The Preventive Effect of Recombinant Human Growth Factor (Rhegf) on the Recurrence of Radiodermatitis

J. Radiat. Res. Regular Paper

The Preventive Effect of Recombinant Human Growth Factor (rhEGF) on the Recurrence of Radiodermatitis

Seung-Hee RYU1, Yeun Hwa KIM2, Sang-wook LEE1 and Joon Pio HONG2*

Recombinant human epidermal growth factor (rhEGF)/Irradiation/ Radiodermatitis/ Recurrence. The effects of topical application of recombinant human epidermal growth factor (rhEGF) on wound healing and the recurrence of radiodermatitis were assessed in the irradiated skin of BALB/c Nu/Nu mice. Mice irradiated with 45 Gy of radiation were divided into 5 groups and treated with 10, 50, and 100 μg/g rhEGF ointment, vehicle alone, or no treatment (control) for 6 months. Wounds were observed initially in all groups and complete healing time (HT100) for initial wound repair did not differ significantly among groups. However, the rate of recurrence over 6 months was significantly lower in the EGF-treated groups than in the control group (p < 0.05). Histological examination showed that treatment with the optimum dose of EGF (50 μg/g) accelerated normal wound healing when compared with the higher dose of EGF (100 μg/g), vehicle alone, or no treatment, with the latter group showing irregular epidermal thickness, poor definition of epidermis and dermis, and unstable dermal structure. Collagen distribution was also significantly increased in mice treated with 50 μg/g rhEGF (p < 0.05) compared with the control or vehicle- treated group. Taken together, these results indicate that treatment with exogenous EGF (50 μg/g dose) can enhance radiation-induced wound repair while preserving structural tissue stability and preventing the recurrence of radiodermatitis.

their ability to prevent radiation-induced skin toxicity and INTRODUCTION increase wound healing; these include topical steroid creams, washing practices, aqueous creams, aloe vera gel, Radiation is often used to treat malignant tumors, either chamomile, almond cream, sucralfate cream, and amifos- alone or in combination with surgery and/or chemotherapy. tine,2,5–7) but none has proven optimal for treating radiation- Despite the advantage of radiotherapy and the improvements induced skin damage. Recently, various growth factors have in techniques, many patients experience moderate to severe been applied in attempts to manipulate the environment of side effects, including xerostomia, diarrhea, mucositis, der- wounds and to enhance wound healing.2) Key elements in matitis, ulceration, and fibrosis.1) Rapidly dividing cells such wound healing, including platelet-derived growth factor as those in the skin, bone marrow and gastrointestinal tract (PDGF), transforming growth factor-β (TGF-β), fibroblast are easily damaged by radiation.2) Ionizing radiation gener- growth factor (FGF), keratinocyte growth factor (KGF), and ates highly reactive free radicals, which damage cells, even epidermal growth factor (EGF), can trigger tissue regenera- in adjacent normal tissue, especially in the skin covering the tion and the remodeling process.8–11) Despite their potential entire body.3) Delayed wound healing of irradiated skin can efficacy in the wound healing process, there have been few cause other severe complications, such as skin necrosis and studies on the wound healing capacity of growth factors, ulceration,2) and non-cancerous skin damage is considered a especially in radiodermatitis. Recombinant human PDGF- dose-limiting factor of radiotherapy that can impair patient BB may be used to treat selected individuals with refractory, quality of life.4) Various interventions have been tested for poorly healing, dermal wounds caused by irradiation,12) and the combination of a topical recombinant PDGF gel and hydrophilic copolymer membranes was found to ameliorate *Corresponding author: Phone: +82-2-3010-3105, chronic ulcerating radiodermatitis.13) Topical and local

Fax: +82-2-3010-3037, application of granulocyte-macrophage colony-stimulating E-mail: [email protected] 1Department of Radiation Oncology, University of Ulsan College of factor (GM-CSF) can facilitate rapid healing of radiation- 14,15) Medicine, Asan Medical Center, Seoul, Korea; 2Department of Plastic induced ulcers or dermatitis, and EGF has been reported Surgery, University of Ulsan College of Medicine, Asan Medical Center, to accelerate recovery from oral or intestinal mucosal dam- Seoul, Korea. age induced by irradiation in animal models.16,17) Our group doi:10.1269/jrr.10010

