대장암 및 전립선암 세포에서 Ginsenoside Rg3에 의한 Docetaxel의 항암 상승효과 및 작용기전 = Ginsenoside Rg3 enhances the susceptibility of colon and prostate cancer cells to docetaxel

국문 초록 (Abstract)

인삼은 예전부터 면역력을 높이고, 영양분을 공급하며, 피로를 줄이는 의약품으로 사용되어 왔다. 인삼의 항암, 항염증, 항증식효과는 진세노사이드에서 유래한 것으로 인삼에서 유래된 성분인  진세노사이드 Rg3(이하 Rg3)에 관하여 항암을 포함한 여러 약리학적 작용에 대한 보고가 있어 이에 대한 연구가 꾸준히 진행되어 왔다. Nuclear factor-kappa B(NF-kB)는 암세포에서 활성화되어 암세포가 항암제에 저항력을 갖도록 작용한다. 본 연구에서는 최근 Rg3가 대장암 및 전립선암 세포에서 항암제에 대한 암세포의 저항력을 증가시키는 인자인 NF-kB의 활성을 억제하여 결과적으로 항암작용을 나타내는지에 대해 연구하였다. Rg3가 NF-kB의 활성을 억제하여 암세포의 증식을 억제하는지 확인하기 위해 대장암세포(SW620 and HCT116) 및 전립선암세포(LNCaP, PC-3 and DU145)에 Rg3를 농도별(25, 50, 75, 100 μM)로 처리하여 NF-kB의 활성을 억제하고 apoptosis를 유도하여 암세포 성장을 저해하는지 관찰했다. Rg3는 농도의존적으로 apoptosis를 유도하고 NF-kB의 활성을 억제하여 암세포의 성장을 억제했다. 대장암 및 전립선암에서의 IC50 값의 절반 농도를 이용하여 Rg3와 항암제를 병용투여하였다. : Rg3 (50 μM)와 docetaxel (5 nM), paclitaxel (10 nM) cisplatin (10 μM) 및 doxorubicin (2 μM) 각각을 병용 투여하는 것이 각 성분을 단독으로 적용했을 때 보다 NF-kB의 활성 억제 및 암세포 성장 저해작용이 더 효과적임을 확인했다. 또한, Rg3와 docetaxel의 병용 투여 시 NF-kB에서 apoptotic 세포사를 유도하는 인자인 Bax, caspase-3, caspase-9의 발현이 증가하고, apoptotic 세포사를 억제하는 인자인 Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP); Cox-2, c-Fos, c-Jun and cyciln B, D1 and E, cyclin-dependent kinase 2 and 4의 발현이 억제되는 것을 확인했다. 병용투여 결과를 수치화하기 위해 combination index(CI)를 이용하여 계산한 결과 모든 cell line에서 CI 값이 1보다 작아 시너지효과를 나타냄을 확인하였다. CI 값이 1보다 작으면 synergism, 1과 같으면 additivity이며 1보다 크면 antagonism 이다. 위의 실험결과로 Rg3가 NF-kB 활성의 억제를 통해 대장암 및 전립선암에 대한 docetaxel 및 다른 항암제의 감수성을 증가시켜 주는 것을 확인할 수 있었다. 따라서 Rg3가 항암제 또는 항암제의 보조제로 유용하게 사용될 수 있을 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Panax(P) ginseng C.A. Mayer has been used in traditional medicine to strengthen immunity, provide nutrition, and reduce fatigue. The anticancer, anti-inflammatory, and anti-proliferative effects of P. ginseng are attributed to ginsenosides found in the plant. Ginsenoside Rg3(Rg3), the active component isolated from P. ginseng, has been interested in using as a cancer preventive or therapeutic agent. Nuclear factor-kappaB (NF-κB) is constitutively activated in the cancer, and gives cancer cells resistance to chemotherapeutic agents. These studies were investigated to know whether Rg3 inhibits activity of NF-κB which is a key transcriptional factor constitutively activated in the colon and prostate cancers, and inhibits cancer cells resistance to chemotherapeutic agents. To investigate whether Rg3 suppresses the activation of NF-κB, and inhibits cancer cell growth, the susceptibility of colon cancer (SW620 and HCT116) cells and prostate (LNCaP, PC-3 and DU145) cells as well as activation of NF-κB were examined by different concentrations at Rg3 (25, 50, 75 and 100 μM). Rg3 inhibited cancer cell growth through induction of apoptosis and decreased NF-κB activity in a dose dependent manner. In further study using half doses of IC50 values of compounds in colon cancer and prostate cancer, it was found that the combinded treatment with conventional agents, such as docetaxel (5 nM), paclitaxel (10 nM), cisplatin (10 μM) and doxorubicin (2 μM), Rg3 (50 μM) was more effective (synergistic effects) in the inhibition of cancer cell growth and induction of apoptosis accompanied with the significant inhibition of NF-κB activity than the cell growth inhibition. It was also found that NF-κB target gene expression of apoptotic cell death proteins such as Bax, caspase-3, caspase-9 was significantly enhanced, but the expression of anti-apoptotic genes as well as cell proliferation marker genes such as Bcl-2, inhibitor of apoptosis protein (IAP-1) and X chromosome IAP (XIAP) such as Cox-2, c-Fos, c-Jun and cyciln B, D1 and E, cyclin-dependent kinase 2 and 4 was significantly inhibited by the combination treatment compared to the expressions by the Rg3 or silgle docetaxel treatment. The synergistic inhibition is well correlated (combination index are less than 1 in all cell types) by calculations of combination effects and the results were expressed as a combination index (CI) with CI < 1, CI = 1 or CI > 1 representing synergism, additivity or antagonism, respectively. The combination significantly inhibited NF-kB activities as well as expression of NF-kB target apoptotic cell death proteins, but decreased anti-apoptotic cell death proteins. These results indicate that ginsenoside Rg3 inhibit NF-κB, and enhance the susceptibility of colon and prostate cancer cells to other chemotherapeutics such as docetaxel. Thus, ginsenoside Rg3 could be useful as an anti-cancer or adjuvant anti-cancer agents.

