臺灣博碩士論文加值系統

本論文研究的目的是在了解在乳突性甲狀腺癌中粒線體DNA（mitochondrial DNA）與BRAFV600E突變（papillary thyroid carcinoma）的關聯，並研究一種糖酵解抑制劑─2-去氧葡萄糖（2-deoxyglucose）在治療乳突性甲狀腺癌和神經母細胞瘤（neuroblastoma）中的角色。乳突性甲狀腺癌是最常見的甲狀腺癌，以BRAFV600E突變最常見。此外，粒線體DNA突變或含量的改變被認為有助於癌細胞對抗癌藥物產生抗藥性。研究結果顯示不到20%病人的乳突性甲狀腺癌病灶組織裡有粒線體DNA D-環突變和4977 鹼基對（base pairs）缺失；而乳突性甲狀腺癌裡的粒線體DNA含量（mtDNA content）增加與BRAFV600E突變有關。我們利用兩株乳突性甲狀腺癌細胞：BCPAP（有BRAFV600E突變）和CG3（無BRAFV600E突變）細胞進行實驗。BCPAP細胞比CG3細胞更依賴有氧糖酵解，乳突性甲狀腺癌的細胞增殖正相關於能量供給。合併使用2-去氧葡萄糖及兩種美國批准可以用在治療晚期乳突性甲狀腺癌的藥物，阿黴素（doxorubicin）和索拉非尼（sorafenib），發現無論是否有BRAFV600E突變，2-去氧葡萄糖可增加乳突性甲狀腺癌細胞對這兩種藥的敏感性，並將其半抑制率（IC50）各下降至單獨使用時的70%及50%以下。因此，同時使用低劑量的2-去氧葡萄糖和較低劑量的阿黴素或索拉非尼，可提供等同於高劑量的阿黴素或索拉非尼的抗癌作用。
研究也探討2-去氧葡萄糖對孩童最常見的顱外實體瘤─神經母細胞瘤的影響，一般認為具有MYCN擴增的神經母細胞瘤較依賴有氧糖酵解也對於治療較有抗藥性。2株神經母細胞瘤細胞被選擇來進行小鼠異種移植實驗：SK-N-DZ 細胞(有MYCN擴增)及SK-N-AS細胞(無MYCN擴增)，並一週兩次於腹腔內注射生理食鹽水或2-去氧葡萄糖治療三週後將腫瘤取出。結果顯示不論有無MYCN擴增，2-去氧葡萄糖會經由降低HIF-1α，PDK1和p-Bad的表現，且在SK-N-AS細胞中抑制c-Myc的表現，進而減緩腫瘤成長。此外，2-去氧葡萄糖促使內皮細胞凋亡及抑制偽足的生成而減少腫瘤血管新生。因此，使用2-去氧葡萄糖治療，可以同時獲得抑制癌細胞及內皮細胞的生長的雙重效益。
綜合以上結果，2-去氧葡萄糖不論在單獨或併用其它抗癌藥，均可作為治療乳突性甲狀腺癌和神經母細胞瘤病人的一種新選擇。

The purpose of this research was to investigate the changes in mitochondrial DNA (mtDNA) associated with the BRAFV600E mutation in papillary thyroid carcinoma (PTC), and the role of 2-deoxyglucose (2-DG), a glycolysis inhibitor, in PTC and neuroblastoma (NB). PTC is the most common form of thyroid cancer, and the BRAFV600E mutation is the most common genetic aberration. MtDNA mutations and the changes of mtDNA content were thought to facilitate chemotherapy resistance in cancer cells. Less than 20% of human PTC shows somatic D-loop mutation and mtDNA 4977 bp deletion. Moreover, an increase of mtDNA content in PTC is associated with the BRAFV600E mutation. The bioenergetic function and response to 2-DG of two PTC cell lines, BCPAP cells (with the BRAFV600E mutation) and CG3 cells (without the BRAFV600E mutation), were studied. BCPAP cells are more glycolytic than CG3 cells, and energy depletion is responsible for inhibiting proliferation of both PTC cells. Chemosensitivity to doxorubicin and sorafenib, two USFDA-approved drugs for treating advanced PTC, increased in both cell lines when combination with 2-DG for 48 h. The IC50 of doxorubicin in BCPAP and CG3 cells fell by more than 30%, and the IC50 of sorafenib in both cells also fell by more than 50% when combined therapy with only 0.0625 mM 2-DG. This suggests that 2-DG with lower doses of either doxorubicin or sorafenib will provide anticancer effects which can otherwise be achieved only with high doses of doxorubicin or sorafenib alone, regardless of the status of the BRAFV600E mutation.
Because of its significantly positive effects on PTC, the effects of 2-DG on NB was investigated. NB is the most common extracranial solid tumor in childhood with wide genetic variations. MYCN amplification in NB is correlated with glycolysis and resistance to treatment. We implanted the MYCN-amplified SK-N-DZ and the MYCN-nonamplified SK-N-AS cell lines into the subcutaneous tissue of NOD/SCID mice with 6 intraperitoneal injections of normal saline or 2-DG (2 doses per week). Our results demonstrated tumor shrinkage was significant in mice treated with 2-DG, regardless of MYCN amplification or not. 2-DG inhibited tumor growth by downregulating HIF-1, PDK1, and p-BAD in both NB xenografts and downregulating c-Myc in SK-N-AS xenografts. Moreover, 2-DG inhibited angiogenesis by apoptosis and suppressing lamellipodia and filopodia formation of endothelial cells. Our findings indicate that 2-DG treatment in mice simultaneously targeted tumor cells and cancerous endothelial cells.
Taken together, our findings indicate that 2-DG, alone or combined with other chemotherapeutic agents, might provide novel and more efficacious therapy for patients with PTC and NB.

