반합성천연물 TMQ0153 (Tetrahydrobenzimidazole)이 일으키는 세포사멸과 만성 백혈병 치료법 연구

학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2020. 2. Marc Francois Diederich.Abstract in Korean (국문초록) 새로운 반합성 천연물 TMQ (Unaromatized Tetrahydrobenzimidazole)는 p-Benzoquinone 과 N-Arylamidines에서 합성된 물질이며, 만성 골수성 백혈병 (CML) 에서 다양한 세포 사멸 형태인 apoptosis, necroptosis 및 Autophagy를 동시에 유발하는 새로운 저분자 천연물질이다. 오랫동안 Programmed cell death에 있어서 apoptosis가 유일한 것으로 여겨져 왔으나 최근에는 necrosis과정에도 programmed necrosis 또는 necroptosis라 불리는 세포사멸이 존재한다는 것이 밝혀졌다. 최근 necroptosis에는 RIP1과 RIP3가 중요 조절인자임이 알려지고 있으나 현재까지 관련 연구의 많은 부분이 미미한 수준이고 많은 연구가 필요한 분야임을 확인할 수 있었다. 또한 기존에 알려지지않은 autophagy가 apoptosis와 necroptosis에 어떤 작용을 하는지에 대한 연구가 필요하며, 특이적이고 결정적인 조절인자의 차이점에 대하여 많은 연구가 필요한 실정이다. 선행연구를 통해 TMQ 시리즈 중 건강한 PBMC (A peripheral blood mononuclear cell) 세포에서는 독성을 일으키지 않으며 여러 종류의 암세포에서 특이적으로 세포독성을 일으키는 TMQ0153을 선별하였다. 따라서 TMQ0153을 통해 낮은 농도에서 유도된 canonical-programmed 세포사멸은 caspase에 의존적이지만 높은 농도에서 유도된 non-canonical-programmed 세포사멸은 caspase와 무관하며 미토콘드리아의 기능장애를 거쳐 ROS를 축적시켜 그 결과 세포스트레스와 다른 미토콘드리아의 손상을 일으킴을 seahorse XFp cell mito stress 및 FACS를 통해 입증하였고, 세포스트레스의 작용으로 necroptosis 세포사멸이 일어나기 전에 autophagy가 먼저 일어남을 공초점 형광 현미경과 관련 단백질 LC3과 p62의 western blotting을 통해 관찰하였다. 또한 autophagy의 핵심 단백질 Beclin-1의 knockdown을 통해 억제시킨뒤 TMQ0153을 투여하였을때 세포사멸이 더 증가하였으며 이는 암세포를 민감하게 (Sensitization)하는 것을 밝혀내었다. 전자 현미경 이미지를 분석함으로써 autophagy가 일어나는 시점에서 미토콘드리아의 팽창 및 autophagosome 의 형성을 추가 관찰하였고, 세포스트레스의 요소로 세포질의 Ca2+ 축적, 세포내 GSH (Glutathione)의 감소 및 Lysosomal membrane potential (LMP) 의 증가를 FACS와 형광 현미경을 통해 분석하였다. 세포스트레스의 증가 및 necroptosis와 관련있는 특이적 PARP-1 (C2-10), RIP1 단백질의 검출, 세포내 ATP의 급격한 감소가 necroptosis의 원인임을 알 수 있었고 이러한 효과는 necrostatin-1에 의해 저해됨을 증명하였다. 이어서 TMQ0153 에 의해 유도되는 necroptosis는 mitochondria membrane permeabilization (MMP) 를 증가시키고 N-Acetyl Cysteine (NAC)에 의해 세포사멸 및 미토콘드리아가 보존되는 것을 입증할 수 있었다. 최근 선행연구에 따르면 RIP3단백질은 necroptosis를 조절 하는 핵심 단백질로 알려져 있는데, 많은 암세포에서 현저히 감소되어 새로운 암세포 사멸 전략으로 활용하는데 한계가 있었다. 또한 암세포의 특징중 하나인 apoptosis에 대한 저항성은 암세포가 항암제에 저항할 수 있게 함으로써, 암세포 사멸 전략의 한계점을 가져왔다. 따라서 이를 극복할 Necroptosis를 일으키는 약물의 기전연구를 통해 약물의 저항성을 극복하는 방법을 제시할 수 있었다. 탈메틸화제 (5-aza-2'-deoxycytidine; 5aza)를 투여하여 암발생으로 감소된 RIP3 단백질을 복구한 뒤 TMQ0153을 투입한 결과, TMQ0153만 투여한 암세포보다 더 많은 necroptosis를 관찰할 수 있었음을 형광현미경을 통해 확인하였다. TMQ0153이 처리된 Necroptosis가 일어난 세포로부터 HMGB1의 방출과 세포 외 ATP의 증가, Ecto-Calreticulin와 ERp57 의 세포막 노출 및 증가를 관찰함으로써 이 천연물질의 면역세포사멸과 이에 따른 중계연구의 가능성을 제시할 수 있었다. 마지막으로 TMQ0153가 콜로니와 In vivo 제브라피쉬 xenograft 모델에서 종양형성의 발달을 저해하는 것을 추가적으로 관찰하였다. 따라서 본 연구를 통해 제시된 다양한 세포사멸의 현상을 조절하는 세포스트레스의 원인 분석 및 항암제의 분자 작용과 세포사멸 제어 기전을 규명하여 효과적인 암 치료제 개발에 활용하고자 한다. 주요어 세포스트레스, 미토콘드리아, 활성산소, 만성 골수성 백혈병, 프로그램된 세포사멸Overall Abstract Tetrahydrobenzimidazole TMQ0153 induces autophagy followed by controlled necrosis via oxidative stress induction in chronic myeloid leukemia cell lines Sungmi Song College of Pharmacy Department of Pharmacy Graduate School Advisor: Prof. Marc Francois Diederich Chronic myeloid leukemia (CML) is characterized by the Philadelphia (Ph) chromosome with a t(9;22)(q34;q11) reciprocal translocation in single hemopoietic stem cells (HSC) and resulting in expression of the BCR-ABL chimeric oncoprotein. This abnormal gene stimulates the production of reactive oxygen species (ROS). ROS levels increase with CML progression and induce BCR-ABL self-mutagenesis. Imatinib and other tyrosine kinase inhibitors (TKIs) such as dasatinib and nilotinib radically improved the diseases