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研究生：	陳乃豪 Chen, Nai-Hao
論文名稱：	探討粒線體DNA 缺陷與gelsolin 表現對癌症抗藥性的影響 A Study in the Effect of Mitochondrial DNA Deletion and Gelsolin Expression in Cancer Drug Resistance
指導教授：	謝達斌 Shieh, Dar-Bin
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	72
中文關鍵詞：	白藜蘆醇、粒線體、DNA 缺陷、口腔癌、抗藥性、細胞毒殺
外文關鍵詞：	Resveratrol, mitochondria, DNA deletion, oral cancer, drug resistance, cytotoxicity
相關次數：	點閱：104 下載：2
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口腔鱗狀細胞癌在全球是十大癌症之一，而在台灣更是在十大癌症當中排行第七名。Cisplatin已知是一種含鉑的有效抗癌藥物之一，廣泛地應用在治療各個部位所發生的惡性腫瘤。然而，病人在進行化學治療時常會演發出抗藥性問題而導致治療過程失敗。關於腫瘤細胞對於Cisplatin產生抗藥性，在過去的研究中發現: 具有抗藥性的癌細胞，Gelsolin蛋白質的表現量相對來說是比較高的，而且會干擾Cisplatin促進細胞凋亡的效果。
粒線體中DNA的缺陷將會使細胞中reactive oxygen species(ROS)濃度增加，並促使正常細胞趨向癌化。腫瘤細胞被發現和正常細胞相比，粒線體DNA具有較多的突變與缺失。近期的研究也指出，腫瘤細胞中粒線體的突變與抗藥性具有密切關係。Resveratrol (RES)與tetrahydroxystilbene glucoside (THSG)都是藥草萃取物，擁有相似的化學結構以及抗氧化特性。由於氧化壓力被視為與腫瘤的生成有關係，因此可以透過RES與THSG的抗氧化特性用以預防和治療癌症。
在本篇研究當中，我們假設粒線體DNA 4977-bp缺陷會影響腫瘤細胞對於化療藥物的敏感性，而RES及THSG在這關係中扮演著重要的角色。我們的結果顯示，具有抗藥性的鼻咽癌細胞株HONE1-CIS6和不具抗藥性的組別HONE-1相比，粒線體DNA 4977-bp缺陷的比例較高，拷貝數較少。而在RES作用後，不論在敏感性或抗藥性的細胞株中，其拷貝數都有明顯的增加，但對4977-bp缺陷的表現則沒有太大的差異。我們也將這兩株細胞株以RES與Cisplatin共同處理後，發現會改善細胞毒殺的效果，促使癌細胞進行細胞凋亡。藉由這些發現，我們期待未來能夠利用天然藥物的特性，發展出安全而有效的藥物。

Oral squamous cell carcinoma is one of the top ten malignancies in the world. According to some reports in Taiwan, oral cancer is ranked seventh leading cause of cancer death. To cure cancer, cisplatin (CDDP) is one of the most potent anti-cancer agents in clinical use for a wide variety of solid tumors. However, chemoresistance is a problem that lowers the therapeutic efficiency and leads to treatment failure. In previous studies, the results indicated that gelsolin (GSN) expression levels were positively associated with chemoresistance in vitro and in vivo. In chemoresistant cells, GSN was highly expressed, and CDDP had no significant effect on inducing apoptosis.
Mitochondrial DNA (mtDNA) defect is known to confer accumulation of intracellular reactive oxygen species (ROS) that contributes the transformation of normal cells to malignant state. Recent study also showed the association between mitochondria mutation and chemoresistant phenotype. Resveratrol (RES) and tetrahydroxystilbene glucoside (THSG) are herbal extract of similar chemical structure known for prominent antioxidant activity. As oxidative stress has been demonstrated to contribute carcinogenesis and tumor progression, RES and THSG have been investigated for their roles in cancer prevention and adjuvant therapy.
In this study, we hypothesize the level of mtDNA 4977-bp deletion would affect chemosensitivity of tumor cells and RES or THSG may play certain role in this regard. Our results showed that 4977-bp deletion was higher in chemoresistant cancer cell (HONE1-CIS6) than their paired chemosensitive line (HONE-1). The total copy number of mtDNA was lower in HONE1-CIS6 than HONE-1. RES treatment significantly increase mtDNA copy number in both cancer cell lines while no significant effect was observed in 4997-bp deletion. The cell viability in two cell lines of RES co-treatment with CDDP was found that there had a great cytotoxic effect to induce apoptosis and enhance the level of apoptotic-related proteins. In the future, we want to dig deeper into the mechanisms of cytotoxic operation and predict the pathway more clearly to confirm our hypothesis.