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口試委員會審定書
誌 謝............................................................................. iii
中文摘要........................................................................... iv
Abstract….......................................................................... vi
Table of Contents................................................................ viii
Directory of Figures............................................................. xiii
Directory of Tables ................................................................xv
Abbreviations..................................................................... xvi
CHAPTER I. INTRODUCTION..............................................................1
1.1 Background ......................................................................1
1.1.1 Metabolic reprogramming and changes of microenvironment in cancers.............1
1.1.2 Alterations of mitochondrial DNA in cancers ...................................2
1.1.3 Papillary thyroid carcinomas and therapy ......................................3
1.1.4 Characteristics of neuroblastoma and therapeutic implication ..................4
1.1.5 Angiogenesis and glycolysis....................................................5
1.1.6 2-Deoxyglucose and its potential applications .................................6
1.2 Study aims and hypotheses .......................................................7
1.2.1 Discovery of the alterations of mtDNA and the relationship with BRAFV600E mutation in PTCs ....................................................................7
1.2.2 Effects of 2-DG on PTCs in vitro ..............................................8
1.2.3 Simultaneously targeting cancer cells and endothelial cells in vivo using 2-DG in mice with NB......................................................................8
CHAPTER II. MATERIALS AND METHODS ...................................................9
2.1 Analysis of mtDNA alterations and BRAFV600E mutation in PTCs ....................9
2.1.1 Patients and samples ..........................................................9
2.1.2 DNA extraction ................................................................9
2.1.3 Determination of the BRAFV600E mutation .......................................9
2.1.4 Direct sequencing analysis of sequence variations in mtDNA D-loop region......10
2.1.5 Detection of the 4,977-bp deletion of mtDNA ..................................11
2.1.6 Determination of mtDNA content................................................11
2.1.7 Extraction of total RNA and real-time PCR analysis ...........................12
2.1.8 Statistical analysis..........................................................13
2.2 Treatment with 2-DG alone or in combination with doxorubicin and sorafenib in PTC cells in vitro .....................................................................14
2.2.1 Cell lines and cell culture...................................................14
2.2.2 Chemicals.....................................................................14
2.2.3 Cell viability assay .........................................................15
2.2.4 Colony formation assay .......................................................15
2.2.5 Glucose uptake analysis ......................................................16
2.2.6 Intracellular ATP content analysis............................................16
2.2.7 Glycolysis assay..............................................................17
2.2.8 Extracellular flux assay......................................................17
2.2.9 Flow cytometric analysis of apoptosis.........................................18
2.2.10 RNA isolation, RT-PCR, qRT-PCR, and primers..................................18
2.2.11 Western blot analysis........................................................18
2.2.12 Statistical analysis ........................................................19
2.3 Effects of 2-DG on NB in vivo ..................................................20
2.3.1 Immunohistochemistry..........................................................20
2.3.2 Animal xenografts ............................................................20
2.3.3 Western blot analysis.........................................................21
2.3.4 Quantifying endothelial cell density after staining with isolectin B4 ........22
2.3.5 Cell proliferation assay .....................................................22
2.3.6 Trypan blue exclusion assay ..................................................23
2.3.7 Flow cytometry to detect apoptosis ...........................................23
2.3.8 Cell migration detected using a wound healing assay and a Boyden chamber......23
2.3.9 Immunofluorescence and laser confocal microscopy..............................24
2.3.10 Statistical analysis.........................................................25
CHAPTER III. RESULTS................................................................26
3.1 Concomitant presence of increased mtDNA content and BRAFV600E mutation was detected in PTCs ...................................................................26
3.1.1 BRAFV600E mutation and somatic mtDNA alterations in PTCs......................26
3.1.2 Correlation between somatic mtDNA alterations and BRAFV600E mutation..........27
3.1.3 The expression of nuclear genes involved in mitochondrial biogenesis .........28
3.2 2-DG inhibited the proliferation of PTC cells and decreased the IC50 of doxorubicin and sorafenib in PTC cells in vitro ....................................29
3.2.1 Mapping bioenergetics in PTC cells ...........................................29
3.2.2 Effect of 2-DG on cell viability in PTC cell lines ...........................29
3.2.3 Effects of 2-DG on the changes of bioenergetic functions of PTC cell lines....30
3.2.4 Effects of 2-DG plus doxorubicin on cell viability and colony formation in PTC cell lines..........................................................................31
3.2.5 Effects of 2-DG plus sorafenib on cell viability and colony formation in PTC cell lines..........................................................................31
3.2.6 Effects of 2-DG plus doxorubicin or sorafenib on apoptosis and metabolic dysfunction in PTC cell lines.......................................................32
3.3 2-DG targeted NB and endothelial cells .........................................33
3.3.1 Differential PDK1 expression in human NB .....................................33
3.3.2 2-DG treated NB tumors in NOD/SCID mice shrank ...............................33
3.3.3 HIF-1, PDK1, c-Myc, and p-Bad expression, but not Bax or Bak expression, fell in 2-DG-treated NB xenografts..........................................................34
3.3.4 The number of tumor vessels in 2-DG-treated AS and DZ xenografts was significantly lower.................................................................34
3.3.5 Cell proliferation and migration were significantly downregulated and apoptosis was upregulated in 2-DG-treated mouse endothelial cells ............................35
3.3.6 Lamellipodia and filopodia formation was inhibited and F-actin filaments were disorganized in 2-DG-treated mouse endothelial cells................................35
CHAPTER IV. DISCUSSION AND CONCLUSIONS..............................................37
4.1 MtDNA content in PTC is increased and is associated with BRAFV600E mutation.....37
4.2 2-DG complements doxorubicin and sorafenib in PTC cells in vitro................39
4.3 2-DG simultaneously inhibits the growth of NB and angiogenesis in vivo .........41
4.4 Conclusion .....................................................................44
CHAPTER V. FUTURE PERSPECTIVES .....................................................45
FIGURES.............................................................................46
TABLES..............................................................................68
REFERENCES .........................................................................73
APPENDIX............................................................................87
Approval of animal study (Approval from Institutional Animal Care and Use Committee) ....................................................................................88
Research Projects...................................................................90
Bibliography........................................................................91
口試委員審查意見回覆.................................................................93