outcome, but TKI-resistance is still considered as a major problem. TKI resistance can be associated with even higher ROS production than in TKI-sensitive cells. So far, a role for redox changes in apoptosis has been established; however, several new modalities of regulated cell death have been recently described, The importance of ROS production as well as cellular stress are being actively investigated. Taken together, we investigate here the role of ROS and redox changes in the activation and execution of regulated necroptosis. We also discuss how cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models.Chapter 1. Redox biology of regulated cell death in cancer: A focus on necroptosis 1 1.1. Abstract 2 1.2. Introduction 3 1.3. Regulated cell death and ROS 8 1.4. Redox cancer therapy 16 1.5. Future direction 21 Chapter 2. Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophay and necroptosis crosstalk in chronic myeloid leukemia 22 2.1. Abstract 23 2.2. Introduction 24 2.3. Material and methods 26 2.4. Results 34 2.5. Discussion 79 References 84 Abstract in Korean (국문초록) 102Docto

The activity of arachidonic acid and gamma-linolenic acid on human gliomas

Despite recent advances in tumour cell biology, the prognosis for patients suffering from malignant glioma remains poor. Although primary glioma rarely metastasises outside the central nervous system (primary being defined as the mass of tumour cells at the original site of the neoplastic event) median survival of adults is less than 1 year after diagnosis.The efficacy of existing therapeutic interventions is limited by poor penetration of chemotherapeutic drugs across the blood brain barrier, the inherent radioresistance of glioma tissue and the infiltrating nature of the tumour. Further progress is likely to be achieved through analysis of the complex biology of these tumours and the development of novel therapeutic strategies. The purpose of this study was to investigate the therapeutic potential of the n-6 essential fatty acids arachidonic acid and gamma-linolenic acid, which may inhibit tumour proliferation by acting as substrates for the production of potentially cytotoxic reactive oxygen intermediates and stimulating apoptotic cell death, both alone and in conjunction with radiation.Experiments were undertaken to investigate the effects of exogenous arachidonic acid and gamma-linolenic acid on cellular peroxidation, proliferation, viability and apoptosis. These investigations were carried out on single cell suspensions of morphologically heterogeneous fresh human glioma tissue and associated normal brain, human phagocytes and the rat C6 glioma cell line. It was shown that oxidative activity was impaired in human glioma tissue. Addition of 4-40μM arachidonic acid and gamma-linolenic acid induced a concentration dependant increase in tumour reactive oxygen intermediate production and apoptotic activity. Although the kinetics of reactive oxygen intermediate formation in the presence of arachidonic acid and gamma-linolenic acid followed an exponential function in both normal and tumour cell preparations, tumour cells showed a significantly higher sensitivity to exogenous essential fatty acid stimulus. The kinetics of this stimulation were grade dependent, with high grade tumours responding in a more rapid and sustained manner in comparison with lower grade tumours. The morphological heterogeneity