초록 ▼

○ 연구내용 및 방법
TB4의 역할 및 기능을 규명하기 위한 연구는 다음과 같은 방법들을 사용하여 수행하였다.
1. 항암제 내성 발현에 대한 TB4의 역할을 규명하기 위하여 ① HeLa 세포에 TB4 유전자를 transfection한 후 안정적으로 발현하는 TB4 과발현 세포주를 제작함. ② TB4가 과발현된 세포의 성장을 wildtype의세포와 비교학나, paclitaxel을 처리한후에 세포사멸정도를 비교함. ③ TB4의 siRNA를 transfection하여 TB4를 konckdown 시킨 후 paclitaxel에 대

○ 연구내용 및 방법
TB4의 역할 및 기능을 규명하기 위한 연구는 다음과 같은 방법들을 사용하여 수행하였다.
1. 항암제 내성 발현에 대한 TB4의 역할을 규명하기 위하여 ① HeLa 세포에 TB4 유전자를 transfection한 후 안정적으로 발현하는 TB4 과발현 세포주를 제작함. ② TB4가 과발현된 세포의 성장을 wildtype의세포와 비교학나, paclitaxel을 처리한후에 세포사멸정도를 비교함. ③ TB4의 siRNA를 transfection하여 TB4를 konckdown 시킨 후 paclitaxel에 대한 세포사멸효과를 비교함. ④ TB4가 과발현된 세포와 wildtype 세포에 paclitaxe을 처리한후 신호전달 단백질들의 변화를 확인하기 위하여 western blot 분석을 수행함.
2. 세포사멸에 미치는 TB4의 역할을 규명하기 위하여 ① 암세포를 paclitaxel로 처리한후 cell lysate를제작하고, Western blot 방법을 이용하여 세포사멸인자들의 변화를 확인함. ② 암세포를 TB4 단백질이 포함되거나 포함되지 않은 상태로 paclitaxel로 처리한 후 cell lysate를 제작하고, western blot 방법을 이용하여 세포사멸 인자들의 변화를 비교함. ③ HeLa 세포주와 TB4유전자를 과발현하는 HeLa 세포주의 paciltaxel에 대한 세포사멸효과를 비교함. ④ TB4의 상대적인 양이 서로 다른 위암 세포주 SNU638과 SNY668을 이용하여 paclitaxel에 대한 세포 사멸효과를 비교함. ⑤ TB4의 siRNA를 transfection하여 TB4를 konckdown 시킨후 paclitaxel 에 대한 세포사멸효과를 비교함.
3. 저산소 유도인자 HIF1 - ${\alpha}$ 발현에 대한 TB4의 역할을 규명하기 위하여 ① B16마우스 melanoma cell을 이용하여 침윤에 대한 연구를 수행하였고, TB4가 과발현된 세포를 이용하여 스크래칭 실험을 통해 세포의 이동에 대한 연구를 수행하여 예비데이타를 확보함. ② 암세포를 paclitaxel로 처리하거나, TB4 단백질을 처리한후 cell lysate를 제작하고, Western blot 방법을 이용하여 VEGF단백질을 분석하거나 RT-PCR을 이용하여 VEGF mRNA를 분석함. ③ TB4 단백질에 의한 HIF-1${\alpha}$발현과 Erk의 활성화를 측정함. 세포배양시 Erk 억제제인 PD98059를 처리한 경우의 paclitaxel에 의한 세포사멸을 PD98059를 처리하지 않은 경우와 비교함. MTT assay와 westernblot을 사용함. ④ TB4 단백질에 의한 HIF-1A의 발현을 측정한 후 paclitaxel에 의한 세포사멸 효과를 normoxia와 hypoxia 조건에서 비교함.
4. TB4 단백질의 구조 분석조건을 다음과 같이 확림하였다. ① TB4는 아미노산이 43개인 5kd 정도의 단백질이므로 TB4 단백질의 직접적인 역할을 연구하고자 TB4 유전자를 E. coli에서 발현시킨 후 affinity column을 이용하여 TB4 단백질을 분리 정제함. ② NMR을 이용하여 TB4 단백질에 대한 NMR 측정 조건을 확림함.
5. TB4 발현 동물 모델을 다음과 같은 방법을 통하여 확보하고 암전이에 대한 예비데이타를 확보하였다. ① TB4의 생리학적인 역할을 확인하는 것이 실험실적 방법을 통한 연구보다 더 중요하고 의미있다고 사료되어 TB4를 과발현하는 transgenic mouse 생산을 위한 targeting vector를 제작함. ② 제작된 TB4 유전자의 targeting vector를 과배란시켜 확보한 수정란에 주사한 후, 대리모에 이식하여 littermate를 확보하고 genotyping을 통하여 TB4를 발현하는 마우스를 선별함. ③ 정상마우스와 TB4 과발현 마우스에 luciferase를 발현하고있는 흑색암 세포를 피하이식한 후 양전자 양상기(PET)와 vernier caliper로 종양의 성장을 측정하고, 최종일에 마우스를 폐로의 전이를 확임함.

