泰莫西芬與BP012W乙醇分離物之協同作用造成強化管狀A型乳腺癌細胞凋亡影響

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研究生：	鄧棨文 Chi-Wen Deng
論文名稱：	泰莫西芬與BP012W乙醇分離物之協同作用造成強化管狀A型乳腺癌細胞凋亡影響 Synergy effects of tamoxifen and the ethanol fractions of BP012W to enhance apoptosis of luminal-A breast cancer cells
指導教授：	蘇立仁 Li-Jen Su
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 系統生物與生物資訊研究所 Graduate Institute of Systems Biology and Bioinformatics
論文出版年：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	79
中文關鍵詞：	乳癌、中草藥、泰莫西芬、合併治療
外文關鍵詞：	Breast cancer, Traditional Chinese Medicines, Tamoxifen, Synergistic anticancer effects
相關次數：	點閱：8 下載：0
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乳癌一直是女性死亡的主要原因，而且死亡人數也是逐年上升。目前最為廣泛使用的化癌藥物，泰莫西芬，作為乳癌的賀爾蒙治療藥物，但若是長期使用容易衍生抗藥性。雖然泰莫西芬效果好，但也會造成像是子宮頸癌和血管栓塞的不良副作用。在中國，傳統中草藥早已用在治療癌症許多年了，其特性相較於化癌藥物，除了毒性較低，也比較少衍生出不好的副作用。本文利用高通量細胞學藥物篩選系統，從60種此類中草藥篩選出蒲公英的萃取物BP012W，BP012W對於管狀A型乳癌細胞株MCF-7表現很強的抑制生長的效果，因此本文我們選擇BP012W做更深入的研究。我們接著用西方墨點法，發現管狀A行乳癌細胞株MCF-7的BAX蛋白質表現量隨著藥物治療而上升，同時Bcl-2的蛋白質表現量則隨之下降。最後我們利用玻璃管柱層析法，將BP012W做藥物分層，依據乙醇濃度變化，0%、20%、40%、60%、80% 和99.9%，並發現只有0% 和20% 仍保持原藥物抑制生長的特性，因此我們將0% 和20% 做合併為EP1，確實發現EP1疊加了0% 和20% 的效果表現最好，最後我們用泰莫西芬做合併治療，發現在管狀A型乳癌細胞株MCF-7，EP1的合併效果比BP012W的合併效果來的好。BP012W和其分成翠取的EP1可能是一個有希望的候選藥物作為乳腺癌治療化療上。

Breast cancer has been ranked the first leading cause of death among female, and the numbers have been rising over the years. Tamoxifen is the most widely used anti-estrogen for the treatment of breast cancer, but resistance often develops due to long-term use. In addition, adverse side effects including uterine cancer and thromboembolic disease have been attributed to the use of tamoxifen. Traditional Chinese Medicines (TCMs) have been applied for the treatment of cancer in China for many years due to low toxicity and side effects. In this study, we screened 60 TCMs and found that BP012W, one of the families of Asteraceae, showed strong growth inhibition toward luminal-A breast cancer cell line MCF-7, thus was chosen for further studies. The western blot analysis indicated that BAX proteins were increased in expression while BCL-2 was decreased in expression. The DIAION column extraction was performed for isolation of pure compounds from BP012W using different concentrations of ethanol, 0%, 20%, 40%, 60%, 80% and 99.9%, and found that the concentrations at 0% and 20% were most effective in inhibiting proliferation of cells than the crude extract and was denoted EP1. Furthermore, BP012W showed stronger growth inhibition of MCF-7 cells when in combination with tamoxifen than applying tamoxifen alone. BP012W may be a promising candidate as a chemosensitizer for breast cancer treatments.

