摘要


癌症起始細胞(Cancer Initiating Cells, CICs)為一群具有幹細胞特性及生成腫瘤能力的亞群癌細胞。癌起始細胞被認為能夠抵抗傳統化療與放射線療法，進而造成腫瘤復發及腫瘤轉移。藥物開發以標靶癌症起始細胞，有助於未來癌症病患之治療。
依據近年來的研究發現高醋醛脫氫酶 (aldehyde dehydrogenase, ALDH)的活性為癌起始細胞之特性。故我們以ALDEFLUOR kit分析細胞內高醋醛脫氫酵素活性自樟芝菌絲體(Antrodia cinnamomea Mycelia, ACM)萃取物中篩選出能標靶頭頸癌症起始細胞的純物質。我們發現樟芝菌絲體萃取物中分離出之純物質4-Acetylantroquinonol B (4-AAQN B)能有效降低細胞內高醋醛脫氫酶活性。在本研究中我將探討4-AAQN B是否能標靶癌起始細胞及探討其對於此癌症起始細胞的影響。4-AAQN B 能夠抑制OECM1與SAS的高醋醛脫氫酶活性以及降低癌症起始幹細胞生物標記的表現如：CD44、uPAR及CCN1。我的研究並發現4-AAQN B 處理下能影響細胞內reactive oxygen species (ROS) 之含量; 而細胞內ROS 之含量亦與癌症起始幹細胞之特性有關。，因此4-AAQN B可做為標靶有活性的癌症起始幹細胞的藥物，可進而結合傳統的化療藥物作為未來癌症治療之方法。

Abstract


Cancer initiating cells (CICs), a subtype of cancer cells, have the enhanced stemness properties and tumorigenic abilities. CICs are also considered to render resistance of chemotherapy or radiotherapy, therefore; it results in recurrence and metastasis. Development of drugs that can specifically target cancer initiating cells, as a therapeutic method to against the CICs, would benefit for future treatment of cancer patients.
Recently, others’ and our discoveries have shown that the elevated activity of aldehyde dehydrogenase (ALDH) is closely related to CICs. Herein, we first used the ALDH assay to screen for purified compounds of the myceluim from Antrodia Cinnamomea (ACM) with inhibitory activity of ALDH in cancer cells. Then, we find out that treatment of 4-acetylantroquinonol B (4-AAQN B), which was isolated from ACM, significantly down regulated the ALDH activity of cancer cells. Further, the anticancer activity of 4-AAQN B by targeting CICs had been characterized.
Our results showed that the ALDH activity and expression of stemness biomarkers including CD44, uPAR and CCN1 of cancer cells were significantly decreased after 4-AAQN B administration. We also observed the reduced cellular viability in 4-AAQN B treated cancer cells. Of note, 4-AQQN B disturbed the intracellular content of reactive oxygen species (ROS) of cancer cell where the subpopulations of cancer cells with differential ROS content have been related to CICs.
The anticancer activity of 4-AAQN B has been reported before. However, we are the first group to confirm that 4-AAQN B can effectively target CICs. The abovementioned findings provide an alternative therapeutics for future cancer treatment in combination with conventional chemotherapy.

Contents
【Signature Page】 I
【Thesis Approval Form】 II
【Acknowledgements】 III
【English Abstract】 IV
【Chinese Abstract】 VI
【Table of Contents】 VIII
【List of Figures】 XI
【List of Figures】 XIII
【Introduction】 1
1.1 Head and neck squamous cell carcinoma (HNSCC) 2
1.2 Head and Neck Cancer initiating cells (HN-CICs) 2
1.3 Identification and characterization of cancer initiating cells 3
1.4 Antrodia Cinnanomea 4
1.5 4-Acetylantroquinonol B (4AAQN B) 5
【Hypothesis and Specific Aims】 5
【Materials and methods】 9
2.1 Cell lines and culture condition 9
2.2 4-Acetylantroquinonol B 10
2.3 Flow cytometry 10
2.4 ALDH assay 12
2.5 Reactive oxygen species (ROS) staining 13
2.6 AnnexinV/PI kit 13
2.7 Cell viability 14
2.8 Western blot 15
2.9 Migration 16
2.10 Anchorage-independent growth 17
2.11 Double stain 18
【Results】 20
3.1 ALDH+ activity, stemness marker, decreased after the treatment of 24 hours 4-Acetylantroqinonol B. 20
3.2 Cell morphology changed after treated 4-Acetylantroquinonol B. 21
3.3 4-Acetylantroquinonol B treatment for 24 hours decreased Cell viability. 21
3.4 CD44 is known as a stemness marker also decreased after 24hr 4-Acetylantroquinonol B treatments in HNSCC cell lines. 22
3.5 Parental cells detected AnnexinV/PI assay after 24 hours 4-Acetylantroquinonol B administrations. 23
3.6 Sphere cells treated 4-Acetylantroquinonol B changed morphology thinly. 23
3.7 4-Acetylantroquinonol B treated 24 hours not decreased cell viability in sphere cells. 24
3.8 Urokinase-type plasminogen activator receptor (uPAR), stemness markers, curtailed after the 24 hours 4-Acetylantroquinonol B administrations in sphere cells. 24
3.9 Stemness markers of CCN1 curtailed after the 24 hours 4-Acetylantroquinonol B administrations in OECM1-Sphere cells. 25
3.10 The cells with high intracellular level of reactive oxygen species (ROSHigh) were shifted toward to another ROS population after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 26
3.11 Anchorage-independent growth was decreased for 24 hours 4-Acetylantroquinonol B treatment of sphere cells. 27
3.12 Sphere cells for 24 hours 4-Acetylantroquinonol B administration curtailed the migratory activity. 27
3.13 AnnexinV/PI assay not increased after 24 hours 4-Acetylantroquinonol B administrations in sphere cells. 28
3.14 The Propidium Iodide (PI+) cells were raised after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 28
3.15 To confirm the stemness biomarkers and differentiation marker in SAS-Sphere cell line of western blotting. 29
3.16 To confirm the ROS expression compared with parental cells and sphere cells. 30
3.17 To confirm the ROS and stemness marker in SAS-Sphere cells. 30
【Discussion】 31

