天然精油香芹酚(carvacrol)或麻醉劑異丙酚(propofol)對於人類正常細胞或非正常細胞中，細胞存活率、細胞週期分佈、活性氧化物，細胞內鈣離子濃度和蛋白酶-3-參與的細胞凋亡效應還不清楚。在此研究中，所使用的細胞株為人類牙齦纖維母細胞(human gingival fibroblast, HGF)，人類口腔癌細胞(human oral cancer cell, OC2)和人類腦神經瘤細胞(human glioblastoma cell, DRTBG-05MG, HGB)。本研究實驗方法包括以水溶性四唑鹽(water soluble tetrazolium salt-1, WST-1)檢測細胞存活率；以膜聯蛋白V (annexin V)/碘化丙啶(propidium iodide, PI)偵測細胞凋亡；以PI染色檢測細胞週期分佈；以膜滲透探針dichlorofluorescein diacetate (DCFH-DA)或hydroethidine (HE)偵測活性氧化物的改變；以流式細胞儀來分析細胞凋亡、細胞週期分佈和活性氧化物的表現；以鈣螢光染料fura-2測量細胞內鈣離子濃度；並以西方墨點法偵測蛋白酶-3的活化。
香芹酚在200 μM至800 μM的濃度依賴效應下造成OC2或HGB細胞死亡， 1,000 μM的香芹酚幾乎殺光OC2或HGB細胞，但在HGF細胞，200 μM 至800 μM的香芹酚並沒有顯著殺死細胞且1,000 μM的香芹酚只降低約63%的細胞存活率。另一方面，異丙酚具有類似香芹酚的細胞毒殺情形，異丙酚在300 μM至600 μM的濃度依賴效應下能造成OC2或HGB細胞死亡而700 μM的異丙酚能幾乎殺死OC2或HGB細胞，但在HGF細胞中，300-600 μM的異丙酚並不會殺死OC2或HGB細胞且700 μM的異丙酚只降低約62%的細胞存活率。香芹酚(200、400或600 μM)或異丙酚(300、400或500 μM)能誘導OC2或HGB細胞凋亡，增加活性氧化物產生，細胞週期阻滯且活化蛋白酶-3。蛋白酶-3抑制劑(DEVD-CHO)能抑制香芹酚或異丙酚誘發的細胞凋亡。
此外，香芹酚在400至1,000 μM的濃度依賴效應下能誘發OC2或HGB細胞鈣離子濃度上升且在去除胞外鈣離子的情形下，細胞內鈣離子的濃度會下降。但在HGF細胞中，1,000 μM的香芹酚並沒有誘發立即性的鈣離子濃度上升。類似的情形也發生在異丙酚，異丙酚在400至1,000 μM的濃度依賴效應下能誘發OC2或HGB細胞鈣離子濃度上升且在去除細胞外鈣離子的情形下，細胞內鈣離子的濃度會下降。但在HGF細胞中，1,000 μM的異丙酚也沒有誘發立即性的鈣離子濃度上升。再者，香芹酚的細胞毒殺效應並不會被細胞內鈣離子螯合劑(BAPTA-AM)降低，卻能部分降低異丙酚的細胞毒殺效應，此外，BAPTA-AM也能部分降低異丙酚對OC2或HGB細胞凋亡的影響。
最後，此研究也探討香芹酚或異丙酚對於OC2和HGB細胞鈣離子上升的機制。由香芹酚或異丙酚誘發細胞外鈣離子內流，不受L-型鈣離子通道抑制劑(L-type voltage gated Ca2+ channel, nifedipine)、鈣庫調控的鈣離子通道抑制劑 (store-operated Ca2+ channel, econazole 或SK&F96365)及蛋白質激酶C (protein kinase C, PKC)活化物 phorbol myristate acetate (PMA)所影響，卻被蛋白質激酶C抑制劑GF109203X抑制。在不含鈣離子的細胞培養液中，先對細胞處以內質網鈣離子幫浦抑制劑(thapsigargin, TG 或 2,5-di-tert-butylhydroquinone, BHQ)，再加入香芹酚或異丙酚後，細胞內的鈣離子濃度並不會上升；反之，先加入香芹酚或異丙酚，再加入內質網鈣離子幫浦抑制劑也出現類似的情形。另外，磷脂酶C (phospholipase C, PLC) 抑制劑U73122也能完全抑制香芹酚或異丙酚所誘發的細胞內鈣離子濃度。
總結這項研究，首先，在HGF細胞中，香芹酚(200-800 μM)或異丙酚(300-600 μM)並沒有引起細胞內鈣濃度的上升且也沒有造成大量細胞死亡。其次，在OC2或HGB細胞中，香芹酚能誘發細胞內鈣濃度的升高且造成活性氧化物參與、鈣離子非依賴和蛋白酶-3-相關路徑的細胞凋亡。第三，在OC2或HGB細胞中，異丙酚能誘發細胞內鈣濃度的升高且造成活性氧化物、鈣離子和蛋白酶-3-相關路徑的細胞凋亡。最後，在 OC2或HGB細胞中，香芹酚或異丙酚誘發鈣離子濃度上升是經由磷脂酶C、蛋白質激酶C依賴途徑和內質網釋放所引起；同時細胞外鈣離子內流則經由非鈣庫調控的鈣離子通道所造成。

