Radioprotective effects of recombinant human epidermal growth factor (rhEGF) in C3H/HeJ mice

Dept. of Medical Science/박사[한글] 목 적: 본 연구에서는 방사선이 조사된 C3H/HeJ마우스에서 재조합표피성장인자(rhEGF)가 방사선에 의한 점막의 손상의 보호에 관여하는지를 알아보고자 하였다.대상 및 방법:　마우스 손상 모델은 C3H/HeJ 마우스에 전신방사선조사 (8, 10 Gy)하여 성립하였다. 실험군은 정상대조군, 10 Gy 방사선 단독그룹, rhEGF 단독투여그룹, 방사선조사와 rhEGF 투여병행군으로 각각 구분하였다. rhEGF의 투여는 1,2,3과 3.5일에 100 ㎍/kg 의 용량을 사용하여 복강에 주사 하였다. 소장점막의 병리 조직학적 검사를 위하여 H&E 염색을 시행하였다. 방사선에 의해 유도되는 세포고사는 DNA terminal transferase nick-end labeling assay 방법으로 Apoptatag kit를 사용하여 시행하였다. PCNA 발현정도를 면역조직화학염색을 통해 측정하였다.결과:　마우스 손상모델에서, 8 Gy이 10 Gy 그룹보다 마우스의 체중이 덜 감소하였다. H&E 염색에서 10 Gy 방사선 투여군의 소낭선세포가 24시간부터 감소하였다. Apoptosis index는 10 Gy 방사선 단독투여군에서 방사선 조사 후 24시간째 의의있게 증가되었다(p<0.05). 방사선조사와 rhEGF 투여병행군에서는 10 Gy 방사선 단독그룹에 비하여 방사선의 의한 세포고사가 감소하였고, 마우스의 체중 감소가 향상됨을 관찰 할 수 있었다.결론: 마우스 모델에서 rhEGF는 방사선 조사 후 소장의 점막손상을 효과적으로 회복시켰다. 점막의 회복을 촉진시키고, 점막을 보호하는 기전에 의하여 rhEGF는 방사선에 의한 세포고사를 감소시킨다. 결과적으로 rhEGF의 처치는 방사선조사 후의 마우스 소장 점막의 세포고사를 감소시켰다. [영문]ObjectIn order to investigate radioprotective effects of recombinant human epidermal growth factor (rhEGF) on radiation-induced mucosal damage in C3H/HeJ mice.Materials and MethodsThe radiation damage model was established that C3H/HeJ mice exposed to a single dose whole body irradiation of 8 Gy, 10 Gy. The groups of treatment model were divided into 4 groups: control, 10 Gy irradiation alone group, rhEGFalone group, and combination group of rhEGF and radiation. The rhEGF was administered 100 ㎍/㎏ intraperitoneally on day 1, 2, 3 and 3.5. Histologic examination was performed with H&E stain in jejunal mucosa. Radiation-induced apoptosis was determined in each group with the Apoptag kit: DNA terminal transferase nick-end labeling method. Tissue sections were evaluated for PCNA expression by immunohistochemical stain.ResultsIn the radiation damage model, the 8 Gy irradiated groups statistically had less weight loss compared to the 10 Gy irradiated group. The number of crypt cells was greatly decreased at 24h after 10 Gy in jejunum crypt by H&E stain. Apoptosis index of jejunum crypt in 10 Gy irradiated group was significantly increased at 24h after irradiation(p<0.05). In the treatment model, the combination group showed significantly improvement the reduction of weight loss and the number of radiation-induced apoptosis compared with 10 Gy irradiated group.ConclusionIt is suggested that rhEGF represents an effective strategy to reduce small intestine mucosal injury of radiation treatment in murine models. By promoting mucosal repair and protecting mucosal layer, rhEGF decreased radiation-induced apoptosis. In conclusion, rhEGF administered treatment decrease apoptosis of small intestine mucosal after the radiation exposure.ope

Enhancement of tumor radioresponse by wortmannin in C3H/HeJ hepatocarcinoma

The objective of this study was to explore whether a specific inhibitor of PI3K, wortmannin, could potentiate the antitumor effect of radiation in vivo, particularly on radioresistant murine tumors. C3H/HeJ mice bearing syngeneic hepatocarcinoma (HCa-I) were treated with 25 Gy radiation, wortmannin, or both. Wortmannin was administered intraperitoneally (1 mg/kg) once daily for 14 days. Tumor response to treatment was determined by a tumor growth delay assay. Possible mechanisms of action were explored by examining the level of apoptosis and regulating molecules. The expression of regulating molecules was analyzed by Western blot for p53 and p21WAF1/CIP1, and immunohistochemical staining for p21WAF1/CIP1, CD31 and VEGF. In the tumor growth delay assay, wortmannin increased the effect of tumor radioresponse with an enhancement factor (EF) of 2.00. The level of apoptosis achieved by the combined treatments was shown to be no more than an additive effect; peak apoptotic index was 11% in radiation alone, 13% in wortmannin alone, and 19% in the combination group. Markedly increased areas of necrosis at 24 h in the combination group were noted. Western blotting showed upregulation of p21WAF1/CIP1 in the combination treatment group, which correlated with low levels of VEGF. Microvascular density was evidently also reduced, based on low expression of CD31. In murine hepatocarcinoma, the antitumor effect of radiation was potentiated by wortmannin. The mechanism seems to involve not only the increase of induced apoptosis but also enhanced vascular injury. Wortmannin, in combination with radiation therapy, may have potential benefits in cancer treatment.ope