Abstract ▼

It is challenging for tumor therapeutics not to spread from one part of the body to
another via the bloodstream or across a body cavity. Thymosin beta-4 (TB4) is an
actin-sequestering protein to enhance cell migration and to interact signaling molecules.
The purpose of this study is to unde

It is challenging for tumor therapeutics not to spread from one part of the body to
another via the bloodstream or across a body cavity. Thymosin beta-4 (TB4) is an
actin-sequestering protein to enhance cell migration and to interact signaling molecules.
The purpose of this study is to understand a function and a role of TB4 on tumor cells
and tumor cell's microenvironment. We investigated molecular mechanisms of TB4 on
tumor cell growth and death by using TB4-expressing stable cell line or TB4 protein. We
also established experimental condition for NMR analysis with TB4 protein and transgenic
mouse model TB4 gene. The results obtained in this project were summarized below.
To investigated the effect of TB4 on paclitaxel-induced tumor cell death, we
overexpressed TB4 in Hela cells (TB4-Hela) and examined the effect of TB4 in
paclitaxel-induced cell death. TB4-Hela cells showed a higher growth rate and a lower
percentage of basal apoptosis than Hela cells. TB4-Hela cells were more resistant to
paclitaxel-induced cell death than Hela cells. TB4 transcript expression with paclitaxel
treatment was dose-dependently increased in Hela cells but that was not in
TB4-Helacells. Small interfering RNA (siRNA) of TB4 inhibited Hela cell growth and
enhanced paclitaxel-induced cell death. Basal ERK phosphorylation was elevated and basal
p38 kinase phosphorylation was reduced in paclitaxel non-treated TB4-Hela cells. When
treated with paclitaxel, cell death and resistance-induction were independent of ERK and
p38 kinase activation. Paclitaxel-resistance of TB4-Hela cells was overcome by the
inhibition of basal ERK activity with PD98059 pre-treatment. The inhibition of basal p38
kinase activity with SB203580 pre-treatment attenuated the paclitaxel-induced Hela cell
death. In conclusion, TB4 induced paclitaxel-resistance through the elevation of basal
level of ERK phosphorylation. Therefore, TB4 could be a novel target to regulate
paclitaxel-resistance.
We investigated the inhibitory effect of TB4 peptides on tumor cell death by the
incubation with paclitaxel. The effect of TB4 peptides was assayed by the measurement of
caspase-3 activity, G2/M arrest, and Bcl-2 phosphorylation. Cell survival rate was increased
and caspase-3 activity was decreased by the treatment with TB4 peptides. In contrast,
small interfering RNA (siRNA) of TB4 inhibited cellviability and augmented caspase-3
activity. A significant change in Bcl-2 phosphorylation was detected by TB4peptide
treatment or by the overexpression of TB4 gene in Hela cells. The reduced population
in G2/M phase by TB4 peptide treatment was correlated with the decreased expression of
cyclin B1. Above data were confirmed in gastric tumor cell lines, SNU 638 (low TB4
level) and SNU 668 (high TB4 level) that was established from clinically isolated gastric
tumors. In conclusion, soluble TB4 peptides produced in cancer cells could be an obstacle
to treat tumor with paclitaxel. Therefore, TB4 could be a novel target to control
paclitaxel-resistance.
Vascular endothelial growth factor (VEGF)and hypoxia-inducible factor 1 (HIF1) are
important regulators of neovascularization. HIF1 regulates VEGF expression at
transcriptional level. Here, we investigated whether paclitaxel-treatment affects VEGF
expression for the development of paclitaxel-resistance. Paclitaxel-treatment induced
dose-dependent cell death and increased VEGF expression. Paclitaxel also induced NF-kB
activation and stabilized HIF1- , which stimulated luciferase activity α of HIF-1a response
element (HRE) on VEGF gene. As paclitaxel-treatment produced reactive oxygen species
(ROS), VEGF expression was increased by H2O2 treatment and reduced by various ROS
scavengers such as N-acetyl-L-cysteine (NAC), pyrrolidine dithiocarbamate (PDTC), and
diphenylene iodonium (DPI). Paclitaxel-induced cell death was aggravated by the
incubation with NAC, DPI, and PDTC. Collectively, it suggests that paclitaxel-induced
VEGF expression could be mediated by paclitaxel-induced ROS production through NF-kB
activation and HIF-1a stabilization, which could affect resistance-induction to antitumor
therapeutics during cancer treatment.
We investigated whether TB4 proteins (TB4P) affect tumor microenvironment by
measuring hypoxia inducible transcription factor (HIF)1-α stabilization in cervical tumor
cells, since TB4P reduced paclitaxel-induced cell death rate. TB4P increased HIF1-α
stabilization and transactivation, which is measured by the increase of hypoxia response
element (HRE)-luciferase activity and target gene, vascular endothelial growth factor
(VEGF) transcription. TB4P also elevated ERK phosphorylation. PD98059, ERK inhibitor
reduced HIF-1aincreased by TB4P. Paclitaxel-induced cell death was inhibited by hypoxia
conditioning that increased HIF1-α stabilization and ERK phosphorylation. PD98059
reversed paclitaxel-induced cell death which was attenuated by hypoxia. Collectively,
TB4P could lead tumor cell microenvironment to hypoxia condition, which might be
resulted in antitumor drug-resistance induction. It suggests that soluble TB4P could be a
novel target to control tumor cell death by regulating tumor cell microenvironment.

목차 Contents

• 제출문...1
• 목차...2
• I. 연구개발결과 요약문...3
• - 한글 요약문...3
• - 영문 요약문...5
• II. 연구개발과제 연구결과...7
• 1. 연구개발과제의 최종 연구개발 목표...7
• 2. 연구개발과제의 연구대상 및 방법...17
• 3. 연구개발과제의 연구개발결과...19
• 4. 연구개발과제의 연구결과 고찰 및 결론...37
• 5. 연구개발과제의 연구성과 및 목표달성도...40
• 6. 연구개발과제의 활용계획...51
• 7. 참고문헌...51
• 8. 첨부서류...52