The effect of the natural essential oil carvacrol or the anesthetic propofol on cell viability, cell cycle distribution, reactive oxygen species (ROS), intracellular free Ca2+ concentrations ([Ca2+]i) and caspase-3-associated apoptosis in human normal cells or non-normal cells is unclear. Human gingival fibroblasts (HGF), human oral cancer cell line (OC2) and human glioblastoma cell line (DRTBG-05MG, HGB) were used in this study. Cell viability was measured by detecting reagent water soluble tetrazolium salt-1 (WST-1). Apoptosis was detected by Annexin V/propidium iodide (PI) staining, cell cycle distribution was detected by PI staining, and ROS was detected by membrane-permeable probe dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) staining. Apoptosis, cell cycle distribution and ROS were analyzed by flow cytometry. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Caspase-3 expression was detected by western blotting.
Carvacrol at 200-800 μM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 1,000 μM carvacrol almost killed all OC2 or HGB cells, but in HGF cells, 200-800 μM carvacrol did not significantly kill cells and 1,000 μM carvacrol decreased only about 63% of cell viability. Similarly, propofol at concentrations between 300 and 600 μM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 700 μM propofol almost killed all OC2 or HGB cells, but in HGF cells, 300-600 μM propofol did not significantly kill cells and 700 μM propofol decreased about 62% of cell viability. In OC2 or HGB cells, carvacrol (200 μM, 400 μM or 600 μM) or propofol (300 μM, 400 μM or 500 μM) induced apoptosis, increased ROS production, evoked cell cycle arrest and activated caspase-3. The caspase-3 inhibitor (DEVD-CHO) partially decreased the apoptotic effect induced by carvacrol or propofol.
On the other hand, in OC2 or HGB cells, carvacrol at concentrations between 400 μM and 1,000 μM induced a [Ca2+]i rise in a concentration-dependent manner and the signal was reduced by removal of extracellular Ca2+. In HGF cells, carvacrol at 1000 μM did not induce immediate [Ca2+]i rises in Ca2+-containing or Ca2+-free medium. Similarly, propofol at concentrations between 400 μM and 1,000 μM induced a [Ca2+]i rise in a concentration-dependent manner in OC2 or HGB cells, but not in HGF cells. This cytotoxic effect was not reversed in carvacrol-treated groups, but was partially reversed in propofol-treated groups when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA-AM) in OC2 or HGB cells. The apoptotic effect of propofol was also partially decreased by BAPTA-AM treatment in OC2 and HGB cells.