of the human glioma preparations was confirmed with immunohistochemical analysis and flow cytometry using monoclonal and polyclonal anti-Glial Acidic Fibrillary Protein (GFAP). GFAP positive cells responded to exogenous arachidonic acid and gamma-linolenic acid with increased reactive oxygen intermediate production, indicating a high sensitivity of glioma cells to essential fatty acid stimulus. Reactive oxygen intermediate production was also investigated in phagocyte preparations of patients undergoing pulmonary resection for lung cancer. It was found that reactive oxygen intermediate generation was stimulated in patient and control phagocytes by exogenous 1 -40μM arachidonic acid and gamma-linolenic acid both pre and post-operatively. Increased reactive oxygen intermediate formation was detected in the cell population identified as leukocytes in preparations of human primary glioma, although this response was less than that of associated tumour. It was also found that surgery was associated with an increase in phagocyte reactive oxygen intermediate at 2 and 7 days post-operatively in lung cancer patients. The interactive effects of arachidonic acid, gamma-linolenic acid and therapeutic radiation were demonstrated in the rat C6 glioma cell line. The rate ofreactive XVI oxygen intermediate production in response to exogenous arachidonic acid and gamma-linolenic acid increased within the first hour, and elevated oxidative activity was detected for up to three hours. However, a different pattern ofreactive oxygen intermediate generation was observed in response to radiation alone. Similarly, an early apoptotic response was observed following exogenous arachidonic acid and gamma-linolenic acid stimulation. In comparison, radiation induced stimulation of apoptosis occurred over the 12 hour period of incubation and was maximal between 6 and 8 hours post-irradiation. An enhanced radiation response was observed when the stimulation of apoptosis induced by essential fatty acid stimulus alone was low, suggesting that essential fatty acids and radiation may interact to potentiate reactive oxygen intermediate generation and apoptosis.In conclusion, this study has provided evidence that glioma tissue has low basal oxidative activity in comparison with associated normal brain, and that addition of exogenous arachidonic acid and gamma-linolenic acid stimulates peroxidative and apoptotic activity in glioma tissue a grade dependant manner. Studies on the cellular heterogeneity of human glioma samples indicate that the stimulation ofreactive oxygen intermediate production by exogenous arachidonic acid and gamma-linolenic acid occurs in GFAP positive cells. This indicates high sensitivity of human glioma to exogenous essential fatty acid stimulus. Phagocyte populations from lung cancer and malignant glioma patients also respond with increased reactive oxygen intermediate production to exogenous arachidonic acid and gamma-linolenic acid, although the magnitude of this increase is less than that observed for tumour cells. In addition, there is evidence ofpotentiation ofthe oxidative and apoptotic response of the rat C6 cell line to exogenous arachidonic acid and gamma-linolenic acid in the presence of therapeutically relevant doses ofradiation. These results are consistent with a clinical role for arachidonic acid and gamma-linolenic acid in the treatment of malignant glioma