6 research outputs found
๋ฐํฉ์ฑ์ฒ์ฐ๋ฌผ TMQ0153 (Tetrahydrobenzimidazole)์ด ์ผ์ผํค๋ ์ธํฌ์ฌ๋ฉธ๊ณผ ๋ง์ฑ ๋ฐฑํ๋ณ ์น๋ฃ๋ฒ ์ฐ๊ตฌ
ํ์๋
ผ๋ฌธ(๋ฐ์ฌ)--์์ธ๋ํ๊ต ๋ํ์ :์ฝํ๋ํ ์ฝํ๊ณผ,2020. 2. Marc Francois Diederich.Abstract in Korean (๊ตญ๋ฌธ์ด๋ก)
์๋ก์ด ๋ฐํฉ์ฑ ์ฒ์ฐ๋ฌผ TMQ (Unaromatized Tetrahydrobenzimidazole)๋ p-Benzoquinone ๊ณผ N-Arylamidines์์ ํฉ์ฑ๋ ๋ฌผ์ง์ด๋ฉฐ, ๋ง์ฑ ๊ณจ์์ฑ ๋ฐฑํ๋ณ (CML) ์์ ๋ค์ํ ์ธํฌ ์ฌ๋ฉธ ํํ์ธ apoptosis, necroptosis ๋ฐ Autophagy๋ฅผ ๋์์ ์ ๋ฐํ๋ ์๋ก์ด ์ ๋ถ์ ์ฒ์ฐ๋ฌผ์ง์ด๋ค. ์ค๋ซ๋์ Programmed cell death์ ์์ด์ apoptosis๊ฐ ์ ์ผํ ๊ฒ์ผ๋ก ์ฌ๊ฒจ์ ธ ์์ผ๋ ์ต๊ทผ์๋ necrosis๊ณผ์ ์๋ programmed necrosis ๋๋ necroptosis๋ผ ๋ถ๋ฆฌ๋ ์ธํฌ์ฌ๋ฉธ์ด ์กด์ฌํ๋ค๋ ๊ฒ์ด ๋ฐํ์ก๋ค. ์ต๊ทผ necroptosis์๋ RIP1๊ณผ RIP3๊ฐ ์ค์ ์กฐ์ ์ธ์์์ด ์๋ ค์ง๊ณ ์์ผ๋ ํ์ฌ๊น์ง ๊ด๋ จ ์ฐ๊ตฌ์ ๋ง์ ๋ถ๋ถ์ด ๋ฏธ๋ฏธํ ์์ค์ด๊ณ ๋ง์ ์ฐ๊ตฌ๊ฐ ํ์ํ ๋ถ์ผ์์ ํ์ธํ ์ ์์๋ค. ๋ํ ๊ธฐ์กด์ ์๋ ค์ง์ง์์ autophagy๊ฐ apoptosis์ necroptosis์ ์ด๋ค ์์ฉ์ ํ๋์ง์ ๋ํ ์ฐ๊ตฌ๊ฐ ํ์ํ๋ฉฐ, ํน์ด์ ์ด๊ณ ๊ฒฐ์ ์ ์ธ ์กฐ์ ์ธ์์ ์ฐจ์ด์ ์ ๋ํ์ฌ ๋ง์ ์ฐ๊ตฌ๊ฐ ํ์ํ ์ค์ ์ด๋ค. ์ ํ์ฐ๊ตฌ๋ฅผ ํตํด TMQ ์๋ฆฌ์ฆ ์ค ๊ฑด๊ฐํ PBMC (A peripheral blood mononuclear cell) ์ธํฌ์์๋ ๋
์ฑ์ ์ผ์ผํค์ง ์์ผ๋ฉฐ ์ฌ๋ฌ ์ข
๋ฅ์ ์์ธํฌ์์ ํน์ด์ ์ผ๋ก ์ธํฌ๋
์ฑ์ ์ผ์ผํค๋ TMQ0153์ ์ ๋ณํ์๋ค.
