대한임상검사과학회지 (Korean Journal of Clinical Laboratory Science)

  • 제52권1호
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  • Pages.69-77
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  • 2020
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  • 1738-3544(pISSN)
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  • 2288-1662(eISSN)
대한임상검사과학회 (Korean Society for Clinical Laboratory Science)
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Hep3B 세포에서 베르베린은 DNA methyltransferase3b 억제를 통해 p53을 발현시켜 세포사멸을 유도

Berberine Induces p53-Dependent Apoptosis through Inhibition of DNA Methyltransferase3b in Hep3B Cells

  • 김대연 (강원대학교 춘천캠퍼스 수의과대학 동물의학종합연구) ;
  • 김선형 (강원대학교 춘천캠퍼스 수의과대학 동물의학종합연구) ;
  • 정희태 (강원대학교 춘천캠퍼스 수의과대학 동물의학종합연구) ;
  • 라창식 (강원대학교 춘천캠퍼스 동물생명과학대학) ;
  • 이기종 (연세대학교 원주캠퍼스 보건과학대학 생명의학연구소) ;
  • 정배동 (강원대학교 춘천캠퍼스 수의과대학 동물의학종합연구)
  • Kim, Dae-Yeon (College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University) ;
  • Kim, Seon-Hyoung (College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University) ;
  • Cheong, Hee-Tae (College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University) ;
  • Ra, Chang-Six (College of Animal Life Sciences, Kangwon National University) ;
  • Rhee, Ki-Jong (Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University at Wonju) ;
  • Jung, Bae Dong (College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University)
  • 투고 : 2020.01.09
  • 심사 : 2020.01.31
  • 발행 : 2020.03.31
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초록

종양 억제 유전자 p53은 인간 간암세포 Hep3B에서는 불활성화되어 있다. 베르베린(berberine)은 암세포의 증식을 억제하는 것으로 보고되어 있다. 우리는 베르베린을 처리한 Hep3B 세포에서 세포사멸이 유도되는지를 조사하였고 이 세포사멸이 p53과 DNA methyltransferase의 발현과 연관되어 있는지를 관찰하였다. MTT 분석을 통하여 세포 생존력을 측정하였다. 세포사멸은 각각 Annexin V flow 세포 분석을 사용하여 측정하였다. 베르베린이 처리된 세포에서 DNMT 효소 활성, mRNA 발현, 단백질 발현 정도가 검사되었으며, p53 단백질 발현은 웨스턴 블롯 분석에 의해 검사되었다. 베르베린 처리는 시간 및 용량 의존적으로 Hep3B세포의 세포사멸을 증가시켰다. 베르베린 처리 시 DNMT3b의 활성 정도, mRNA 발현 그리고 단백질 발현 정도가 모두 감소되었다. 이와는 대조적으로, Hep3B에서는 비활성인 p53 단백질의 발현은 DNMT3b의 감소와 동시에 증가했다. ERK의 발현은 변화가 없었으나, P-ERK의 발현은 농도 의존적으로 감소하는 것으로 나타났다. 이러한 결과는 Hep3B 세포에 베르베린의 처리는 DNMT3b의 발현을 감소시켜서 종양 억제 유전자인 p53의 증가를 유도할 수 있고, 이를 통해서 세포사멸을 증가 시킬 수 있다는 것을 나타낸다. 이는 베르베린이 간암 세포의 증식 억제에 효과적으로 작용할 수 있음을 보여준다.

The tumor suppressor gene, p53, is inactivated in the human hepatocellular carcinoma cells line, Hep3B. Berberine has been reported to inhibit the proliferation of cancer cells. This study examined whether apoptosis was induced in berberine-treated Hep3B cells and observed the association between apoptosis and the expression of p53 and DNA methyltransferase (DNMT). The cell viability was measured using an MTT assay. Apoptosis of Hep3B was measured using annexin V flow cytometry. Berberine-treated cells were examined for their DNMT enzymatic activity, mRNA expression, and protein synthesis. The p53 levels were examined by Western blot analysis. The berberine treatment resulted in increased Hep3B cell death and apoptosis in a time- and dose-dependent manner. The DNMT3b activity, mRNA expression, and protein levels all decreased after the berberine treatment. In contrast, the p53 protein levels increased with a concomitant decrease in DNMT3b. No change in the expression of ERK was observed, but the P-ERK levels decreased in a dose dependent manner. These results indicate that a treatment of Hep3B cells with berberine can reduce the expression of DNMT3b, leading to an increase in the tumor suppressant gene p53 and an increase in cell apoptosis. This shows that berberine can effectively suppress the proliferation of liver cancer cells.

키워드

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