EGCG-induced selective death of cancer cells through differential regulation of the autophagy-dependent p62-mediated antioxidant survival pathway

다국어 초록 (Multilingual Abstract)

The decision of whether (-)-epigallocatechin-3-gallate (EGCG), a major component of the polyphenol in green teas, acts as an antioxidant or a prooxidant within cells depends on the type of cells as well as its treatment time and concentration. Even though it has been accepted that EGCG induces selective death of cancer cells without adversely affecting normal cells, less attention has been paid to the molecular mechanism underlying the contradictory effects of EGCG as a signaling molecule for cell survival and an apoptotic molecule in normal cells and cancer cells, respectively. Here, the exact nature of the impact of EGCG on selective killing of cancer cells through differential regulation of the antioxidant pathways via autophagy was explored. Cancer cells of various natures underwent apoptotic cell death under the conditions of oxidative stress induced by EGCG while there was no clear evidence of apoptosis in normal cells, implying that the contradictory effect of EGCG between normal cells and cancer cells is not limited to specific type of cells. In addition, the expression levels of SQSTM1/p62, NRF2 and HO-1 increased significantly in MRC5 under the EGCG-induced oxidative stress while the three proteins were all downregulated in HeLa and HCT116. The increased level of NRF2 in MRC5 (EGCG-MRC5) treated with EGCG was due to the increase in its stability by being released from KEAP1 through competitive binding of the increased p62 to KEAP1, not to the transcriptional activation, and the complex of p62 and KEAP1 was increased within inclusion bodies in EGCG-MRC5 but was decreased in EGCG-HeLa with no noticeable evidence of inclusion bodies. Moreover, autophagic flux was blocked for prolonged timeframe of 72 hr after EGCG treatment in MRC5 but was driven in HeLa at least from 18 hr after EGCG treatment, suggesting that EGCG can induce autophagy in a different time and manner between normal cells and cancer cells. It was also found that the differential induction of autophagy was attributed to differential regulation of the AKT and/or AMPK-mTOR-ULK1 signaling pathways between normal and cancer cells under the EGCG-induced oxidative stress. EGCG induced positive regulation of the p62-KEAP1-NRF2-HO-1 antioxidant defense pathway mainly through activation of the AKT-mTOR-ULK1 S556 and S758 in MRC5 while it did negative regulation of the p62-mediated antioxidant pathway in HeLa through fine-tuning of the AMPK-mTOR-ULK1 S758 signaling pathways, leading to the selective death of cancer cells. Furthermore, RNA interference-mediated silencing of 67LR, which is upregulated specifically in a wide range of cancer cells as a cell-surface EGCG receptor, induced the abrogation of EGCG-induced apoptotic death through restoration of p62 expression mainly by downregulation of AMPK in HeLa, suggesting that the differential responsiveness to EGCG between normal cells and cancer cells may be determined by whether 67LR promotes downstream signaling pathways for induction of the p62-mediated antioxidant pathway via autophagy. Overall, it is apparent that the selective apoptotic death of cancer cells is induced by the differential autophagy-dependent regulation of the p62-mediated antioxidant survival pathway via fine-tuning of AKT, AMPK, mTOR and ULK1 between normal cells and cancer cells at least in our experimental setting. Our findings may help to provide molecular insights into not only the differential regulation of the p62-mediated antioxidant survival pathway via autophagy between normal cells and cancer cells but also development of better-tailored cancer therapies without affecting normal cells.

국문 초록 (Abstract)

