Effect of epicatechin against radiation-induced oral mucositis: in vitro and in vivo study

Abstract

Purpose: Radiation-induced oral mucositis limits the delivery of high-dose radiation to head and neck cancer. This study investigated the effectiveness of epicatechin (EC), a component of green tea extracts, on radiation-induced oral mucositis in vitro and in vivo.

Experimental design: The effect of EC on radiation-induced cytotoxicity was analyzed in the human keratinocyte line HaCaT. Radiation-induced apoptosis, change in mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation and changes in the signaling pathway were investigated. In vivo therapeutic effects of EC for oral mucositis were explored in a rat model. Rats were monitored by daily inspections of the oral cavity, amount of oral intake, weight change and survival rate. For histopathologic evaluation, hematoxylin-eosin staining and TUNEL staining were performed.

Results: EC significantly inhibited radiation-induced apoptosis, change of MMP, and intracellular ROS generation in HaCaT cells. EC treatment markedly attenuated the expression of p-JNK, p-38, and cleaved caspase-3 after irradiation in the HaCaT cells. Rats with radiation-induced oral mucositis showed decreased oral intake, weight and survival rate, but oral administration of EC significantly restored all three parameters. Histopathologic changes were significantly decreased in the EC-treated irradiated rats. TUNEL staining of rat oral mucosa revealed that EC treatment significantly decreased radiation-induced apoptotic cells.

Conclusions: This study suggests that EC significantly inhibited radiation-induced apoptosis in keratinocytes and rat oral mucosa and may be a safe and effective candidate treatment for the prevention of radiation-induced mucositis.

Figure 1. Effect of epicatechin on viability of the HaCaT cells after treatment with radiation.

The HaCaT cells were exposed to a single dose of radiation (20 Gy), various concentrations of EC (0–200 µM) or radiation plus EC. At 5 days interval after irradiation, cell viability was measured by MTT assay. The data represent the mean ± SD of three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, compared to radiation alone.

Figure 2. Protective effect of epicatechin against radiation toxicity in HaCaT cells.

(A) TUNEL assay of treated HaCaT cells. EC reduced radiation-induced apoptosis of HaCaT cells. Scale bar = 50 µm. (B) Quantification of percentage of apoptosis seen in flow cytometry of Annexin V-FITC and PI double stained HaCaT cells. The data represent the mean ± SD of three independent experiments. ** p<0.01, by one-way ANOVA.

Figure 3. Effect of epicatechin on radiation-induced mitochondrial membrane potential in the HaCaT cells.

(A) Cells were irradiated or not (20 Gy) in the absence or presence of EC (100 µM), and stained with JC-1 (red fluorescence). Increase of green fluorescence in irradiated cells suggest loss of MMP. (B) The MMP change was objectively measured using the flow cytometry FACScan. The data represent the mean ± SD of three independent experiments. ** p<0.01, *** p<0.001. Scale bar = 50 µm.

Figure 4. Effect of epicatechin on radiation-induced intracellular ROS generation in HaCaT cells and radiation-induced NOX3 expression in rat oral mucosa.

(A) Intracellular ROS generation was measured in HaCaT cells treated with 20 Gy of radiation in the presence or absence of EC (200 µM). The level of intracellular ROS was measured by FACScan flow cytometry using the peroxide sensitive fluorescent probe, DCFDA. (B) The results (mean ± SD) were calculated as a percent of the control group (not exposed to radiation). The data represent the mean ± SD of five independent experiments. ** p<0.01. (C) Immunohistochemistry of NOX-3 in control, radiation alone, EC alone and radiation plus EC treated rat oral mucosa sections from each experimental group were stained with anti-NOX-3 antibody. Scale bar: 100 µm.

Figure 5. Inhibition of radiation-induced phosphorylation of MAPKs and caspase-3 dependent pathway by epicatechin treatment in HaCaT cells.

The cells were pretreated with 200 µM of EC followed by the addition of 20 Gy radiation. Immunoblot of treated HEI-OC1 cells stained with antibodies against p38, JNK, ERK, and cleaved caspase-3.

Figure 6. Effect of epicatechin on oral intake and body weight of irradiated rats.

(A) Effect of EC on rat oral intake. On the 1^st day after irradiation, the oral intake of irradiated rats began to decrease. The oral intake in the rats in the RT+EC group started to increase on the 9^th day after irradiation and was markedly increased on the 11^th day. The mean oral intake of the RT+EC group was similar to that of the control group between the 11^th and 23^rd day (P = 0.09, Mann-Whitney U test). (B) Effect of EC on rat body weight after irradiation. On 2^nd day after irradiation, the body weight of irradiated rats began to decrease. The RT+EC group displayed a statistically significant lower weight loss than the RT group on the 5^th, 6^th and 7^th day (*p<0.05). The rats in the RT+EC group started to gain weight on the 10^th day and reached their original weight on the 17^th day. Weight gain velocity was not significantly different as compared with the control group (p = 0.75).

Figure 7. Effect of epicatechin on histopathologic findings of irradiated rats.

(A) Gross photographic images and histopathologic images of oral mucosa on the 4^th day after irradiation. Each paraffin-embedded specimen was sectioned at a thickness of 5 µm. (H&E staining: x100 and x400). Tongue tip ulceration (arrow). (B) The gross photographic images and histopathologic images (H&E staining: x100 and x400) of oral mucosa on the 9^th day after irradiation. (C) The gross photographic images and histopathologic images (H&E staining: x100 and x400) of oral mucosa on the 23^rd day after irradiation.

Figure 8. Protective effect of epicatechin against radiation in rat oral mucosa, as indicated by TUNEL staining for apoptosis.

(A) The 4^th day after irradiation, (B) The 9^th day after irradiation, (C) The 23^rd day after irradiation. TUNEL staining of rat mucosa revealed EC treatment significantly decreased radiation-induced TUNEL-positive cells (arrow). Scale bar = 50 µm.