中文摘要    i
Abstract    ii
Table of contents    iii
List of tables    v
List of figures    vi
List of supplements tables    vii
List of supplements figures    viii
1. Introduction    1
1-1. Breast cancer    1
1-2. Molecular genetics of breast cancer and targeted therapy    2
2-1. Cell line & drugs    5
2-2. Preparation of BP012W    5
2-3. Preparation active compound fractionation and isolation form BP012W    7
2-4. Cell viability measurement    8
2-5. Analysis of apoptosis by flow cytometry assay    8
2-6. Preparation of protein extracts    9
2-7. Western blot assay    9
2-8. Preparation of RNA extracts    11
2-9. Microarray hybridization    12
2-10. Data analysis    12
2-11. Differential expression analysis    13
2-12. Function and pathway analysis    13
3. Results    14
3-1. In vitro cytotoxicity study by alamar blue assay    14
3-2. Active compound fractionation and Isolation from BP012W    16
3-3. Synergistic anti-proliferative of BP012W and tamoxifen    19
3-3-1. Promotion of tamoxifen-induced apoptosis by BP012W    19
3-3-2. Apoptosis analysis by flow cytometry    21
3-3-3. Differential gene expression and bioinformatics analysis    23
3-4. Bioinformatics analysis    25
3-4-1. Genome-wide expression profiling    25
3-4-2. Data quality assurance    25
3-4-3. Differential expression analysis    26
3-4-4. Function and pathway analysis    27
4. Discussion    38
5. Reference    39
6. Supplements    41
7. Appendix    51

 
List of tables

Table 1. The extraction of chemical compounds from BP012W using different concentrations of ethanol.     18
Table 2. Quality Assessment of Microarray Gene Expression Data.     26
Table 3. Enriched biological term and biological function analysis of the genes selected as differentially expressed between Tam-treated and untreated MCF-7cells.     28
Table 4. Enriched biological term and biological function analysis of the genes selected as differentially expressed between EP1-treated and untreated MCF-7cells.     29
Table 5. Enriched biological term and biological function analysis of the genes selected as differentially expressed between Tam+EP1-treated and untreated MCF-7cells.     30
Table 6. Enriched biological term and biological function analysis of the genes selected as differentially expressed between EP2-treated and untreated MCF-7cells.     31
Table 7. Enriched biological term and biological function analysis of the genes selected as differentially expressed between Tam+EP2-treated and untreated MCF-7cells.     32
 
List of figures

Figure 1. A detailed extraction procedure for TCM was established. Water-soluble compounds for BP012 was extracted by condenser.     6
Figure 2. Active compound fractionation and Isolation form BP012W.     7
Figure 3. The different effectivenesses of BP012W on suppressing breast cancer subtypes.     15
Figure 4. Effect of crude BP012W and its ethanol extracts on the viability of MCF-7 cells.     17
Figure 5. Enhancement of tamoxifen-induced apoptosis in breast cancer cell lines by BP012W and isolated compounds.     20
Figure 6. Promotion of tamoxifen-induced apoptosis by BP012W and EP1.     22
Figure 7. Western blot analysis of BP012W and EP1 treated alone or combination with tamoxifen for 72 hrs.     24
Figure 8. Functional analysis for MAPK and APK expression levels in different treatment on MCF-7 cell lines.     33
Figure 9. Function analysis for expression of TGF-β to inhibit tumorigenesis while treatment with Tam+EP1.     34
Figure 10. Function analysis for negative regulation of SMAD protein complex assembly switching off the biological response.     35
Figure 11. Function analysis for overexpressing immune response genes had better prognosis.     36
Figure 12. Apoptosis pathway from KEGG pathway.     37

List of supplements tables

Table S1. Estimated New Female Breast Cancer Cases and Deaths by Age, US, 2015*.    41
Table S2. Experiment buffer reagents for drugs screening.    42
Table S3. Molecular subtpyes of breast cancer and systemic treatment recommendations for subtypes.    43
Table S4. RNA QA/QC information.     44
Table S5. Quality control by Expression Console™ 1.3.1.187     45
Table S6. Complete summary table of functional analysis     51

 
List of supplements figures

Figure S1. That is RNA quality information for array analysis.    41
Figure S2. Effect of crude BP004W and its methanol extracts on the viability of MCF-7 cells.    47
Figure S3. Isolation from BP012W by separatory funnel with organic solvents.    43
Figure S4. Tam-R cells were derived by long-term exposure to tamoxifen and grown under the same conditions as wild-type MCF-7 cells.     49
Figure S5. Western blot analysis of tamoxifen treated alone or combination with ethanol extracts form BP012W for 72 hrs.     50
                                

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