A novel combination treatment of armed oncolytic adenovirus expressing IL-12 and GM-CSF with radiotherapy in murine hepatocarcinoma

In this study, a novel combination treatment of armed oncolytic adenovirus expressing interleukin 12 (IL-12) and granulocyte-macrophage colony-stimulating factor (GM-CSF) with radiation was investigated for antitumor and antimetastatic effect in a murine hepatic cancer (HCa-I) model. Tumor bearing syngeneic mice were treated with radiation, armed oncolytic virus Ad-ΔE1Bmt7 (dB7) expressing both IL-12 and GM-CSF (armed dB7), or a combination of both. The adenovirus was administered by intratumoral injection 1 × 10(8) PFU per tumor in 50 µl of PBS four times every other day. Tumor response to treatment was determined by a tumor growth delay assay. Metastatic potential was evaluated by a lung metastasis model. To understand the underlying mechanism, the level of apoptosis was examined as well as the change in microvessel density and expression of immunological markers: CD4+, CD8+ and Cd11c. The combination of armed dB7 and radiation resulted in significant growth delay of murine hepatic cancer, HCa-1, with an enhancement factor of 4.3. The combination treatment also resulted in significant suppression of lung metastasis. Increase of apoptosis level as well as decrease of microvessel density was shown in the combination treatment, suggesting an underlying mechanism for the enhancement of antitumor effect. Expression of immunological markers: CD4+, CD8+ and Cd11c also increased in the combination treatment. This study showed that a novel combination treatment of radiotherapy with armed oncolytic adenovirus expressing IL-12 and GM-CSF was effective in suppressing primary tumor growth.ope

Recombinant human epidermal growth factor (rhEGF) protects radiation-induced intestine injury in murine system

This study was to investigate whether rhEGF protects radiation induced intestine injury without compromising antitumor effect of radiation in murine system. A radiation induced intestinal injury model was established in mice by whole body irradiation. Using this model, 4 groups were set; control, rhEGF (100 µg/kg intraperitoneally), radiation (10 Gy), and a combination (rhEGF and radiation). The level of apoptosis and proliferation were analyzed by TUNEL assay and proliferation cell nuclear antigen (PCNA) immunohistochemical staining, respectively, as well as observation of survival and body weight change. A tumor growth delay assay was performed using murine syngeneic tumors; one radioresistant tumor, HCa-I and one radiosensitive tumor, MCa-K. In the radiation induced intestinal injury model, the 10 Gy group had significantly more weight loss with less number of crypt cells and higher apoptosis than the 8 Gy group. Using 10 Gy model, radioprotective effect of rhEGF was tested. Addition of rhEGF improved not only the body weight loss but also survival following radiation. It also induced suppression of apoptosis as well as increase of PCNA expression and recovery of villi. rhEGF did not enhance the tumor growth after radiation exposure in the tested tumors. These findings suggest that combination of exogenous rhEGF and radiation can be a new anticancer strategy by protecting radiation-induced intestinal injury without alleviating antitumor effect of radiationope

Identification of proteins indicating radiation-induced hepatic toxicity in cirrhotic rats

Radiation therapy (RT) has been emerging as one of the palliative treatments for locally advanced hepatocellular carcinoma (HCC). However, hepatic toxicity is a major obstacle in radiotherapy for HCC. The purpose of this study is to identify proteins indicating radiation-induced hepatic toxicity in cirrhotic rats, which can be used as possible biomarkers. Liver cirrhosis was induced in Wistar rats with thioacetamide (TAA) 0.3 g/L in drinking water for 9 weeks. The development of liver cirrhosis was observed histologically. Radiation hepatic injury was induced by treating 1/3 of the liver with 10 Gy single dose radiation. To find out commonly expressed proteins, liver tissue and serum were analyzed using two-dimensional electrophoresis and quadrupole time of flight mass spectrometry. Identified proteins were validated using western blotting. Histological examination showed that the degree of hepatic fibrosis increased by radiation in liver cirrhosis. It was associated with a decrease in the proliferation of cell nuclear antigen and an increase of apoptosis. The proteomic analysis of liver tissue and serum identified 60 proteins which showed significant change in expression between the TAA-alone and TAA-plus-radiation groups. Among these, an increase of heparanase precursor and decrease of hepatocyte growth factor were shown commonly in liver tissue and serum following radiation. Hepatic fibrosis increased following radiation in cirrhotic rats. These proteins might be useful in detecting and monitoring radiation-induced hepatic injury.ope