(The) Anti-tumor Effect of Orostachys japonicus Extracts on Cholangiocarcinoma Cell Line SNU-1079

다국어 초록 (Multilingual Abstract)

Orostachys japonicus A. Berger (O. japonicus) extracts have shown various biological activities including anti-inflammatory, neuroprotective, anti-ulcerative, anti-oxidant, and anti-tumor effects. Particularly, it has been recognized as a potential anti-tumor agent against various tumor cells. In the present study, the anti-tumor effect of O. japonicus extracts on intrahepatic cholangiocarcinoma cell line SNU-1079 was investigated. Cells were treated with various concentrations of O. japonicus DW and EtOH extracts (0-300 ㎍/㎖) for 24, 48, or 72 h. Cell viability was evaluated through PMS/MTS assay, and the apoptosis rate was examined through ELISA assay and flow cytometry analysis. The mRNA expression of apoptosis- and cell cycle-related genes (Bcl-2, Mcl-1, Bax, Survivin, Cyclin D1, and p21) was evaluated using real-time PCR, and the caspase activity was examined using immunoblot analysis. O. japonicus extracts inhibited cell proliferation and increased apoptosis rate in both ELISA assay and flow cytometry analysis. O. japonicus extracts decreased Bcl-2, Mcl-1, Survivin, and Cyclin D1 mRNA expression and increased Bax mRNA level. O. japonicus extracts also increased Caspase-3 activation. Overall, O. japonicus DW extracts were more effective than EtOH extracts. Taken these results together, O. japonicus inhibited cell proliferation and induced apoptosis in SNU-1079 cells via mitochondria-mediated intrinsic pathway, which leads to Caspase-3 activation. The results indicate that O. japonicus is a potential therapeutic herb with anti-tumor effect against intrahepatic cholangiocarcinoma.

국문 초록 (Abstract)

와송(瓦松)은 항염증, 신경보호, 항궤양, 항산화 및 항암작용이 있는 것으로 알려져 있으며 특히 최근 연구에서는 다양한 암세포들에 대한 항암효과가 보고되고 있다. 본 연구에서는 와송(瓦松) 증류수 추출물과 에탄올 추출물이 간내 담도암 세포주인 SNU-1079 에 미치는 영향을 관찰하였다. 와송(瓦松) 추출물이 세포의 활성도에 미치는 영향은 PMS/MTS 비색분석법으로 분석하였고 세포자멸은 ELISA 방법과 flow cytometry 방법을 통해 측정하였다. 세포자멸과 세포주기에 관련된 유전자인 Bcl-2, Mcl-1, Bax, Survivin, Cyclin D1 및 p21 의 mRNA 발현 정도는 real-time PCR 법으로 측정하였으며 Caspase-3 의 단백질 발현 양상은 immunoblot 법으로 분석하였다. 그 결과, 와송(瓦松) 추출물은 세포 활성도를 감소시켰고, 세포자멸의 내인성 경로에 관련된 유전자 및 Caspase-3 의 발현을 조절하여 세포자멸을 증가시키는 것으로 관찰되어, 간내 담도암에 대한 와송(瓦松)의 활용가능성을 확인할 수 있었다.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and Methods 3
• 1. Preparation of Orostachys japonicus A. Berger extract (DW & EtOH) 3
• 2. Cell culture 3
• 3. Cell viability assay 3
• 4. ELISA assay 4
• 5. FACS analysis 4
• 6. RNA extraction and real-time PCR 4
• 7. Immunoblot analysis 5
• 8. Statistical analysis 6
• III. Results 7
• 1. Effects of Orostachys japonicus on cell proliferation 7
• 2. Effects of Orostachys japonicus on the apoptosis rate using ELISA assay 9
• 3. Effects of Orostachys japonicus on the apoptosis rate using FACS analysis 11
• 4. Effects of Orostachys japonicus on the mRNA expression of apoptosis-related genes 14
• 5. Effects of Orostachys japonicus on Caspase-3 activation 21
• IV. Discussion and Conclusion 23
• References 28
• Appendix 33
• Abstract 35
• 국문초록 36