목차 (Table of Contents)

• I. INTRODUCTION 1
•    1. Cancer developments 1
•    2. Cancer cell death 3
•    3. NF-κB 6
• 4. Transcription factors as a target for cancer prevention 12
• 5. Ginsenosides 14
•    6. Docetaxel 15
• Ⅱ. MATERIALS AND METHODS 18
•    1. Chemicals 18
• 2. Isolation and identification of ginsenoside Rg3 18
•    3. Cell culture 19
•    4. Cell viability assay 19
• 5. Western blot analysis 20
• 6. Gel electromobility shift assay 21
• 7. Transfection and assay of luciferase activity 22
•    8. Detection of apoptosis 23
•    9. Cell cycle analysis by flow cytometry 23
• 10. Statistical analysis 24
• Ш. RESULTS 25
• 1. Isolation and identification of ginsenoside Rg3 25
• 1-1. Isolation of ginsenoside Rg3 25
• 1-2. Identification of ginsenoside Rg3 27
• 2. Inhibition of NF-κB by ginsenoside Rg3 enhances the susceptibility of colon cancer cells to docetaxel 31
• 2-1. Effect of the combination of ginsenoside Rg3 with docetaxel on the NF-κB activation in colon cancer cells 31
• 2-2. Rg3 sensitizes colon cancer cells to docetaxel 37
• 2-3. Rg3 augments docetaxel-induced the expression of apoptotic regulatory proteins 41
• 2-4. Effect of the combination of Rg3 with other chemotherapeutics on cancer cell growth and NF-κB activity 43
• 3. Combination of ginsenoside Rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of NF-kB 46
• 3-1. Effect of the combination of Rg3 with docetaxel on the NF-kB activation in prostate cancer cell 46
• 3-2. Rg3 sensitizes prostate cancer cells to docetaxel for growth inhibition 52
• 3-3. Rg3 augmented docetaxel-induced G0/G1-phase cell cycle arrest and decreased the levels of G0/G1 regulatory proteins 57
• 3-4. Rg3 augmented docetaxel-induced the expression of apoptotic regulatory proteins 59
• 3-5. Effect of the combination of Rg3 with other chemotherapeutics on prostate cancer cell growth and NF-kB activity 62
• 3-6 Combination index(CI) between ginsenoside Rg3 and docetaxel 66
• Ⅳ. DISCUSSION 67
•   
• Ⅴ. REFERENCES 76
•  
• Ⅵ. SUMMARY IN KOREAN 103