녹차에 함유된 폴리페놀의 주성분인 (-)-epigallocatechin-3-gallate (EGCG)가 세포 내에서 항산화제로 작용하는지 산화제로 작용하는지 여부는 세포의 종류와 처리시간, 농도에 따라 결정된다. EGCG가 정상세포에 악영향을 미치지 않으면서 암세포의 선택적 사멸을 유도한다는 것이 널리 알려졌으나, EGCG가 정상세포에서 세포 생존을 위한 신호 분자와 암세포에서 세포 사멸을 위한 신호 분자로서 모순된 효과를 내는 메커니즘에 대한 연구는 많지 않다. 이에 따라 본 논문에서는 EGCG에 의해 나타나는 자가포식(autophagy)을 통한 항산화 경로의 조절에 의해 나타나는 암세포의 선택적인 사멸 과정을 연구하였다. 100 μM에서 24시간 동안 EGCG에 의해 유도된 산화 스트레스 조건에서 다양한 성질의 암세포가 세포사멸을 겪었으나, 정상세포에서는 확실한 세포사멸이 보이지 않았으며, 이는 특정 세포 유형에 국한되지 않고 여러가지 세포에서 일반적으로 발생하였다. 또한 인간 폐 섬유아세포 세포주인 MRC5의 경우 EGCG에 의해 유도된 산화 스트레스 환경에서 SQSTM1/p62, NRF2, HO-1의 발현량이 유의하게 증가한 반면, 인간 자궁경부암세포인 HeLa 및 인간 결장암 세포주인 HCT116에서는 세 단백질이 모두 감소하였다. EGCG를 처리한 MRC5(EGCG-MRC5)에서 NRF2의 증가는 전사 활성화가 아닌 증가된 p62와 KEAP1의 경쟁적인 결합으로 인해 NRF2가 KEAP1에서 방출됨에 따른 안정성 증가가 원인이며 p62와 KEAP1은 EGCG-MRC5에서 inclusion body를 형성하며 증가했지만 EGCG-HeLa에서는 inclusion body를 형성하지 않고 감소하였다. 또한 MRC5에서는 EGCG가 처리된 72시간 동안 autophagy 과정이 차단되거나 중단되었지만 HeLa에서는 EGCG 처리 후 적어도 18시간 이후부터 다시 autophagy 과정이 일어나는 것을 확인할 수 있었고, 이를 통해 EGCG는 정상세포와 암세포에서 서로 다른 시간과 방식으로 autophagy를 유도함을 알 수 있었다. 추가적으로 EGCG에 의한 산화스트레스 환경에서 AKT-mTOR-ULK1 및/또는 AMPK-mTOR-ULK1 신호경로가 정상세포와 암세포 내에서 서로 다른 방식으로 조절되어 autophagy가 서로 다르게 일어남을 밝힐 수 있었다. MRC5의 경우 EGCG에 의해 AKT-mTOR-ULK1 S556 및 S758가 활성화되어 p62-KEAP1-NRF2-HO-1의 항산화 방어 작용이 증가한 반면, 암세포에서 EGCG는 AMPK-mTOR-ULK1 S758 신호 경로의 미세조정을 통한 선택적인 사멸을 일으킨다. 67LR은 세포 표면 EGCG 수용체로서 대부분의 암세포에서 높은 발현량을 나타내고 지질뗏목 군집화(Lipid-raft clustering) 유도와 관련이 있다고 알려져 있다. 이 단백질을 HeLa에서 억제시킬 경우, EGCG에 의해 증가하던 AMPK의 발현량이 줄어들게 되고, 줄어들던 p62 발현을 회복시켜 세포사멸이 일어나지 않게 됨을 확인할 수 있었다. 그에 따라서 정상세포와 암세포에서 서로 다른 EGCG에 대한 반응을 보이는 이유는 67LR에 의한 autophagy를 통한 p62 매개 항산화 경로의 활성화 여부에 따른 것이라 판단할 수 있었다. 정리해보면, 우리의 실험 환경에서 AKT, AMPK, mTOR 및 ULK1의 미세 조정을 통해 정상세포와 암세포에서 서로 다른 autophagy 의존적 p62 매개 항산화 작용이 일어나게 되고, 이에 따라 암세포 특이적 세포사멸이 유도된다는 것을 확인할 수 있었다. 본 연구 결과를 통해 정상세포와 암세포 사이의 서로 다른 autophagy를 통한 p62 매개 항산화 생존 경로 조절의 확인 외에도 정상 세포에 영향을 미치지 않는 효과적인 맞춤형 암 치료법의 개발에 대한 효과적인 접근법을 얻을 수 있을 것이다.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 18
• 2.1 Cell culture and reagents 18
• 2.2 WST-1 cell viability assay 18
• 2.3 Quantitative real-time PCR (RT-qPCR) 19
• 2.4 Small interfering RNA (siRNA)-mediated knockdown 20
• 2.5 Construction of overexpression vectors and transfection 20
• 2.6 Nucleus/cytoplasm fractionation 21
• 2.7 Western blot assay 22
• 2.8 Immunoprecipitation 22
• 2.9 Fluorescence microscopy image analysis 23
• 2.10 CRISPR/Cas9-mediated AMPKα1 knockout 24
• 2.11 Phosphorylated protein microarray 24
• 2.12 Statistical analysis 25
• 3. RESULTS 29
• 3.1 EGCG-induced selective killing of cancer cells through apoptotic pathway 29
• 3.2 EGCG-induced differential expression of HO-1 between normal cells and cancer cells 33
• 3.3 Differential regulation of the KEAP1-NRF2-HO-1 antioxidant pathway between normal cells and cancer cells under EGCG-induced oxidative stress 38
• 3.4 Differential regulation of the p62-KEAP1-NRF2-HO-1 pathway between normal cells and cancer cells under EGCG-induced oxidative stress 44
• 3.5 Differential autophagy-dependent expression of p62 between normal cells and cancer cells under the EGCG-induced oxidative stress 52
• 3.6 Differential regulation of mTOR between normal and cancer cells under the EGCG-induced oxidative stress 62
• 3.7 Regulation of AKT and AMPK, upstream signaling pathways for differential phosphorylation of mTOR between normal and cancer cells under the EGCG-induced oxidative stress 68
• 3.8 Roles of AMPK for differential regulation of the ULK1-mTOR signaling pathway between normal and cancer cells 76
• 3.9 67LR-mediated differential induction of the antioxidant survival pathway between normal cells and cancer cells under the conditions of EGCG-induced oxidative stress 80
• 4. DISCUSSION 89
• REFERENCES 123
• 국문초록 149