【Reference】 35






【List of Figures】 XI
Figure. 1 The stemness marker ALDH+ activity decreased after the treatment of 4-Acetylantroqinonol B in HNSCC cell lines. 40
Figure. 2 24hr 4-Acetylantroquinonol B treated in HNSCC cell lines cause cell morphology changed. 42
Figure. 3 The Cell viability was decreased after 24 hours and 48 hours 4-Acetylantroquinonol B treatments in HNSCC cell lines. 43
Figure. 4 Stemness biomarker CD44 was decreased after 24hr 4-Acetylantroquinonol B treated in HNSCC cell lines. 44
Figure. 5 The apoptosis cell death detected after 24 hours 4-Acetylantroquinonol B treated in HNSCC cell lines. 45
Figure. 6 24hr 4-Acetylantroquinonol B treated in Sphere cells would not change cell morphology. 46
Figure. 7 After 24 hours 4-Acetylantroquinonol B treated in sphere cells were not influenced the cell viability. 47
Figure. 8 Stemness biomarker uPAR were curtailed after the 24 hours 4-Acetylantroquinonol B administration in sphere cells. 48
Figure. 9 Stemness biomarker CCN1 were curtailed after the 24 hours 4-Acetylantroquinonol B administrated in OECM1-Sphere cells. 49
Figure. 10 The cells with high amount of intracellular reactive oxygen species (ROSHigh) diminished after 24hr 4-Acetylantroquinonol B treatments in sphere cells. 50
Figure. 11 The anchorage-independent growth was decreased after treated 24 hours 4-Acetylantroquinonol B in sphere cells. 51
Figure. 12 The migration capability was decreased after 24 hours hours 4-Acetylantroquinonol B treated in sphere cells. 52
Figure. 13 AnnexinV/PI stained of apoptosis cells death was not increased for 24 hours 4-Acetylantroquinonol B treatments in sphere cells. 53
Figure. 14 24 hours 4-Acetylantroquinonol B administration in sphere cells were increased Propidium Iodide positive (PI+) cells. 54
Figure. 15 Stemness biomarkers Oct4 and Nanog were reduced slightly and differentiation marker Involucrin was raised after the 24 hours 4-Acetylantroquinonol B administrations in SAS-Sphere cell line of western blotting. 55
Figure. 16 Measured reactive oxygen species (ROS) population in contrast to parental cells and sphere cells. 56
Figure. 17 Reactive oxygen species and stemness biomarker double stained in SAS-Sphere cells. 57










【List of Tables】 XIII
Table. 1 Cancer is top one major factor for death in Taiwan and oral cancer is the fifth common cancer. 58
Table. 2 Head and neck cancer congaing several organs. 59
Table. 3 Cancer stem cells as a therapeutic target. 60
Table. 4 Microarray of stemness markers CD44, uPAR, and CCN1 compared parental cells and sphere cells in our lab. 61
Table. 5 Reactive oxygen species (ROS) expressed in cancer stem cells. There were ROSLow and ROSHigh populations. 62
Table. 6 uPAR interacted with integrin and down regulate several signaling pathways. 63
Table. 7 CCN1 interacted with integrin and down regulate several signaling pathways. 64
Table. 8 memGrp78 expression in SAS-Parental cells, OECM1-Parental cells and SAS-Sphere cells. 65
Table. 9 HSC cell viability decreased to baseline under 4-Acetylantroquinonol B treated 40 hours. 66
Table. 10 Conclusion of all experiments under 4-Acetylantroquinonol B administration. 67

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