J. Radiat. Res. Advance Publication. This article may be modified at the final publication. 2 S.-H. Ryu et al. has reported that EGF stimulated epithelialization of a opinion of two trained researchers. Recurrence was assessed chronic radiation ulcer in a patient who had a mastectomy in all groups for up to 6 months. EGF or vehicle ointment and radiation therapy 23 years previously.18) These studies, was applied until the termination of the experiment. however, have been conducted in few patients, with very limited conditions. Moreover skin wounds resulting from Evaluation of morphological changes irradiation often recur after long periods of time due to At sacrifice, the dorsal buttock skins of the mice were abnormal pathological changes during the early stages of fixed in 10% neutral buffered formalin and embedded in par- healing. We hypothesized that EGF treatment of the radia- affin. The tissue sections were stained with hematoxylin and tion-induced skin wounds would enhance the normal healing eosin (H&E) and Masson’s trichrome reagent (Dako, CA, process and lower the recurrence rate without abnormal USA) to evaluate morphological changes. The percentage of pathologic changes. We therefore tested the effects of 6 trichrome stained area was calculated with an image analyzer months of rhEGF treatment on radiation-induced dermatitis at 100× magnification. and recurrence in BALB/c Nu/Nu mice. All values were expressed as mean ± standard deviation, and the Student’s t-test was used for comparisons between MATERIALS AND METHODS vehicle and treatment groups.

Experimental animals and radiation RESULTS Male BALB/c Nu-Nu mice (6 weeks old, average body weight 20 ± 2 g) were obtained from OrientBio Inc. (Republic 100% healing time (HT100) of Korea) and adapted for 1 week before experiments. Mice Acute radiodermatitis was effectively induced in BALB/c were immobilized during irradiation using a mixture of Nu-Nu mice within 1 week after 45 Gy of fractionated irra- ketamine (100 mg/kg) and xylazine (10 mg/kg) injected diation. Complete wound healing time, expressed as mean intramuscularly. The buttock regions of these anesthetized HT100 was 12.3 ± 1.5 days in the control group, 12.33 ± 2.1 mice were irradiated with a total of 45 Gy over 3 consecutive days in the vehicle-treated group, and 12–15 days in the days (15 Gy per day) with a 4 MV photo beam generated by three rhEGF-treated groups (Fig. 1). a linear accelerator (CLINAC 600C, Varian, Palo Alto, CA, USA) at dose rate of 2 Gy/min. During irradiation, the bod- Recurrence of radiodermatitis ies of the mice were covered with a 1.5-cm thick bolus to Measurements of the frequency of recurrence of radioder- provide an adequate radiation dose build-up on the skin sur- matitis for 6 months after irradiation showed that, in the con- face. Irradiated mice were subsequently divided randomly trol and vehicle-treated groups, radiodermatitis recurred into 5 groups of 6 mice per group and treated with 10, 50, 3.33 ± 0.58 and 3.00 ± 1.00 times per mouse, respectively and 100 μg/g rhEGF ointment, vehicle alone, or no treatment (control) for 6 months. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Asan Medical Center, University of Ulsan.

EGF treatment Human recombinant EGF (Nepidermin®) ointment was provided by Daewoong® Pharmaceutical Company (Seoul, Republic of Korea). Briefly, EGF was mixed with ointment base at concentrations of 10, 50, and 100 μg/g, and 0.1 g of each ointment was topically applied to the skin twice daily, at 10 AM and 4 PM, after irradiation. The quantities of EGF applied on each application to each wound area were 1, 5, and 10 μg, respectively.

Measurement of 100% healing time (HT100) and frequency of recurrence Fig. 1. Comparison of 100% healing time (HT100) of radiation- Digital images of the irradiated skin areas of each mouse induced skin wounds treated with various concentration of rhEGF. were taken every day, and evaluated for epithelialization Following irradiation, 0.1 g of ointment containing various concen- with an image analyzer (Image measurement Standard trations of EGF or vehicle alone was topically applied to the but- v4.01, Bersoft, Puerto Plata, Dominican Republic) to assess tock regions twice daily for 6 months. Optical images were used to healing time. HT100 was defined as the time of full epithe- evaluate epithelialization and to calculate HT100. Each bar shows lialization of a wound, as determined by the single-blinded the mean ± standard deviation for that group.

J. Radiat. Res. Advance Publication. This article may be modified at the final publication. rhEGF Inhibits the Recurrence of Radiodermatitis 3

