미생물학회지 (Korean Journal of Microbiology)

  • 제42권4호
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  • Pages.319-325
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  • 2006
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지

아가리쿠스로부터 분리한 $\beta$-glucan과 그 올리고당류의 HT-29 인체 대장암 세포에 대한 항암 활성에 관한 연구

Study on the Anti-HT-29 Human Colon Cancer Activity of $\beta$-Glucans and Their Enzymatically Hydrolyzed Oligosaccharides from Agalicus blazei Murill

  • 이미영 (인제대학교 의생명공학대학 임상병리학과) ;
  • 김기훈 (인제대학교 의생명공학대학 임상병리학과) ;
  • 김예운 (인제대학교 의생명공학대학 임상병리학과) ;
  • 장헌길 (인제대학교 의생명공학대학 임상병리학과) ;
  • 이동석 (인제대학교 의생명공학대학 임상병리학과)
  • Lee, Mi-Young (Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University) ;
  • Kim, Ki-Hoon (Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University) ;
  • Kim, Yea-Woon (Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University) ;
  • Chang, Hun-Gil (Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University) ;
  • Lee, Dong-Seok (Department of Biomedical Laboratory Science and Biohealth Products Research Center, Inje University)
  • 발행 : 2006.12.30
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초록

칡을 첨가하여 배양한 아가리쿠스버섯(Agaricus blaxei Murill)으로 부터 열수추출, gel filtration chromatography, DEAE ion exchange chromatography를 통하여 아가리쿠스 $\beta$-glucan (AG)을 추출하였다. 추출한 아가리쿠스 $\beta$-glucan에 Bacillus megaterium 유래의 endo-$\beta$-(1$\rightarrow$6)-glucanase를 처리하여 올리고당류(AO)를 얻었다. 이렇게 얻은 AG와 AO를 이용하여 HT-29 인체 대장암 세포에 대한 항암 활성을 조사한 결과, 암세포의 성장 억제 효과는 시료의 처리 농도에 의존적으로 증가하였으며, apoptosis assay에서 암세포의 apoptosis 유발이 농도에 의존적으로 증가되었다. 또한, 암세포의 세포 주기를 분석한 결과, apoptosis 발생을 뜻하는 G0 (sub-G1)기와 G1기의 비율이 증가한 반면 S기와 G2/M기는 대조군에 비해 감소되었다. 이러한 결과를 바탕으로 암세포의 apoptosis 증가에 대한 AO의 작용이 어떤 유전자와 연관이 있는지를 알아보기 위하여 cDNA microarray를 통해 유전자의 발현율을 검색한 결과, apoptosis의 내부 외부 경로에 영향을 주는 유전자(TNESE9, TNFRSF9, FADD, CASP8, BAD, CRADD, CASP9 등)의 발현이 증가되었고 세포 분열 주기의 진행과 관련된 유전자(CCND2와 CDK2)의 발현이 감소되었으며, 세포 분열 주기를 지체시키는 유전자 (CDKN2A)의 발현은 증가되었다. 또한, 사이토카인을 암호하는 유전자 (IL6, IL18, IL6R 등)와 tumor suppressor와 관련된 유전자 (CEACAM1, TP53BP2, IRF1및 PHB)의 발현이 2배 이상 증가된 것을 확인할 수 있었다. 따라서 HT-29 인체 대장암세포에 대한 AO의 성장 억제 작용은 G0/Gl기를 지체시켜 암세포 증식을 억제하고 apoptosis에 의해 암세포를 사멸시키는 항암 활성을 나타내는 것으로 확인되었으며, 특허 AO가 AG보다 현저한 활성을 보였다. 더 나아가 아가리쿠스 $\beta$-glucan (AG)과 올리고당류 (AO)는 항암 활성을 가진 대체 의약 소재로 개발될 수 있을 것으로 기대된다.

[ $\beta$ ]-Glucans (AG) were prepared from Agaricus blazei cultured in the medium fortified with the roots of Pueraria spp. by repeated extraction with hot water, gel filtration chromatography and DEAE ion exchange chromatography. Oligosaccharides (AO) were derived from the hydrolysis of AG by an endo-$\beta$-(1$\rightarrow$6)-glucanase from Bacillus megaterium. The anti-HT-29 human colon cancer activity of AG or AO was investigated using MTT assay, apoptosis assay, cell cycle analysis, and cDNA microairay. AG and AO both inhibited proliferation and growth of HT-29 cells, and stimulated apoptosis of the cells in a dose-dependent manner. In cell cycle analysis, treating HT-29 cells with AG or AO resulted in the increase of cells in the G0 (sub-G1) and G1 phase. Especially, AO was more effective in inducing G0/G1 cell cycle arrest than AG. To screen the genes involved in the increase of apoptosis, the gene expression profile of the HT-29 cells treated with AO was examined by cDNA microarray. While several genes involved in cell cycle progression (CCND2 and CDK2) were down-regulated, many genes involved in apoptosis (TNFSF9, TNFRSF9, FADD, CASP8, BAD, CRADD, CASP9 etc), cell cycle inhibitor (CDKN2A), immune response (IL6, IL18, IL6R etc), and tumor suppressor (CEACAM1, TP53BP2, IRF1, and PHB) were up-regulated. These results suggest that AO could inhibit the proliferation and growth of HT-29 cells by G0/G1 cell cycle arrest and induction of apoptosis.

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