In OC2 and HGB cells, carvacrol- or propofol-induced Ca2+ signal was not altered by L-type voltage-gated Ca2+ channel blocker nifedipine, store-operated Ca2+ channel blocker econazole or SK&F96365) and protein kinase C (PKC) activator phorbol myristate acetate (PMA), but was inhibited by PKC inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol- or propofol-induced [Ca2+]i rises. Incubation with carvacrol or propofol also abolished TG or BHQ-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol- or propofol-induced [Ca2+]i rises.
Together, first, in HGF cells, carvacrol (200-800 μM) or propofol (300-600 μM) did not induce [Ca2+]i rises and cell death. Second, in OC2 or HGB cells, carvacrol induced [Ca2+]i rises and cell death that might involve ROS- and caspase-3-associated apoptosis. Third, in OC2 or HGB cells, propofol induced [Ca2+]i rises and cell death that might involve ROS-, Ca2+- and caspase-3-associated apoptosis. Lastly, in OC2 or HGB cells, carvacrol or propofol induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, non store-operated Ca2+ channels.

摘要 i
Abstract iii
List of abbreviations vi
1. Introduction 1
1.1. Alkyl phenol group. 1
1.2. Carvacrol 2
1.3. Propofol (2,6-diisopropylphenol) 3
1.4. The characteristics of apoptosis 4
1.5. The apoptotic pathway 5
1.6. Cell cycle and the relationship between cell cycle and apoptosis 7
1.7. ROS and the role of ROS in apoptosis 10
1.8. Ca2+ signaling and its role in apoptosis 12
1.9. Caspase family in apoptosis 13
2. Aim 15
3. Materials and methods 16
3.1. Chemicals 16
3.2. Cell culture 16
3.3. Solutions 17
3.4. [Ca2+]i measurements 18
3.5. Cell viability assays 19
3.6. Alexa ®Flour 488 annexin V/propidium iodide (PI) staining for apoptosis 20
3.7. Cell cycle distribution by PI staining 21
3.8. Measurements of intracellular ROS levels 22
3.9. Assessment of caspase-3 activation by Western immunoblotting 23
3.10. Statistics 24
4. Results 26
Effects of carvacrol/propofol on cell viability of HGF, OC2 or HGB cells 26
4.1. Carvacrol decreased the cell viability in HGF, OC2 or HGB cells 26
4.2. Propofol decreased the cell viability in HGF, OC2 or HGB cells 26
Effects of carvacrol/propofol on apoptosis of HGF, OC2 or HGB cells 26
4.3. Carvacrol induced apoptosis in HGF, OC2 or HGB cells 27
4.4. Propofol induced apoptosis in HGF, OC2 or HGB cells 27
Effects of carvacrol/propofol on cell cycle distribution of HGF, OC2 or HGB cells 28
4.5. Carvacrol affected cell cycle distribution in HGF, OC2 or HGB cells 28
4.6. Propofol affected cell cycle distribution in HGF, OC2 or HGB cells 29
Effects of carvacrol/propofol on ROS production of HGF, OC2 or HGB cells 29
4.7. Carvacrol induced ROS production in HGF, OC2 or HGB cells 29
4.8. Propofol induced ROS production in HGF, OC2 or HGB cells 30
Effects of carvacrol/propofol on [Ca2+]i of HGF, OC2 or HGB cells 30
4.9. Carvacrol evoked [Ca2+]i in HGF, OC2 or HGB cells 30
4.10. Propofol evoked [Ca2+]i in HGF, OC2 or HGB cells 32
The relationship between chelating Ca2+ with BAPTA-AM and carvacrol/propofol-induced cell death in OC2 or HGB cells 33
4.11.1. Chelating Ca2+ with BAPTA-AM did not reverse carvacrol-induced cell death in OC2 or HGB cells 33