Abstract	I
中文摘要	III
Acknowledgements	V
Contents	VI
Introduction	1
1	Cancer	1
1.1	Cancer	1
1.2	Therapy of cancer	1
1.3	Head and neck cancer	3
1.4	Therapy of head and neck cancer	4
2	Chemotherapeutic drugs	4
2.1	Cisplatin	4
2.2	Chemotherapy and cell death	5
2.3	Drug resistance in cancer	6
2.4	Drug resistance in head and neck cancer	7
3	Mitochondria and mitochondrial DNA	8
3.1	Mitochondria	8
3.2	Mitochondrial DNA	8
3.3	Dysfunction of mitochondria in cancer	9
3.4	Mitochondrial DNA copy number	10
3.5	Mitochondrial DNA 4977-bp deletion mutations	10
4	Reactive oxygen species in cancer	11
5	Herbal drug extracts	12
5.1	Therapeutic applications of herbal medicines in cancer	12
5.2	Resveratrol and tetrahydroxystilbene glucoside	13
6	Rationale	14
Materials and Methods	16
1	Cell Lines and Cell Culture	16
1.1	Cell Culture (Subculture of Adherent Cells)	16
1.2	Freezing Cells	17
1.3	Thawing Frozen Cells	19
1.4	Counting Cells	19
2	DNA Extraction	20
3	Nucleic Acid Quantitation	21
4	Quantitative Real-Time PCR (Polymerase Chain Reaction)	22
5	Protein Sample Preparation and Protein Isolation	24
6	Protein Assay	25
7	Western Blot	26
7.1	SDS-Polyacrylamide Gel Electrophoresis	26
7.2	Electrotransfer	27
7.3	Antibody Staining (Immnobloting)	28
8	MTT Assay	30
9	Functional Mitochondrial Activity Assay	31
10	Cellular Reactive Oxygen Species Detection Assay	32
10.1	ROS Staining	32
10.2	Flow Cytometry	33
Results	34
1 Analysis of the mitochondrial profiles in HONE-1 and HONE1-CIS6	34
2 Mitochondrial copy number and 4977-bp deletion in HONE-1 and HONE1-CIS6 treatment with CDDP	34
3 Mitochondrial copy number and 4977-bp deletion in HONE-1 and HONE1-CIS6 after treatment with RES	35
4 Cytotoxicity of THSG and RES in HNC cell lines, HONE-1 and HONE1-CIS6 compared with normal cell lines	36
5 Cytotoxicity of RES in HNC cell lines, HONE-1 and HONE1-CIS6 after 24, 48 and 72hrs treatment	36
6 Cytotoxicity of RES with CDDP co-treatment in chemosensitive cancer cell line HONE-1 after 24, 48 and 72hrs treatment	37
7 Cytotoxicity of RES with CDDP co-treatment in chemoresistant cancer cell line HONE1-CIS6 after 24, 48 and 72hrs treatment	38
8 Expression level of protein activation or downregulation in HNC cell lines, HONE-1 and HONE1-CIS6	38
9 Measurement of intracellular ROS concentration in HNC cell lines, HONE-1 and HONE1-CIS6	39
Discussion	41
Summary	48
Reference	49
Figures and Legends	57
Figure 1: Analysis of the mitochondrial profiles in HONE-1 and HONE1-CIS6.	57
Figure 2: Mitochondrial copy number and 4977-bp deletion in HONE-1 and HONE1-CIS6 treatment with CDDP.	59
Figure 3: Mitochondrial copy number and 4977-bp deletion in HONE-1 and HONE1-CIS6 after treatment with RES.	60
Figure 4: Cytotoxicity of THSG and RES in HNC cell lines, HONE-1 and HONE1-CIS6 compared with normal cell lines.	61
Figure 5: Cytotoxicity of RES in HNC cell lines, HONE-1 and HONE1-CIS6 after 24, 48 and 72hrs treatment.	62
Figure 6: Cytotoxicity of RES with CDDP co-treatment in chemosensitive cancer cell line HONE-1 after 24, 48 and 72hrs treatment.	63
Figure 7: Cytotoxicity of RES with CDDP co-treatment in chemoresistant cancer cell line HONE1-CIS6 after 24, 48 and 72hrs treatment.	64
Figure 8: Expression level of protein activation or downregulation in HNC cell lines, HONE-1 and HONE1-CIS6.	65
Figure 9: Measurement of intracellular ROS concentration in HNC cell lines, HONE-1 and HONE1-CIS6. Intracellular ROS concentrations were quantified with DCFDA.	68
Appendix	69
Appendix 1: THSG Modulates mtDNA Copy Number/4977-bp Deletion in Cybrid Cells containing defective Mitochondrial DNA.	69
Appendix 2: Cytotoxicity of THSG in Normal Epithelial Cell Lines, HUVEC and VERO after 24, 48 and 72hrs Treatment.	70
Appendix 3: Cytotoxicity of RES in Normal Epithelial Cell Lines, HUVEC and VERO after 24, 48 and 72hrs Treatment.	71
Appendix 4: Cytotoxicity of THSG in HNC Cell Lines, HONE-1 and HONE1-CIS6 after 24, 48 and 72hrs Treatment.	72


                                    

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