Withanolide compounds (Tubocapsanolide A, B, and C)由Tubocapsium anomalum萃取，而它過去已被報導出具有抑制細胞生長的特性，且也被運用在臨床治療上甚至是一般民間用藥。然而他在細胞中的作用機制卻仍有些還未被研究清楚。因此我們將Tubocapsanolide A, B and C分別處理在大腸癌細胞中，HT29和HCT116，利用MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay 得到可抑制50%細胞的藥物濃度 (IC50)。在這三種化合物之中我們發現Tubocapsanolide A (TA)的抑制效果最好，因此我們之後則利用TA作為此篇論文的研究。結果我們發現在加入TA後可造成caspase 3和PARP的活化，伴隨著處理的時間和劑量的增加細胞凋亡(Apoptosis)的現象也會增加。對於有正常的p53功能的細胞HCT116似乎也對TA引發的細胞凋亡的作用也較p53突變的HT29來的敏感。另一方面我們也利用較低劑量(1μM)的TA分別處理在HCT116和HT29中，也利用了流式細胞儀觀察到TA使HCT116的細胞週期停在G1；而HT29停在G2/M期。這些或許也指出p53參與了TA作用機制。另外也發現TA也會造成sub-G1的累積和reactive oxygen species (ROS)的增加。一旦我們加入抗氧化劑N-Acetyl-L-cysteine (NAC)以後，可使原本被TA所引發的細胞毒殺作用被擷抗。在我們的結果顯示了TA可促進細胞凋亡並且引發細胞內ROS的產生，造成細胞損壞使細胞粒線體膜電位的改變並且活化caspase等現象，最後造成細胞凋亡。

The withanolid compounds (Tubocapsanolide A, B, and C) were purify from Tubocapsium anomalum and shown with cell growth inhibitory property. However, the molecular mechanisms of withanolide type compounds on the cell have not been fully clarified. Tubocapsanolide A, B, and C (TA, TB, and TC) of antiproliferative activity on human colorectal adenocarcinoma cells, HT29 and HCT116, were tested and the inhibitory concentration of 50% cell viability (IC50) for these compounds was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. We found that TA has the best inhibitory effect in these compounds. Therefore we utilized TA to study the mechanism of cell toxicity. The caspase 3 and PARP cleavage experiment results indicated that TA induced apoptosis was time and dose dependent. Cells with functional p53 (HCT116) are more sensitive to TA compared to the mutant p53 cells (HT29). With low dose TA treatment, HCT116 and HT29 are arrested at G1 and G2/M phase respectively. We also found that TA induced sub-G1 accumulation and reactive oxygen species (ROS) release with flow cytometry analysis. Pretreatment of N-Acetyl-L-cysteine (NAC), an antioxidant agent, can reverse the antiproliferation effect by TA. Our results indicated that TA can induce cell apoptosis and intracellular ROS generation. The ROS triggers cells damage and decreases the mitochondria membrane potential and thus induce cell apoptosis.

CHINESE ABSTRACT I
ENGLISH ABSTRACT II
ABBREVIATION III
1 INTRODUCTION 1
1.1General background information 1
1.2 General statement about withanolide compounds 2
1.3 p53 and cancer therapy 8
1.4 Cell death 9
2 MATERIALS AND METHODS 11
2.1 Withanolide compounds (Tubocapsanolide A, B, and C) 11
2.2 Cell lines and cell culture methods 11
2.3 MTT cell proliferation assay 11
2.4 Drug treatment assay 12
2.5 Protein preparation and quantification 13
2.6 Western blot analysis 14
2.7 Flow cytometric analysis 16
2 .7.1 Measurement of DNA content (cell cycle and sub-G1 accumulation) 16
2 .7.2 Annexin V-FITC and PI dual-color staining 16
2 .7.3 Measurement of intracellular ROS 17
2.8 Cell counting and dye exclusion viability assay 17
2.9 Nuclear staining with DAPI 17
2.10 Statistical analysis 17
3 RESULTS 18
3.1 The cytotoxic effect of Tubocapsanolide compounds (Tubocapsanolide A, B, and C) 18
3.2 Tubocapsanolide A induced cell apoptosis 18
3.3 Tubocapsanolide A induced reactive oxygen species (ROS) generation 19
3.4 Tubocapsanolide A induced nuclear condensation 20
3.5 Caspase activation is involved in Tubocapsanolide A mechanism 21
3.6 Tubocapsanolide A induced cell cycle arrest in HT29 and HCT116 21
3.7 p53 is involved in Tubocapsanolide A mechanism 22
4 DISCUSSION 23
4.1 Cytotoxic effect of Tubocapsanolide A 23
4.2 Low concentration (1 μM) of Tubocapsanolide A induced cell cycle arrest 24
4.3 Tubocapsanolide A induced reactive oxygen species (ROS) generation and apoptosis 25
i. Middle concentration (5 μM) 25
ii. High concentration (15 μM) 25
5 CONCLUSIONS 27
6 REFERENCES 29
7 FIGURES AND TABLES 35
Figure 1. Structure of Tubocapsanolide compounds (Tubocapsanolide A, B, and C). 1
Figure 2. Structure of withanolide compounds were reported in references. 4
Figure 3. The major functional domains of the p53 protein. 8
Figure 4. Wt-p53 versus mutant p53: two sides of the same coin. 9
Figure 5. Simplified representation of the two main signalling pathways of programmed cell death. 10
Figure 6. Major pathways to caspase-dependent and caspase-independent cell death 10
Figure 7. The cytotoxic effect for Tubocapsanolide compounds (Tubocapsanolide A, B, and C) in colorectal cancer cells. 31
Figure 8. Tubocapsanolide A induced cell apoptosis. 34
Figure 9. Tubocapsanolide A induced sub-G1 accumulation. 36
Figure 10. Tubocapsanolide A induced reactive oxygen species (ROS) generation. 39
Figure 11. Antioxidant repressed reactive oxygen species (ROS) generation and increased cell proliferation. 46
Figure 12. Tubocapsanolide A induced nuclear condensation. 47
Figure 13. Caspase activation is involved in Tubocapsanolide A mechanism. 53
Figure 14. Tubocapsanolide A induced cell cycle arrest. 55
Figure 15. Western blot analysis of p53, p21, 15, and p27 in HCT116 cells with dose course of TA treatment. 61
Figure 16. The structure of compound 5 from W. Somnifera leaf. 23
Figure 17. The structure of compound 5 from Datura metel L. (Solanaceae). 24
Table 1. The IC50 of Tubocapsanolide compounds for HT29 cytotoxicity
33
Table 2. The IC50 of Tubocapsanolide A for HT29 and HCT116 cytotoxicity 33
Table 3. Tubocapsanolide A induced cell cycle arrest. 60

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