4.11.2. Chelating Ca2+ with BAPTA-AM partially reversed propofol-induced cell death in OC2 or HGB cells 34
The relationship between chelating Ca2+ with BAPTA-AM and propofol-induced apoptosis in OC2 or HGB cells 35
4.11.3. Chelating Ca2+ with BAPTA-AM partially inhibited propofol-induced apoptosis in OC2 or HGB cells 35
The mechanism of carvacrol/propofol-induced [Ca2+]i rises in OC2 or HGB cells 35
4.11.4. To confirm that carvacrol- or propofol-induced [Ca2+]i rises involved Ca2+ influx in OC2 or HGB cells 35
4.11.5. Modulations of carvacrol- or propofol-induced [Ca2+]i rises in OC2 or HGB cells 36
4.11.6. Internal stores of carvacrol- or propofol-induced [Ca2+]i rises in OC2 or HGB cells 37
4.11.7. The role of PLC on carvacrol- or propofol-induced [Ca2+]i rises in OC2 or HGB cells 38
Effects of carvacrol/propofol on caspase-3 activation of OC2 or HGB cells 39
4.12. Carvacrol induced caspase-3 activation in OC2 or HGB cells 39
4.13. Propofol induced caspase-3 activation in OC2 or HGB cells 39
The relationship between caspase-3 inhibitor (DEVD-CHO) and carvacrol/propofol-induced cell death in OC2 or HGB cells 40
4.14.1. DEVD-CHO partially reversed carvacrol-induced death in OC2 or HGB cells 40
4.14.2. DEVD-CHO partially reversed propofol-induced death in OC2 or HGB cells 40
The relationship between caspase-3 inhibitor (DEVD-CHO) and carvacrol/propofol-induced apoptosis in OC2 or HGB cells 41
4.14.3. DEVD-CHO partially inhibited carvacrol-induced apoptosis in OC2 or HGB cells 41
4.14.4. DEVD-CHO partially inhibited propofol-induced apoptosis in OC2 or HGB cells 40
5. Conclusion 42
6. Discussion 43
List of figures
Figure 1.1. Alkyl phenol group including carvacrol or propofol 54
Figure 1.2. The features of cell death by apoptosis 55
Figure 1.3. Typical receptor-mediated and mitochondria-mediated apoptotic pathways 56
Figure 1.4. The overview of cell cycle 57
Figure 1.5. The apoptotic pathway induced by ROS or [Ca2+] 58
Figure 2.1. Effect of carvacrol on cell viability in HGF, OC2 or HGB cells 59
Figure 2.2. Effect of propofol on cell viability in HGF, OC2 or HGB cells 60
Figure 3.1. Carvacrol-induced apoptosis in HGF, OC2 or HGB cells (dot plots) 61
Figure 3.2. Carvacrol-induced apoptosis in HGF, OC2 or HGB cells (bar plots) 62
Figure 3.3. Propofol-induced apoptosis in HGF, OC2 or HGB cells (dot plots) 63
Figure 3.4. Propofol-induced apoptosis in HGF, OC2 or HGB cells (bar plots) 64
Figure 4.1. Effect of carvacrol on cell cycle distribution in HGF cells 65
Figure 4.2. Effect of carvacrol on cell cycle distribution in OC2 cells 66
Figure 4.3. Effect of carvacrol on cell cycle distribution in HGB cells 67
Figure 4.4. Effect of propofol on cell cycle distribution in HGF cells 68
Figure 4.5. Effect of propofol on cell cycle distribution in OC2 cells 69
Figure 4.6. Effect of propofol on cell cycle distribution in HGB cells 70
Figure 5.1. Carvacrol-induced ROS (H2O2 or O2-) production in HGF cells 71
Figure 5.2. Carvacrol-induced ROS (H2O2 or O2-) production in OC2 cells 72
Figure 5.3. Carvacrol-induced ROS (H2O2 or O2-) production in HGB cells 73
Figure 5.4. Propofol-induced ROS (H2O2 or O2-) production in HGF cells 74
