THE CANCER PREVENTIVE SEED PEPTIDE LUNASIN FROM RYE IS BIOAVAILABLE AND BIOACTIVE : 호밀로부터 암 예방적 종자 펩타이드(lunasin)의 생물학적 이용성과 생물학적 특성에 관한 연구

다국어 초록 (Multilingual Abstract)

Lunasin is a unique 43-amino acid peptide that has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model against oncogenes and chemical carcinogens. In search for new sources of lunasin and to better understand the role of cereals in cancer prevention, we report here the properties of lunasin from rye. Lunasin content was screened in 15 cultivars of rye. Bioavailability was measured by in vitro digestibility assay using pepsin and pancreatin and feeding rats with lunasin-enriched rye (LER). Inhibition of HAT (histone acetyl transferase) and nuclear localization in mammalian cells were used to measure lunasin bioactivity. Lunasin present in rye crude protein preparation is stable to pepsin and pancreatin in in vitro digestion. The liver, kidney and blood of rats fed lunasin-enriched rye (LER) show the presence of lunasin in western blot. Lunasin extracted from these tissues inhibits the activities of HATs confirming that the peptide is intact and bioactive. When added exogenously, lunasin purified from rye internalizes in the nuclei of mouse fibroblast cells. We conclude that lunasin in rye is bioavailable and bioactive and that consumption of rye may play an important role of cancer prevention in rye-consuming populations.

국문 초록 (Abstract)

43개의 독특한 아미노산 구조를 가진 항암 펩타이드인 lunasin은 발암유전자 와 화학적 발암물질에 의해 야기되는 동물세포 및 마우스 피부암 유발을 억제한다고 보고되었다.
본 논문은 lunasin을 함유한 새로운 원천을 찾기 위해 화본과 작물인 호밀로부터 lunasin을 동정하고, 분리 정제된 lunasin의 생물학적 특성에 대해 보고한다. 생물학적 안정성과 유용성은 pepsin과 pancreatin을 이용한 in vitro digestibility assay와 lunasin 고 함유 호밀 단백질 (LER)이 구강투여 된 마우스를 이용하여 평가 하였다. Lunasin의 화학 방어적 항암활성은 histone acetyl transferase인 yGCN5와 p/CAF의 억제활성 및 동물세포의 핵 내 내재력을 통하여 평가하였다. 호밀 내 lunasin은 동물 소화효소인 pepsin과 pancreatin에 분해되지 않고 안정하였고, lunasin 고 함유 호밀 단백질 (LER)이 구강투여 된 마우스의 간, 신장 및 혈액에서 lunasin이 검출 되었다. 그리고 호밀과 동물 조직으로부터 분리, 정제된 lunasin은 histone acetyl transferase(HAT)인 yGCN5와 p/CAF의 활성에 대해 강력한 억제활성을 보였으며, 동물세포 와 동물 조직내 핵과 결합하였다.
본 연구를 통해 호밀 종자 내 lunasin은 생물학적으로 안정되고 항암 활성을 가진다고 사료된다. 또한 호밀의 섭취는 화학방어적 항암활성에 있어서 중요한 역할을 하리라 사료된다.

목차 (Table of Contents)

• Ⅰ. Introduction = 1
• Ⅱ. Materials and Methods = 3
• 1. Materials = 3
• 1-1. Reagents = 3
• 1-2. Seeds = 3
• 2. Methods = 4
• 2-1. Isolation, purification and identification of lunasin from rye = 4
• 2-2. In vitro digestion with SGF and SIF of lunasin in crude protein extracted from rye = 6
• 2-3. Isolation and purification of lunasin from tissues of rats fed with lunasin-enriched rye (LER) = 6
• 2-4. Inhibition assay of HAT activity = 7
• 2-5. Immunostaining of lunasin in internalization experiment = 8
• Ⅲ. Results = 10
• 1. Screening of lunasin peptide from plants of Gramineae origin = 10
• 2. Contents of lunasin from rye cultivars = 12
• 3. Purification and Identification of Lunasin = 14
• 4. Effect of rye lunasin on HAT activities = 16
• 5. Expression of lunasin and inhibition of HAT activity in developing rye seeds = 18
• 6. In vitro digestion of rye crude protein with SIF and SGF = 20
• 7. Immunostaining of rye lunasin in mouse fibroblast cell line NIH 3T3 = 22
• 8. Bioactivity of lunasin purified from the blood, kidney and liver of rats fed with LER = 24
• Ⅳ. Discussion = 27
• Ⅴ. References = 29
• Korean Summary = 32