臺灣博碩士論文加值系統

大腸癌是惡性腫瘤中三大死亡原因之一。根據先前研究顯示，Metarhizium Anisopliae 的二次代謝產物黑殭菌素B (Destruxin B)具有抑制人類大腸癌細胞株 HT-29 生長的功效。本實驗目的為比較destruxin B 在動物體內、體外抗大腸癌細胞活性的能力，以及研究其在動物體內抑制大腸癌的可能機制。根據Chen 等人(1999) 的研究分離純化出黑殭菌素，並且利用分析型HPLC、ESI-MASS 以及 1H NMR 確定其成分。不同濃度的黑殭菌素B 作用於HT-29 細胞株有其明顯的抑制效果，並呈現劑量相關趨勢。在48 小時作用後，黑殭菌素B 對於HT-29 細胞株的細胞半抑制濃度為(IC50 )為2μM。在動物實驗結果中顯示，低劑量的黑殭菌素B (0.6 mg/kg/day) 能夠抑制荷瘤老鼠 (xenograft) 腫瘤生長。無論是以黑殭菌素B 或是臨床大腸癌用藥5-FU治療兩周後，caspase-3 可被活化。因此，黑殭菌素B 可望成為治療人類大腸癌腫瘤的新一代藥物。

Colon carcinoma is one of the most common malignant diseases worldwide. Previous studies reported destruxins B, one of the secondary metabolites of Metarhiziu anisopliae has potent growth suppression effect on human HT-29 adenocarcinoma cells. The purpose of this study is to compare, in vitro and in vivo, the anti-colon cancer activities of destruxin B and investigate the in vivo anti-colon cancer pathway of destruxin B. The destruxins were isolated and purified according to the previous report (Chen et al., 1999) and characterized by HPLC, ESI-MASS and 1H NMR spectroscopies. A significant inhibition in cell viability was observed upon treatments with various concentrations of destruxin B and was in a dose-dependent manner. The IC50 of destruxin B at 48 hours was 2μM on HT-29. At the end of the experiment, Destruxin B, at low dose concentration ( 0.6 mg/kg/day) could inhibit tumor growth in a xenograft mice model and the caspase-3 is activated after 2 weeks treatment of destruxin B. In conclusion, destruxin B was suggested potentially to be explored as a new therapeutic agent against human colorectal carcinoma cancer.

中文摘要..................................................I
ABSTRACT................................................ II
LIST OF TABLES & FIGURES .............................. VII
GLOOSARY ................................................IX
1. INTRODUCTION.......................................... 1
1.1 DESTRUXINS........................................... 1
1.1.1 Metarhizium anisopliae and it’s secondary metabolism ..... 1
1.1.2 Destruxins......................................... 1
1.1.3 In vitro studies of destruxins .................... 4
1.2 COLON CANCER......................................... 5
1.2.1 Epidemiology of colorectal cancer in Taiwan........ 6
1.2.2 Chemotherapy for colon cancer ..................... 8
1.2.3 Prevention of colon cancer......................... 9
1.3 AIMS AND OBJECTIVES................................. 10
2. MATERIALS & METHODS.................................. 11
2.1 CHEMICALS AND REAGENTS ............................. 11
2.2 PRODUCTION OF DESTRUXINS............................ 11
2.3 PURIFICATION OF DESTRUXIN B ........................ 12
2.4 CELL LINES AND CELL CULTURE ........................ 14
2.5 CELL VIABILITY ASSAY ............................... 15
2.6 PREVENTION AND TUMOR XENOGRAFTS .................... 15
2.6.1 Body weight changing rate (BCRC) ................. 17
2.6.2 Tumor increasing rare ............................ 17
2.7 TREATMENT OF XENOGRAFT-BEARING MICE ................ 17
2.8 EVALUATION OF CASPASE-3 ACTIVITY ................... 18
2.9 WESTERN BLOTTING.................................... 18
2.10 STATISTICAL ANALYSIS .............................. 19
3. RESULTS.............................................. 20
3.1 DESTRUXIN B ISOLATION AND PURIFICATION.............. 20
3.2 DESTRUXIN B ANALYSIS ............................... 23
3.3 DESTRUXIN B DEGRADATION ASSAY....................... 25
3.4 CELL VIABILITY ASSAY ............................... 26
3.5 DESTRUXIN B IN VIVO TEST............................ 27
3.5.1 Prevention........................................ 27
3.5.2 Treatment......................................... 31
3.6 CASPASE-3 ACTIVITY ................................. 40
3.7 WESTERN BLOTTING.................................... 44
4. DISSCUSION & CONCLUSION ............................. 46
5. REFERENCES........................................... 51
APPENDIX ............................................... 59
Table 1 Destruxin B anticancer in vitro ability ....................................... 5
Table 2 FDA approved medicine for colon cancer.................................. 9
Fig. 1 Chemical structures of the five destruxins isolated and purified
from Metarhizium anisopliae. ......................................................... 3
Fig. 2 The stage of colon cancer.............................................................. 6
Fig. 3 The mortality of colon cancer for male and female in Taiwan. .... 7
Fig. 4 Age-specific incidence and mortality of colon cancer.................. 8
Fig. 5 The flow chart of Destruxin B production and purification. ...... 14
Fig. 6 Preparative reversed-phase HPLC chromatographic plot of the
crude extract sample. ..................................................................... 21
Fig. 7 HPLC analysis of purified DB from the preparative HPLC....... 22
Fig. 8 Positive ESI mass spectrum of Destruxin B............................. 23
Fig. 9 1H NMR spectrum (500 MHz) of Destruxin B in Acentonitrile. 24
Fig. 10 HPLC analysis of Destruxin B content storage at 4℃. ............ 25
Fig. 11 Effects of Destruxin B on the cell viability in HT-29 human colon
carcinoma cells. ............................................................................. 27
Fig. 12 The preventative effects of destruxin B and Aspirin on tumor growth and body weight change of human xenograft Balb/c nude
mice. .............................................................................................. 30
Fig. 13 The body weights of treatment groups...................................... 34
Fig. 14 The body weight changing rate (BWCR) of treatment groups. 35
Fig. 15 Spleen weights of treatment groups.......................................... 36
Fig. 16 Tumor increasing rates of treatment groups. ............................ 38
Fig. 17 Normalized tumor volume of HT-29 xenograft........................ 39
Fig. 18 pNA calibration curve for caspase-3 colorimetric quantification
assay............................................................................................... 41
Fig. 19 Caspase-3 activity of HT-29 xenograft treated with Destruxin B.
....................................................................................................... 43
Fig. 20 Effect of destruxin B on apoptosis of HT-29 xenograft tumor. 45

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