Directory of Figures
Figure 1. Four representative somatic D-loop mutation sequences in PTCs (T) compared to paired non-tumorous tissues (N). ................................................47
Figure 2. Alterations in the mtDNA content..........................................48
Figure 3. Alterations in the mRNA expression of mtTFA, mtSSB, NRF-1, PGC-1 and NDUFS2. ....................................................................................49
Figure 4. Basal bioenergetics in BCPAP and CG3 papillary thyroid carcinoma (PTC) cell lines. .............................................................................50
Figure 5. Measuring basal glycolysis and oxidative metabolism in BCPAP and CG3 papillary thyroid carcinoma (PTC) cell lines by Seahorse X-24 flux analyzer.........51
Figure 6. Susceptibility of PTC cells to 2- DG......................................52
Figure 7. Effects of 2-DG on the changes in ATP production of PTC cells.............53
Figure 8. Effect of 2-DG on the expression of glycolytic genes in PTC cells.........54
Figure 9. Effects of 2-DG on the changes in bioenergetics of PTC cells..............55
Figure 10. Effects of doxorubicin on the viability of PTC cells treated with or without 2-DG........................................................................56
Figure 11. Effects of sorafenib on the viability of PTC cells treated with or without 2-DG................................................................................57
Figure 12. Effects of 2-DG, doxorubicin, or both on apoptosis in PTC cells..........58
Figure 13. Effects of 2-DG, doxorubicin, or both on metabolic dysfunction in PTC cells...............................................................................59
Figure 14. Immunohistochemical staining of PDK1 expression in human NB..............60
Figure 15. Treatment with 2-DG significantly inhibited NB xenograft tumor growth in NOD/SCID mice.......................................................................61
Figure 16. 2-DG downregulated the expression of HIF-1α, PDK1 and c-Myc in NB xenografts..........................................................................62
Figure 17. 2-DG downregulated the expression of Bad and pBad in NB xenografts.......63
Figure 18. 2-DG reduced tumor vessels in both AS and DZ xenografts after 6 doses of 2-DG treatment. ......................................................................64
Figure 19. 2-DG significantly suppressed cell proliferation and induced apoptosis in mouse endothelial cells.............................................................65
Figure 20. 2-DG decreased wound closure and inhibited cell migration in mouse endothelial cells. .................................................................66
Figure 21. 2-DG suppressed lamellipodia and filopodia formation and caused disorganization of F-actin filaments in mouse endothelial cells. ...................67


Directory of Tables
Table 1. Sequences of the oligonucleotide primers used in this study................69
Table 2. Sequences of the oligonucleotide primers used in this study................70
Table 3. Summary of the somatic mutation in the D-loop region of mtDNA in 89 papillary thyroid carcinomas compared to non-tumorous tissues in the same patient.............71
Table 4. Association between the alterations of mtDNA and BRAFV600E mutation in papillary thyroid carcinomas .......................................................72

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