간암 세포주 HepG2에 대한 대황 (Rheum palmatum L.) 추출물의 항암효과 = Anticancer effect of Rheum palmatum L. on human liver cancer HepG2 cells

다국어 초록 (Multilingual Abstract)

Rheum palmatum L. (Dae-Whang) has been demonstrated to possess anti-tumor activity. However, the mechanisms of the Rheum palmatum L.-produced biological effects remained unknown. This study was performed for the investigation of anticancer effects of methanol extract of Rhei palmatum L. (MeOH-RP) on a human liver cancer cell line (HepG2). To study the cytotoxic effect of (MeOH-RP) on HepG2 cells, the cells were treated with various concentrations of MeOH-RP and then cell viability was determined by XTT reduction method and trypan blue exclusion assay. MeOH-RP reduced proliferation of HepG2 cells in a dose-dependent manner at 24 h and 48 h treatment. MeOH-RP induced the activation of caspase-3, -8, and -9 and the cleavage of poly ADP-ribose polymerase (PARP), a substrate for caspase-3. Furthermore, treatment with MeOH-RP resulted in internucleosomal DNA fragmentation, evidenced by the formation of a DNA ladder on agarose gel, a hallmark of cells undergoing apoptosis.
MeOH-RP downregulated Bcl-2, upregulated Bax, and increased the release of cytochrome c from the mitochondria into cytosol in a dose-dependent manner. These results suggest that MeOH-RP induces apoptosis through the mitochondrial dysfunction mechanism, and that cytochrome c is a key factor in MeOH-RP-induced apoptosis in HepG2 cells.
To explore whether the activation of caspase-3 was required for induction of apoptosis by MeOH-RP, HepG2 cells were co-treated with caspase-3 inhibitor and MeOH-RP incubation with the inhibitor of caspase-3 (Ac-DEVD-CHO) significantly blocked MeOH-RP-triggered apoptosis in HepG2 cells. This datum demonstrates that activation of caspase-3 is involved in MeOH-RP-induced apoptosis.
These results suggest that MeOH-RP is potentially useful as a chemotherapeutic agent in human liver cancer.

목차 (Table of Contents)

• Ⅰ. 서론 = 1
• Ⅱ. 실험재료 및 방법 = 3
• 1. 재료 = 3
• 1) 약재 = 3
• 2) 시약 = 3
• 2. 방법 = 5
• 1) 세포배양 = 5
• 2) XTT Assay = 5
• 3) Trypan Blue Exclusion Assay = 5
• 4) 현미경 관찰 = 6
• 5) Caspase-3 activity assay = 6
• 6) Western blot analysis = 6
• Ⅲ. 실험성적 = 8
• 1. 대황 추출물에 의한 세포의 증식 억제 효과 = 8
• 2. 대황 추출물에 의한 HepG2 세포의 형태적 변화 = 11
• 3. 대황 추출물에 의한 HepG2의 세포사멸 = 13
• 4. Mitochondrial pathway를 통한 세포사멸 = 15
• 5. Caspases를 매개로 한 세포사멸 = 17
• Ⅳ. 고찰 = 20
• Ⅴ. 결론 = 24
• 참고문헌 = 26
• Abstract = 29