臺灣博碩士論文加值系統

研究背景：儘管已有許多抗癌藥物正廣泛使用，但癌症在國內外仍是主要的疾病死亡原因，因此，研發與合成新穎的抗癌藥物是刻不容緩的。前人文獻中發現，許多對於癌細胞具有抑制作用的藥物都具有���釭熊硎c，能使細胞週期停滯、細胞凋亡而達到抗癌的作用；但卻在癌症治療時會造成病人體重下降或器官傷害等副作用。
研究目的：本研究著重於發展一具有���陬硎c的新穎衍生物1,4-bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene，簡稱為SK228，以細胞及前臨床動物模式探討其對癌細胞的毒殺作用及其機制，並確定其為不造成體重下降或器官傷害的癌症專一性藥物。
研究結果：我們發現在SK228處理下，能有效的對多株肺癌細胞以及食道癌細胞產生毒殺作用，對正常肺細胞則沒有明顯細胞毒性影響，且在處理24及48小時後，可分別由內膜phophatidylserine外翻以及Flow cytometry實驗中發現細胞週期停滯在G2/M時期且走向細胞凋亡；另外我們也發現SK228會藉由結合上DNA minor groove、嵌入DNA、及引發自由基產生而造成DNA損傷。由西方墨點法結果顯示，調控細胞週期通過M時期所需的cyclin B1有不正常的表現量上升，以及作為M時期指標的histone H3 phosphoserine 10表現量上升，顯示SK228主要使細胞週期停滯在M時期，由細胞分子模擬對接實驗中也發現SK228有潛力能結合上tubulin的colchicine結合位。另外在細胞凋亡途徑的觀察中發現，調控內在性/粒線體路徑細胞凋亡的caspase 9及caspase 3有裂解活化的現象，而調控外在性細胞凋亡的caspase 8則沒有改變，顯示SK228處理下是以內在性/粒線體路徑使細胞走向細胞凋亡；進一步檢查調控粒線體外膜通透性的BCL-2蛋白家族成員的表現，更確認了內在性/粒線體路徑被活化，而釋放出粒腺體細胞色素c，引起細胞凋亡。同時在前臨床動物模式實驗結果顯示，SK228在活體也能有效的抑制A549腫瘤生長，且沒有顯著的血清生化學指標變異、主要臟器組織傷害或體重減輕等副作用。動物腫瘤組織之TUNEL分析及活化態caspase 3之免疫組織染色顯示，SK228處理在活體腫瘤中亦能誘導癌組織細胞之細胞凋亡。
結論：本研究為首篇以細胞及動物模式證明，新穎���陬硎c的衍生物SK228具有不造成體重下降或器官傷害的癌症專一性藥物。實驗結果顯示，SK228透過引發DNA傷害使細胞週期停滯在M時期，並誘導內在性/粒線體路徑使細胞走向細胞凋亡，我們認為，SK228有潛力作為新穎的抗癌藥物；未來我們將會針對SK228於細胞骨架microtubule的影響，以及對於抑制癌細胞轉移的能力作深入的探討。

Background. Previous studies have shown the antimitotic effect on various malignancies of indole compounds, which cause cell cycle arrest and cell apoptosis. However, clinical use of conventional drugs with indole structure exhibits many undesirable side effects. Therefore, it is necessary to seek for more efficient and less harmful indole compounds for cancer therapy.
Purpose. This study developed a novel indole compound 1,4-bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene, named SK228. Its effects and mechanism on anticancer growth were examined in cell and animal models.
Results. The present study found that SK228 significantly inhibited cell growth of lung cancer cell lines such as A549, H1299, and CL1-1 and esophageal carcinoma cell lines such as CE48T and KYSE150, without affecting the growth of normal lung cell line IMR90. After treating with SK228 for 24 hours, externalization of inner cell membrane phosphatidylserine with the increase of sub-G1 phase at 48 hours were observed by fluorescent microscope and Flow cytometry, indicating that SK228 induced cell apoptosis. The study further verified that SK228 induced DNA damage by Comet assay, and characterized that SK228-induced damage resulted from DNA minor groove binding, DNA intercalating, and reactive oxygen species production from mitochondria. Western blot analyses showed that SK228 treatment induced abnormally sustained protein level of cyclin B1 and upregulated mitosis marker histone H3 phosphoserine 10, indicating SK228 arrested cell at M phase. Furthermore, SK228 was predicted to bind on colchicine-binding site of tubulin by molecular docking. The increased cytochrome c release into cytosol along with the increase of caspase 3 and caspase 9 cleaved forms without affecting the pro-caspase 8. In addition, the expression levels of BCL-2 family regulators of mitochondrial outer membrane permeabilization were also affected. These results indicated that SK228 induced intrinsic/mitochondrial-mediated apoptosis pathway. Animal study showed that SK228 remarkably reduced tumor size in nude mice bearing human A549 lung carcinoma tumor xenograft without significant side effects examining by serum biochemistry, hematology tests, and histological sections of major organs. TUNEL assay and immunohistochemistry of cleaved caspase 3 in tumors from SK228-treated animals confirmed that SK228 inhibited cancer cell growth by initiating apoptosis.
Conclusion. The present study provides first evidence that SK228 shows cancer cell-specific cytotoxicity by mitochondria-mediated apoptosis resulting from DNA damage. In addition, SK228 demonstrates in vivo anti-tumor activity against human xenograft in murine model. SK228 has potential to be tested as a pharmaceutical compound for cancer treatment. The effects of SK228 on microtubule and cancer cell metastasis will be further examined.

