRF2) protein expression. Western blots were performed using NRF2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies. (D) Protein levels of NRF2 in fractionated nuclear or cytosolic lysates treated with 20 Gy of radiation in the presence or absence of NAC (10 mM) were determined by western blot analysis. (E) NRF2 protein levels were detected by immunocytochemistry. Cells were exposed to a single dose of radiation (20 Gy), NAC (10 mM) or radiation+NAC. After 24-hour incubation, immunocytochemistry was performed with an NRF2 antibody. (F) NRF2 mRNA level was measured using real-time polymerase chain reaction. Asterisks indicate statistically significant differences. *p < 0.05.

• 

  Fig. 3. Protective effect of N-acetyl cysteine (NAC) against radiation-induced autophagy in the HaCaT cells. (A) Western blot analysis with antibodies against Beclin-1, p62, and LC3B. (B) Immunocytochemistry staining of LC3B (green), F-actin (red), and nucleus (blue) in cells exposed to a single dose of radiation (20 Gy), NAC (10 mM) or radiation+NAC for 24 hours. LC3B puncta-positive cells were counted in five random fields (n=3). Scale bars=100 μm. Values are presented as mean±standard deviation (SD). ***p < 0.001. (C) Transmission electron microscopy analysis of morphological changes in autophagic vesicles in HaCaT cells. Scale bars=5,000 nm. (D) Quantitative real-time polymerase chain reaction was used to quantify the effect of NAC on radiation-induced autophagy-related genes, ATG5, LC3B, and p62. Results are presented as mean±SD of three independent experiments. *p < 0.05.

• 

  Fig. 4. Protective effect of N-acetyl cysteine (NAC) against histopathologic effects in irradiated rats. (A) Photographic images of buccal mucosa on day 23 after irradiation. Rats in the radiation group were found to have more tongue mucositis (the tip of the tongue is not fully healed). However, the MUCOMIST(NAC) treatment group is clean except for the tip of the tongue. (B) Survival rate and time of death. There was no statistical significance between the treatment and non-treatment groups. (C) Histopathologic images (H&E staining, ×400) of buccal mucosa and tongue on day 23 after irradiation. Scale bars=200 μm.

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  Fig. 5. Protective effect of N-acetyl cysteine (NAC) against irradiation of rat tongue. (A) Immunohistochemical analysis. Rat tongue sections from each experimental group (control, MUCOMIST, radiation, radiation+MUCOMIST) were stained with LC3B and NRF2 antibody. Scale bars=200 μm. (B) NRF2 and LC3B expression was detected by western blot. All the western blotting experiments were performed under the same conditions. (C) Relative expression levels of mRNA of NRF2 and LC3B were determined by real-time polymerase chain reaction analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

Reference

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