๋ฐ๋ผ์ TMQ0153์ ํตํด ๋ฎ์ ๋๋์์ ์ ๋๋ canonical-programmed ์ธํฌ์ฌ๋ฉธ์ caspase์ ์์กด์ ์ด์ง๋ง ๋์ ๋๋์์ ์ ๋๋ non-canonical-programmed ์ธํฌ์ฌ๋ฉธ์ caspase์ ๋ฌด๊ดํ๋ฉฐ ๋ฏธํ ์ฝ๋๋ฆฌ์์ ๊ธฐ๋ฅ์ฅ์ ๋ฅผ ๊ฑฐ์ณ ROS๋ฅผ ์ถ์ ์์ผ ๊ทธ ๊ฒฐ๊ณผ ์ธํฌ์คํธ๋ ์ค์ ๋ค๋ฅธ ๋ฏธํ ์ฝ๋๋ฆฌ์์ ์์์ ์ผ์ผํด์ seahorse XFp cell mito stress ๋ฐ FACS๋ฅผ ํตํด ์
์ฆํ์๊ณ , ์ธํฌ์คํธ๋ ์ค์ ์์ฉ์ผ๋ก necroptosis ์ธํฌ์ฌ๋ฉธ์ด ์ผ์ด๋๊ธฐ ์ ์ autophagy๊ฐ ๋จผ์ ์ผ์ด๋จ์ ๊ณต์ด์ ํ๊ด ํ๋ฏธ๊ฒฝ๊ณผ ๊ด๋ จ ๋จ๋ฐฑ์ง LC3๊ณผ p62์ western blotting์ ํตํด ๊ด์ฐฐํ์๋ค. ๋ํ autophagy์ ํต์ฌ ๋จ๋ฐฑ์ง Beclin-1์ knockdown์ ํตํด ์ต์ ์ํจ๋ค TMQ0153์ ํฌ์ฌํ์์๋ ์ธํฌ์ฌ๋ฉธ์ด ๋ ์ฆ๊ฐํ์์ผ๋ฉฐ ์ด๋ ์์ธํฌ๋ฅผ ๋ฏผ๊ฐํ๊ฒ (Sensitization)ํ๋ ๊ฒ์ ๋ฐํ๋ด์๋ค. ์ ์ ํ๋ฏธ๊ฒฝ ์ด๋ฏธ์ง๋ฅผ ๋ถ์ํจ์ผ๋ก์จ autophagy๊ฐ ์ผ์ด๋๋ ์์ ์์ ๋ฏธํ ์ฝ๋๋ฆฌ์์ ํฝ์ฐฝ ๋ฐ autophagosome ์ ํ์ฑ์ ์ถ๊ฐ ๊ด์ฐฐํ์๊ณ , ์ธํฌ์คํธ๋ ์ค์ ์์๋ก ์ธํฌ์ง์ Ca2+ ์ถ์ , ์ธํฌ๋ด GSH (Glutathione)์ ๊ฐ์ ๋ฐ Lysosomal membrane potential (LMP) ์ ์ฆ๊ฐ๋ฅผ FACS์ ํ๊ด ํ๋ฏธ๊ฒฝ์ ํตํด ๋ถ์ํ์๋ค. ์ธํฌ์คํธ๋ ์ค์ ์ฆ๊ฐ ๋ฐ necroptosis์ ๊ด๋ จ์๋ ํน์ด์ PARP-1 (C2-10), RIP1 ๋จ๋ฐฑ์ง์ ๊ฒ์ถ, ์ธํฌ๋ด ATP์ ๊ธ๊ฒฉํ ๊ฐ์๊ฐ necroptosis์ ์์ธ์์ ์ ์ ์์๊ณ ์ด๋ฌํ ํจ๊ณผ๋ necrostatin-1์ ์ํด ์ ํด๋จ์ ์ฆ๋ช
ํ์๋ค. ์ด์ด์ TMQ0153 ์ ์ํด ์ ๋๋๋ necroptosis๋ mitochondria membrane permeabilization (MMP) ๋ฅผ ์ฆ๊ฐ์ํค๊ณ N-Acetyl Cysteine (NAC)์ ์ํด ์ธํฌ์ฌ๋ฉธ ๋ฐ ๋ฏธํ ์ฝ๋๋ฆฌ์๊ฐ ๋ณด์กด๋๋ ๊ฒ์ ์
์ฆํ ์ ์์๋ค.