to assess collagen distribution in the dermis. Both the control and vehicle-treated groups showed irradiation-induced loss of collagen fibers in the dermal area (Fig. 4). Topical treatment with 50 μg/g EGF ointment increased collagen deposition, with specimens from this group showing much more dense dermal structure than any of the other groups. Treat- ment with low dose (10 μg/g) EGF ointment resulted in smoother collagen distribution than in either the control or vehicle-treated groups, whereas treatment with high-dose (100 μg/g) EGF ointment resulted in a pattern similar to that of the control group. When we measured the percentages of trichrome stained areas in the samples, relative to total area, we found positive staining in 26.58 ± 7.89% and 23.79 ± 4.62% of the control and vehicle-treated tissue specimens, respectively. Treatment with 10 μg/g EGF slightly increased Fig. 2. Effect of rhEGF on the rate of radiodermatitis recurrence ± over 6 months. Topical treatment with rhEGF ointment signifi- the trichome stained area, to 33.26 10.69%, but the differ- cantly inhibited the recurrence of skin wounds after initial wound ence was not statistically significant, and treatment with μ healing, compared with the control and vehicle-treated groups. high-dose (100 g/g) EGF resulted in positive staining sim- There was no difference between the latter two groups. Each bar ilar to that observed in control- or vehicle-treated skin. In shows the mean ± standard deviation for that group, with p values contrast, treatment with 50 μg/g EGF markedly and signifi- shown over each bar. cantly increased collagen deposition, to 41.36 ± 10.47% (p < 0.05).

(Fig. 2). EGF treatment, however, significantly reduced DISCUSSION recurrence rates, to 0.25 ± 0.50 times per mouse in the group treated with 10 μg/g EGF ointment (p < 0.001 compared We have shown here that topical application of rhEGF for with the control group), 0 in the group treated with 50 μg/g 6 months after irradiation with 45 Gy decreased the recur- EGF ointment (p < 0.001), and 1.25 ± 0.96 times per mouse rence of radiodermatitis and normalized epidermal structure in the group treated with 100 μg/g EGF ointment (p < 0.05) in mouse skin. This was confirmed by histopathological (Fig. 2). analysis, in that mice treated with 50 μg/g EGF did not show the characteristics of radiation injured skin, such as loose Histopathological evaluations connections between the epidermal and subcutaneous layers, To assess morphological changes, skin biopsies were pre- thin epidermal layers, and irregular dermal structures. These pared and examined after H&E staining. All wounds were findings also indicate that topical treatment with EGF accel- re-epithelialized and covered with stratum corneum and epi- erates normal wound healing by activating cell proliferation dermal cells. Irradiated skin samples showed impairments in and epidermal regeneration in radiation-induced skin the epidermal layer and disorganized compartments between wounds. the epidermis and dermis, as well as loose connections Radiation-induced skin damage often occurs in non-can- between the dermis and subcutaneous layer (Fig. 3A). Vehicle- cerous tissues as a direct result of ionizing radiation.1) Early treated skin showed stable features of the granular and adverse effects of irradiation may include erythema, pig- spinous layers but the thickness of the epidermal layer was mentation, epilation, dry and moist desquamation, and ero- irregular (Fig. 3B). In contrast, skin treated with the lowest sions, whereas the late adverse effects of irradiation may dose of EGF (10 μg/g) showed more stable morphology of include aggravated wound reactions such as ulceration, der- the epidermis and dermis, despite showing irregular epidermal necrosis, atrophy, telangiectasia, and fibrosis.19) mal thickness (Fig. 3C). Skin treated with 50 μg/g EGF oint- Impaired wound healing is often seen in late stages after ment showed characteristics typical of stratified squamous irradiation and may remain chronic, with delayed or non epithelium and formed dense collagen fibers (Fig. 3D). healing of ulcerations and infections.2) Because wound heal- Unexpectedly, the highest dose of EGF tested (100 μg/g) ing is a complex process involving multiple cells, inflamma- showed patterns similar to that of the irradiated control tory mediators, cytokines, growth factors, and various other group, including disorganized compartments between the factors, manipulating this process remains a challenge. granular, spinous and basal layers in the epidermis. Although the healing process that occurs following radiation-induced damage is not the same as that following inci- Collagen distribution sional wounds, the two processes share several steps such as Skin samples were also stained with Masson’s trichrome epidermal regeneration, fibroblast proliferation, and col-

J. Radiat. Res. Advance Publication. This article may be modified at the final publication. 4 S.-H. Ryu et al.

Fig. 3. Histopathological evaluation of mouse skin samples. Skin samples were stained with H&E staining and photographed at × 200 magnification. A: Irradiated control group, B: vehicle-treated group, C: 10 μg/g EGF-treated group, D: 50 μg/g EGF-treated group, E: 100 μg/g EGF-treated group. Bold-headed arrows indicate irregular epithelium and thin arrows indicate the thickness of the epidermis and dermis.

Fig. 4. Representative images of collagen deposition, as assessed by Masson’s trichrome staining. Images captured under × 100 magnification. A: Irradiated control group, B: vehicle-treated group, C: 10 μg/g EGF-treated group, D: 50 μg/g EGF-treated group, and E: 100 μg/g EGF-treated group. Collagen fibers were stained blue.