Figure 5.5. Propofol-induced ROS (H2O2 or O2-) production in OC2 cells 75
Figure 5.6. Propofol-induced ROS (H2O2 or O2-) production in HGB cells 76
Figure 6.1. Effect of carvacrol on [Ca2+]i in HGF, OC2 or HGB cells 77
Figure 6.2. Effect of propofol on [Ca2+]i in HGF, OC2 or HGB cells 78
Figure 6.3. Concentration-response plots of carvacrol- or propofol-induced [Ca2+]i responses in OC2 or HGB cells 79
Figure 6.4. Effect of BAPTA-AM on carvacrol-induced cell death in OC2 or HGB cells 80
Figure 6.5. Effect of BAPTA-AM on propofol-induced cell death in OC2 or HGB cells 81
Figure 6.6. Effect of BAPTA-AM on propofol-induced apoptosis in OC2 cells 82
Figure 6.7. Effect of BAPTA-AM on propofol-induced apoptosis in HGB cells 84
Figure 6.8. Carvacrol or propofol induced [Ca2+]i rises involved Ca2+ influx from extracellular space in OC2 or HGB cells 86
Figure 6.9. Carvacrol induced [Ca2+]i rises through PKC activation and non-store-operated Ca2+ channels in OC2 or HGB cells 87
Figure 6.10. Propofol induced [Ca2+]i rises through PKC activation and non-store-operated Ca2+ channels in OC2 or HGB cells 88
Figure 6.11.1. Carvacrol pretreatment abolished the TG- or BHQ-induced [Ca2+]i rise, and that TG or BHQ pretreatment abolished carvacrol-induced [Ca2+]i rises in OC2 cells 89
Figure 6.11.2. Carvacrol pretreatment abolished the TG- or BHQ-induced [Ca2+]i rise, and that TG or BHQ pretreatment abolished carvacrol-induced [Ca2+]i rises in HGB cells 90
Figure 6.12.1. Propofol pretreatment abolished the TG- or BHQ-induced [Ca2+]i rise, and that TG or BHQ pretreatment abolished propofol-induced [Ca2+]i rises in OC2 cells 91
Figure 6.12.2. Propofol pretreatment abolished the TG- or BHQ-induced [Ca2+]i rise, and that TG or BHQ pretreatment abolished propofol-induced [Ca2+]i rises in HGB cells 92
Figure 6.13. ATP induced [Ca2+]i rises in OC2 or HGB cells, but not in HGF cells 93
Figure 6.14. Carvacrol or propofol induced [Ca2+]i rises through PLC activation in OC2 or HGB cells 94
Figure 7.1. Carvacrol induced caspase-3 activation in OC2 cells and HGB cells 95
Figure 7.2. Propofol induced caspase-3 activation in OC2 cells and HGB cells 96
Figure 7.3. Effect of DEVD-CHO on carvacrol-induced cell death in OC2 or HGB cells 97
Figure 7.4. Effect of DEVD-CHO on propofol-induced cell death in OC2 or HGB cells 98
Figure 7.5. Effect of DEVD-CHO on carvacrol-induced apoptosis in OC2 cells 99
Figure 7.6. Effect of DEVD-CHO on carvacrol-induced apoptosis in HGB cells 101
Figure 7.7. Effect of DEVD-CHO on propofol-induced apoptosis in OC2 cells 103
Figure 7.8. Effect of DEVD-CHO on propofol-induced apoptosis in HGB cells 105
Figure 7.9. A novel model for carvacrol/propofol-induced [Ca2+]i rises and apoptosis in OC2 or HGB cells 107
Table 1. Effects of carvacrol in vitro or in vivo studies 108
Table 2. Effects of propofol in vitro or in vivo studies 109
References 110
Appendix 1
Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells 127
Appendix 2
Investigation of 2,6-diisopropylphenol (propofol)-evoked Ca2+ movement and cell death in human glioblastoma cells 136
Publications 147

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