Melatonin induced apoptosis against AML via inhibition of miR-589

국문 초록 (Abstract)

멜라토닌은 항산화, 항염증, 조혈, 항암 효과가 발표되고 있다. 하지만 급성 골수성 백혈병에 대한 연구는 전무하여 본 연구를 수행하였다. 세포 독성 실험을 이용하여 정상세포주에서는 독성이 거의 나타나지 않는 농도에서 급성 골수성 백혈병 세포주에서 항암 효과를 확인하였다. 멜라토닌은 c-PARP, p-ATF4, CHOP의 발현량을 증가시켰다. 또한 LIVE/DEAD cell assay를 통하여 죽은 세포의 수가 멜라토닌 처리군에서 현저히 높은 것을 확인하였다. 또한, 멜라토닌 처리군에서 LIF, PI3K/AKT, Mcl-1, c-Jun 그리고Nrf2가 효과적으로 억제되었다. 활성산소는 멜라토닌 처리군에서 유의하게 증가하였으며, 증가된 활성산소와 감소된 세포 생존률은 NAC 전처리군에서 유의하게 역전되는 것을 확인하였다. 발암miRNA인 miR-589은 멜라토닌에 의하여 유의하게 억제되었다. 이 모든 결과들을 종합해보면 멜라토닌은 정상세포에서는 독성이 없고 급성 골수성 백혈병 세포주에서 독성이 있는 것을 근거로 하여 암 특이적으로 항암효과가 있으며 이와 관련된 세부조절기전을 조절하여 급성 골수성 혈액암의 치료제로써의 가능성을 제시하였다.

다국어 초록 (Multilingual Abstract)

Melatonin (MLT) has been reported to have anti-oxidant effect, anti-inflammatory effect, hematopoietic, oncostatic properties. However, its underlying anti-cancer mechanism is still unknown. In this study, the mechanism of MLT was investigated against acute myeloid leukemia cells, including THP-1 (acute myeloid leukemia cell line) and U937 (acute myeloid leukemia cell line) cells. MLT exploited cytotoxic effect in U937 and THP-1 cells but not in normal cells such as IEC-6 (normal intestinal epithelial cell line) and MDBK cells (normal kidney cell line). MLT increased pro-apoptotic protein such as cleaved Poly (ADP-ribose) polymerase (PARP), C/EBP-homologous protein (CHOP), activating transcription factor 4 (ATF4) and attenuated expression of leukemia inhibitory factor (LIF), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), c-Jun, induced myeloid cell leukemia differentiation protein (Mcl-1) and nuclear respiratory factor 2 (Nrf2). MLT induced excessive reactive oxygen species (ROS) generation in a concentration-dependent manner which is attenuated by N-Acetyl-L-cysteine (NAC) pretreatment. Also, onco-miRNA, miR-589 was significantly suppressed by MLT in a dose dependent manner. Consequently, MLT revealed the potential of clinical application in AML.

목차 (Table of Contents)

• Ⅰ. Introduction ···························································································· 1
• Ⅱ. Materials and Methods ·············································································· 4
• 1. Chemicals and reagents ······································································ 4
• 2. Cell culture ····················································································· 4
• 3. Cytotoxicity assay ··········································································· 4
• 4. Western blot analysis ········································································· 4
• 5. Live and dead cell assay ······································································ 5
• 6. Measurement of ROS ········································································· 5
• 7. Quantitative real-time PCR analysis ························································ 6
• 8. Transfection assay ············································································ 6
• 9. Statistical analysis ············································································· 7
• Ⅲ. Results ································································································ 8
• 1. MLT exerted the cytotoxic effect in AML cells……………………………………… 8
• 2. MLT increased apoptosis in U937 and THP-1 cells cells···································· 10
• 3. MLT decreased the expression of PI3K/AKT signal pathways in U937andTHPcells····················· 13
• 4. MLT increased the ROS generation U937 and THP-1 cells ··························· 15
• 5. ROS Scavenger NAC Attenuated MLT induced Cytotoxicity in U937 and THP-1 Cells ····························· 17
• 6. MLT Decreased expression level of miR-589 ··········································· 19
• Ⅳ. Discussion ························································································· 21
• Ⅴ. References ·························································································· 24
• Abstract in Korean ···················································································· 29