J. Radiat. Res. Advance Publication. This article may be modified at the final publication. rhEGF Inhibits the Recurrence of Radiodermatitis 5

findings indicate that the ability of 50 μg/g of EGF ointment to completely inhibit the recurrence of radio-dermatitis was due to an efficient and proper healing process during the initial stages of wound repair. These findings were confirmed by histopathological examination of skin biopsy samples from these mice. Six months after irradiation, tissue samples from control and vehicle-treated mice showed severe alterations in skin structure, including an impaired epidermal layer, irregular dermal structure, and unstable subcutaneous layer, although the skin surfaces appeared relatively normal. In contrast, specimens from mice treated with 50 μg/g EGF ointment showed normal skin structure, characterized by thick epithelium, a def- inite delineation of the epidermis from the dermis, and a Fig. 5. Percentage of collagen deposition in irradiated skin densely composed dermis. Moreover, decreased collagen 1) wounds treated with rhEGF. The percentage of the trichrome deposition, one of the long-term effects of radiotherapy, stained area in each sample was determined using an image ana- was observed in control and vehicle-treated mice, but not in lyzer. Each bar represents mean ± standard deviation. Collagen EGF-treated mice. The dense extracellular matrix of the deposition was significantly increased in the 50 μg/g EGF-treated reticular dermis is accomplished by fibroblasts within the group compared with the control group (p < 0.05). dermis.30) Collagen, the main extracellular matrix compo- nent, is responsible for the strength and elasticity of the skin.1) Because collagen is synthesized by fibroblasts, the lagen deposition. Thus materials or agents that increase cell impairment of fibroblasts in radiation-induced wounds will proliferation may be promising candidates for wound heal- have a negative impact on collagen synthesis, resulting in ing. Growth factors may be effective in wound repair, due to delayed wound healing. These results indicate that EGF their ability to stimulate cell proliferation and their ability to treatment stimulates the proliferation of fibroblasts that chemo-attract immune system cells.2) The EGF treatment actively synthesize collagen during the wound healing pro- regimen described here has been used in wound care, both cess and further strengthens dermal structure. Although in vitro20) and in vivo.21–24) In clinical trials, EGF has been accumulated collagen may be considered a histological fea- found to accelerate the wound healing time of partial thick- ture of fibrosis, collagen synthesis is also the key step in ness skin,25) and to have positive effects on diabetic ulcer- wound repair and possibly a major factor in achieving long- ations and chronic skin wounds.26–28) However, the effects of term stability of skin structure. EGF on radiation-induced skin wounds have not yet been Another goal of this study was to determine the dose tested clinically. range of EGF inducing the most efficient response. Different We found that the initial irradiation-induced wounds concentrations of EGF have been used in other animal stud- developed equally in all five groups, with no differences in ies. For example, 0.1–10 μg/ml of EGF induced a therapeu- 23) the time to achieve complete healing (HT100). Wound recur- tic response in a partial thickness burn pig model although rence rates, however, were significantly decreased in all treatment with 10 μg/ml of rhEGF has been reported to three rhEGF-treated groups compared with the control and delay wound healing in an excisional wound model vehicle-treated groups. Because recurrence or sudden break- compared with treatment with 0.01 to 1 μg/ml EGF.31) We down of the skin may appear clinically long after irradiation, previously reported that the most efficient dose facilitating we used a 6-month model to assess the effects of long-term the re-epithelialization of partial thickness wounds was 1–5 treatment.1) The average number of recurrences per mouse in μg/g rhEGF.21) These divergent results may have been due the control and vehicle-treated groups were 3.33 ± 0.58 and to differences in types of wounds and species. Human 3.00 ± 1.00, showing the strength of this radiation wound recombinant EGF may have a reduced affinity to mouse than model. Undesirable changes in the skin during radiotherapy to human or pig EGF receptors; thus, a higher dose would may alter the concentrations or balance of immune-related be required in mice to induce the same therapeutic activity. cytokines. For example, the concentrations of PDGF and However, doses greater than the optimum range worsen the EGF were decreased in chronic wounds, suggesting that wound healing process. For example, high concentrations of these deficiencies may be related, at least in part, to impaired rhEGF did not show any positive effects in radiation-induced wound healing.23,29) In addition, radiation-induced fibroblast oral or intestinal mucositis,16,17) and we have shown here that dysfunction may be responsible for poor wound healing. mice treated with the highest dose of EGF (100 μg/g) Thus, better wound healing can reduce the incidence of late showed similar morphological patterns to those in irradiated, complications resulting from early wound repair steps. Our untreated control mice. Concentrations of growth factors

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