์ต๊ทผ ์ ํ์ฐ๊ตฌ์ ๋ฐ๋ฅด๋ฉด RIP3๋จ๋ฐฑ์ง์ necroptosis๋ฅผ ์กฐ์ ํ๋ ํต์ฌ ๋จ๋ฐฑ์ง๋ก ์๋ ค์ ธ ์๋๋ฐ, ๋ง์ ์์ธํฌ์์ ํ์ ํ ๊ฐ์๋์ด ์๋ก์ด ์์ธํฌ ์ฌ๋ฉธ ์ ๋ต์ผ๋ก ํ์ฉํ๋๋ฐ ํ๊ณ๊ฐ ์์๋ค. ๋ํ ์์ธํฌ์ ํน์ง์ค ํ๋์ธ apoptosis์ ๋ํ ์ ํญ์ฑ์ ์์ธํฌ๊ฐ ํญ์์ ์ ์ ํญํ ์ ์๊ฒ ํจ์ผ๋ก์จ, ์์ธํฌ ์ฌ๋ฉธ ์ ๋ต์ ํ๊ณ์ ์ ๊ฐ์ ธ์๋ค. ๋ฐ๋ผ์ ์ด๋ฅผ ๊ทน๋ณตํ Necroptosis๋ฅผ ์ผ์ผํค๋ ์ฝ๋ฌผ์ ๊ธฐ์ ์ฐ๊ตฌ๋ฅผ ํตํด ์ฝ๋ฌผ์ ์ ํญ์ฑ์ ๊ทน๋ณตํ๋ ๋ฐฉ๋ฒ์ ์ ์ํ ์ ์์๋ค. ํ๋ฉํธํ์ (5-aza-2'-deoxycytidine; 5aza)๋ฅผ ํฌ์ฌํ์ฌ ์๋ฐ์์ผ๋ก ๊ฐ์๋ RIP3 ๋จ๋ฐฑ์ง์ ๋ณต๊ตฌํ ๋ค TMQ0153์ ํฌ์
ํ ๊ฒฐ๊ณผ, TMQ0153๋ง ํฌ์ฌํ ์์ธํฌ๋ณด๋ค ๋ ๋ง์ necroptosis๋ฅผ ๊ด์ฐฐํ ์ ์์์์ ํ๊ดํ๋ฏธ๊ฒฝ์ ํตํด ํ์ธํ์๋ค. TMQ0153์ด ์ฒ๋ฆฌ๋ Necroptosis๊ฐ ์ผ์ด๋ ์ธํฌ๋ก๋ถํฐ HMGB1์ ๋ฐฉ์ถ๊ณผ ์ธํฌ ์ธ ATP์ ์ฆ๊ฐ, Ecto-Calreticulin์ ERp57 ์ ์ธํฌ๋ง ๋
ธ์ถ ๋ฐ ์ฆ๊ฐ๋ฅผ ๊ด์ฐฐํจ์ผ๋ก์จ ์ด ์ฒ์ฐ๋ฌผ์ง์ ๋ฉด์ญ์ธํฌ์ฌ๋ฉธ๊ณผ ์ด์ ๋ฐ๋ฅธ ์ค๊ณ์ฐ๊ตฌ์ ๊ฐ๋ฅ์ฑ์ ์ ์ํ ์ ์์๋ค. ๋ง์ง๋ง์ผ๋ก TMQ0153๊ฐ ์ฝ๋ก๋์ In vivo ์ ๋ธ๋ผํผ์ฌ xenograft ๋ชจ๋ธ์์ ์ข
์ํ์ฑ์ ๋ฐ๋ฌ์ ์ ํดํ๋ ๊ฒ์ ์ถ๊ฐ์ ์ผ๋ก ๊ด์ฐฐํ์๋ค. ๋ฐ๋ผ์ ๋ณธ ์ฐ๊ตฌ๋ฅผ ํตํด ์ ์๋ ๋ค์ํ ์ธํฌ์ฌ๋ฉธ์ ํ์์ ์กฐ์ ํ๋ ์ธํฌ์คํธ๋ ์ค์ ์์ธ ๋ถ์ ๋ฐ ํญ์์ ์ ๋ถ์ ์์ฉ๊ณผ ์ธํฌ์ฌ๋ฉธ ์ ์ด ๊ธฐ์ ์ ๊ท๋ช
ํ์ฌ ํจ๊ณผ์ ์ธ ์ ์น๋ฃ์ ๊ฐ๋ฐ์ ํ์ฉํ๊ณ ์ ํ๋ค.
์ฃผ์์ด
์ธํฌ์คํธ๋ ์ค, ๋ฏธํ ์ฝ๋๋ฆฌ์, ํ์ฑ์ฐ์, ๋ง์ฑ ๊ณจ์์ฑ ๋ฐฑํ๋ณ, ํ๋ก๊ทธ๋จ๋ ์ธํฌ์ฌ๋ฉธOverall 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 (๊ตญ๋ฌธ์ด๋ก) 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 4-40ฮผM 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 -40ฮผM 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