本研究探討天然產物白藜蘆醇(resveratrol)對人類口腔癌細胞株(human oral cancer cells, OC2)細胞內鈣離子濃度和細胞存活率的影響。本研究利用鈣離子螢光染劑fura-2來偵測胞內鈣離子濃度，人類口腔癌細胞株OC2加入濃度5-20 μM之resveratrol後，其鈣離子的濃度的變化隨resveratrol的濃度增加而上升。當移去細胞外培養液中的鈣離子後，鈣離子的訊號則部分下降。由resveratrol誘發細胞外之鈣離子信號內流，受nifiedipine和蛋白質激酶(protein kinase C, PKC)抑制劑（GF109203X）抑制所影響。當去除細胞外鈣離子，培養在不含鈣離子的細胞培養液中，細胞加入2,5-di-tert-butylhydroquinone (BHQ) (一種內質網上鈣離子幫浦抑制劑)完全抑制resveratrol誘導鈣離子濃度的上升。反之，加入了resveratrol的細胞培養液中，抑制大部分BHQ誘導的鈣離子濃度上升。磷脂酶C (phospholipase C, PLC)抑制劑(U73122)抑制大部分resveratrol誘導的鈣離子濃度上升。Resveratrol在濃度20-100 μM下，以濃度所依賴的方式造成細胞毒殺性死亡。在細胞液中加入BAPTA/ AM (鈣離子螯合劑），這種細胞毒殺作用並沒有改變。Annexin V/Propidium (PI) (偵測細胞凋亡的螢光染劑)的染色數據表示，resveratrol在濃度20 μM和40 μM誘導細胞凋亡。另一方面，在濃度20 μM和40 μM的resveratrol處理下也引起細胞週期的阻滯。總之，在人類口腔癌細胞株中加入resveratrol後誘導鈣離子濃度上升，是藉由內質網釋放之磷脂酶C依賴路徑，且鈣離子經由蛋白質激酶C和nifidepine影響的鈣離子通道進入而上升。Resveratrol誘導細胞死亡可能涉及細胞凋亡。

This study examined whether the natural product resveratrol altered cytosolic Ca2+ concentration ([Ca2+]i) and viability in OC2 human oral cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Resveratrol at concentrations of 5-20 μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Resveratrol-induced Ca2+ signal was inhibited by nifedipine and protein kinase C (PKC) inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished resveratrol-induced [Ca2+]i rise. Conversely, incubation with resveratrol largely inhibited BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 largely inhibited resveratrol-induced [Ca2+]i rise. At concentrations of 20-100 μM, resveratrol caused cytotoxicity in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide (PI) staining data suggest that resveratrol between 20 μM and 40 μM induced apoptosis. At concentrations of 20 μM and 40 μM, resveratrol also caused cell cycle arrest. Collectively, in human oral cancer cells, resveratrol induced a [Ca2+]i rise by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, nifidepine-sensitive Ca2+ channels. Resveratrol induced cell death that might involve apoptosis.

目 錄
論文審定書...............................................................................................................i
誌謝..........................................................................................................................ii
中文摘要...................................................................................................................iii
英文摘要...................................................................................................................iv
List of abbreviations…………………………………………………………...................v
1. Introduction……………………………………………………………….....................1
1.1 Resveratrol...……………………………………………………………....................1
1.2 Ca2+ as a pivotal intracellular signal in cells………………………......................1
1.3 Apoptosis…………………………………………………………………...................3
1.4 The OC2 human squamous cell line……………………………............................5
2. Aims…………………………………………………………………….........................7
3. Materials and methods.………………………………………………….....................8
3.1 Chemicals……………………...............................................................................8
3.2 Cell culture……………………………………………………...................................8
3.3 Solutions used in [Ca2+]i measurements…………..............................................8
3.4 [Ca2+]i measurements…………………………………………………….................8
3.5 Cell viability assays……………………………………………………......................9
3.6 Alexa Flour 488 Annexin V/PI staining for detection of apoptosis..............….10
3.7 Measurements of subdiploidy nuclei by flow cytometry......................................10
3.8 Statistics……………………………………………………………………................11
4. Results……………………………………………………………………………..........12
4.1 Effect of resveratrol on [Ca2+]i…………………………………………..................12
4.2 Effect of resveratrol on Mn2+ influx…………......................................................12
4.3 Effect of resveratrol-induced Ca2+ influx pathways………………………............12
4.4 Intracellular Ca2+ store of resveratrol-induced rise [Ca2+]i…………..................13
4.5 A role of phospholipase C in resveratrol-induced rise [Ca2+]i…………..............13
4.6 Relationship between resveratrol-induce [Ca2+]i rise and cell death…..............14
4.7 The role of apoptosis in resveratrol-induced cell death…………………..............14
5. Discusion………………………………………………………………………..............16
6. Conclusion……………………………………….......................................................18
References…………………………………………………………………………............19
Figure legends………………………………………………………………………...........27
Figure. 1-1 Structure of resveratrol………………………………………………............27
Figure. 1-2 Major Ca2+ entry pathways…………………............................................28
Figure. 1-3 Apoptosis (programmed cell death)………………………………..............29
Figure. 1-4 Extrinsic and intrinsic apoptotic pathways…………………………............30
Figure. 2-1 Resveratrol induced a [Ca2+]i rise in a concentration-dependent
manner in OC2 cells……………………………………………………...........................31
Figure. 2-3 Effect of Ca2+ channel modulators on resveratrol-induced [Ca2+]i rise..33
Figure. 2-4 Intracellular Ca2+ stores of reveratrol-induced Ca2+ release...………....34
Figure. 2-5 Effect of U73122, a phospholipase C inhibitor, on resveratrol
-induced Ca2+ release…………………………............................................................35
Figure. 2-6 Resveratrol reduced cell viability in a concentration dependent
manner and BAPTA-AM loading did not affect resveratrol
-induced cytotoxicity in OC2 cells……………………………………….........................36
Figure. 2-7 Apoptosis induced by resveratrol measured by Annexin V/PI staining……………………………………………………………………..........................37
Figure. 2-8 Resveratrol between 20 μM and 40 μM caused cell cycle arrest in
OC2 cells……………………………………………………………..................................38

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