Introduction---------------------------------------------------------1
I.Clinical Significance of Lung Cancer-------------------------------1
II.Clinical Significance of Esophageal Cancer------------------------2
III.Overview of Cell Cycle-------------------------------------------4
i.Cell cycle---------------------------------------------------------4
ii.Cyclins and cyclin-dependent kinases (CDKs)-----------------------5
iii.Cyclin-dependent kinases inhibitors (CKIs)-----------------------6
iv.Cell cycle checkpoint---------------------------------------------7
v.Cell cycle and cancer----------------------------------------------8
IV.Overview of Apoptosis---------------------------------------------9
i.Apoptosis----------------------------------------------------------9
ii.Caspases (cysteine-dependent aspartate-specific proteases)-------10
iii.BCL-2 family (B-cell lymphoma 2)--------------------------------11
iv.Pathways of apoptosis--------------------------------------------12
v.Apoptosis and cancer----------------------------------------------14
V.Reactive Oxygen Species (ROS)-------------------------------------14
VI.Compounds with Indole Structure----------------------------------15
VII.1,4-bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene (SK228)-------17
Specific Aims-------------------------------------------------------18
Materials and Methods-----------------------------------------------19
I.Materials---------------------------------------------------------19
i.Cell lines--------------------------------------------------------19
ii.1,4-bis(di(5-hydroxy-1H-indol-3-yl)methyl)benzene (SK228)--------19
iii.Animal model----------------------------------------------------20
II.Methods----------------------------------------------------------20
i.Cell culture------------------------------------------------------20
ii.Compound cytotoxicity assay/MTT assay----------------------------20
iii.Early apoptosis detection/Phosphatidylserine (PS) staining------21
iv.Analysis of cell cycle distribution------------------------------22
v.Determination of DNA damage/Comet assay---------------------------22
vi.DNA minor groove binding effect/Hoechst dye 33342 displacement assay--------23
vii.DNA intercalating measurement/Plasmid DNA circle-linear assay--------------24
viii.Determination of intracellular reactive oxygen species (ROS)---25
ix.Western blot analysis--------------------------------------------25
x.Molecular Docking-------------------------------------------------27
xi.Subcutaneous implantation of cancer cells in animals and monitoring of in vivo anti-tumoral activity after drug treatment--------27
xii.TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay------------------------------------------------------29
xiii.Immunohistochemistry-------------------------------------------30
Results-------------------------------------------------------------31
I.SK228 sufficiently inhibits cell growth of various lung cancer cell lines and esophageal carcinoma cell lines---------------------31
II.SK228 inhibits cancer cell growth through G2/M phase arrest and apoptosis--------------------------------------------------------31
III.SK228 causes DNA damage through binding on DNA minor groove, intercalating to DNA, and induction of ROS production--------------32
IV.SK228 alters p53/p21 and cyclin B1 protein level and arrests cell cycle at M phase-----------------------------------------------34
V.SK228 treatment activates intrinsic/mitochondrial-mediated apoptosis pathway------------------------------------------------------36
VI.SK228 remarkably reduces tumor growth in vivo without significant side effects---------------------------------------------------36
VII.SK228 induces apoptosis in animal tumor tissue------------------38
Discussion----------------------------------------------------------39
References----------------------------------------------------------44
Figures-------------------------------------------------------------51

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