Nutr Res Pract.  2018 Feb;12(1):41-46. 10.4162/nrp.2018.12.1.41.

Radioprotective effects of delphinidin on normal human lung cells against proton beam exposure

• Kim HM
• Kim SH
• Kang BS

• KMID: 2429017
• DOI: http://doi.org/10.4162/nrp.2018.12.1.41

Abstract

BACKGROUND/OBJECTIVES
Exposure of the normal lung tissue around the cancerous tumor during radiotherapy causes serious side effects such as pneumonitis and pulmonary fibrosis. Radioprotectors used during cancer radiotherapy could protect the patient from side effects induced by radiation injury of the normal tissue. Delphinidin has strong antioxidant properties, and it works as the driving force of a radioprotective effect by scavenging radiation-induced reactive oxygen species (ROS). However, no studies have been conducted on the radioprotective effect of delphinidin against high linear energy transfer radiation. Therefore, this study was undertaken to evaluate the radioprotective effects of delphinidin on human lung cells against a proton beam.
MATERIALS/METHODS
Normal human lung cells (HEL 299 cells) were used for in vitro experiments. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay assessed the cytotoxicity of delphinidin and cell viability. The expression of radiation induced cellular ROS was measured by the 2"²-7"²-dicholordihydrofluorescein diacetate assay. Superoxide dismutase activity assay and catalase activity assay were used for evaluating the activity of corresponding enzymes. In addition, radioprotective effects on DNA damage-induced cellular apoptosis were evaluated by Western blot assay.
RESULTS
Experimental analysis, including cell survival assay, MTT assay, and Western blot assay, revealed the radioprotective effects of delphinidin. These include restoring the activities of antioxidant enzymes of damaged cells, increase in the levels of pro-survival protein, and decrease of pro-apoptosis proteins. The results from different experiments were compatible with each to provide a substantial conclusion.
CONCLUSION
Low concentration (2.5 µM/mL) of delphinidin administration prior to radiation exposure was radioprotective against a low dose of proton beam exposure. Hence, delphinidin is a promising shielding agent against radiation, protecting the normal tissues around a cancerous tumor, which are unintentionally exposed to low doses of radiation during proton therapy.

Keyword

Delphinidin; radiation protective agent; proton therapy; reactive oxygen species

MeSH Terms

Apoptosis
Blotting, Western
Catalase
Cell Survival
DNA
Humans*
In Vitro Techniques
Linear Energy Transfer
Lung*
Pneumonia
Proton Therapy
Protons*
Pulmonary Fibrosis
Radiation Exposure
Radiation Injuries
Radiotherapy
Reactive Oxygen Species
Superoxide Dismutase
Catalase
DNA
Protons
Reactive Oxygen Species
Superoxide Dismutase

Figure

• 

  Fig. 1 Isodose curve for lung cancer radiotherapy. GTV, gross tumor volume (yellow); CTV, clinical target volume (red); PTV, planning target volume (blue); TV, treated volume (green); IV, irradiation volume (purple); OAR, organs at risk. In many cases, right from the planning stage, organs at risk, which is a normal tissue, are unavoidably included in the irradiation volume for cancer treatment.

• 

  Fig. 2 Chemical structure of delphinidin chloride (Molecular weight: 338.7 g/mol).

• 

  Fig. 3 Cytotoxicity of delphinidin in HEL 299 cells. The cells were treated with varying concentrations of delphinidin (0.01–5 µM/mL) in culture medium for 24 h. The cell viabilities were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Results are expressed as the percentages of control by means ± SD of eight independent experiments.

• 

  Fig. 4 Radioprotective effect of delphinidin against proton beam exposure. (A) ROS scavenger effects of delphinidin in proton-exposed HEL 299 cells. ROS scavenger effect of delphinidin was measured by the 2′-7′-dicholordihydrofluorescein diacetate (DCF-DA) assay. Results are expressed as the intensity of DCF-DA fluorescence. (B) Effects of delphinidin on superoxide dismutase (SOD) activity in proton-exposed HEL 299 cells. (C) Effects of delphinidin on catalase (CAT) activity in proton-exposed HEL 299 cells. The measured data are expressed as mean ± SD. Values with different letters are significantly different from each other at P < 0.05 by Duncan's multiple range tests. CG, Control group; EG, exposed group without pretreatment; EG-LDp, exposed group with low concentration of delphinidin; EG-HDp, exposed group with high concentration of delphinidin.

• 

  Fig. 5 Effects of delphinidin on the protein expression of Bcl-2, Bad, PARP-1, cleaved PARP-1 and cleaved caspase-3 in proton-exposed HEL 299 cells. Whole cell lysates were prepared and analyzed by western blotting using the specific antibodies Bcl-2, Bad, PARP-1, cleaved PARP-1 and cleaved caspase-3. Equal loading of protein was confirmed by stripping the blot and re-probing with β-actin antibody. Data shown here are from a representative experiment repeated three times with similar results.

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