小紅莓(Doxorubicin)又稱阿黴素(Adriamycin)，為一廣泛所使用的肺癌化療用藥。然而，阿黴素於臨床運用上所面對的重大難題為其藥物抗性的產生。先前的研究指出阿黴素處理下激活的 p38 有絲分裂原活化蛋白質激酶 (p38 MAPK) 可助於細胞存活或造成死亡。為了能夠深入了解 p38 MAPK 於阿黴素造成的藥物敏感性的作用，我們以 CL3 非小細胞肺癌細胞株來探討此問題。我們使用 MTT 比色法，發現阿黴素誘發細胞死亡與處理的藥劑量及時間成正相關。 從免疫點墨實驗中得知， 阿黴素激活CL3的 p38 MAPK 生物訊號是暫時性的。加入一 p38 MAPK 抑制劑SB202190共同處理時，會顯著加強阿黴素誘導的細胞毒性。利用流式細胞儀偵測法，我們發現 SB202190 可以顯著地增加阿黴素誘發的細胞凋亡。以專一性的小干擾 RNA (si-p38α/β) 轉染細胞，移除內生之 p38 MAPK 異構體 α 及 β 型，同樣可以增強阿黴素誘導的細胞凋亡。在SB202190共處理或轉染 si-p38α/β 下，可以增強阿黴素所誘發的硫光胺酸蛋白酶－3活化。 阿黴素可誘導抗凋亡因子 Bcl-2 蛋白與前凋亡因子 Bim 蛋白量的增加，然而同時處理 SB202190 狀況下可明顯降低前者，但非後者之蛋白質量。 阿黴素也可以誘發 Bcl-2(Ser70) 的磷酸化但在 SB202190 影響下卻只有些微的降低。SB202190 共處理降低阿黴素誘發Bcl-2 蛋白質的現象，也在粒腺體胞器萃取液中獲得驗證。總括而言，這篇研究指出 p38 MAPK 的活化促使粒腺體蛋白 Bcl-2 量上升，且可能影響Bcl-2(Ser70)的磷酸化，因而協助CL3 肺癌細胞抵抗阿黴素造成的細胞凋亡。

Doxorubicin, also called adriamycin, is a widely used therapeutic agent in lung cancer. However, the development of drug resistance is a major problem for clinical applications of doxorubicin. Activation of the p38 mitogen-activated protein kinase (MAPK) has been reported to contribute to survival or apoptosis following doxorubicin treatment. To gain insight into the role of p38 MAPK in doxorubicin sensitivity, we explore this issue in a non-small cell lung cancer cell line, CL3. We found that doxorubicin dose- and time-dependently induces cell death by using MTT assay. Doxorubicin transiently activates the p38 MAPK signaling as measured by immunoblotting. Co-administering SB202190, a 38 MAPK inhibitor, markedly enhanced the doxorubicin-induced cytotoxicity. Using a flow cytometry-based methodology, we found that SB202190 significantly increased the doxorubicin-induced apoptosis. Depleting α or β isoforms of p38 MAPK by transfection of specific small interfering RNAs (si-p38α/β) also augmented the apoptosis caused by doxorubicin. SB202190 co-treatment or introducing si-p38α/β could enhance the doxorubicin-induced caspase-3 activation. Doxorubicin increased the protein levels of anti-apoptotic Bcl-2 and apoptotic Bim, and SB202190 co-treatment markedly reduced the former but not the later. Doxorubicin also induced the amounts of phospho-Bcl-2(Ser70) that could only be slightly decreased by SB202190. The down-regulation of doxorubicin-induced Bcl-2 by SB202190 co-treament was verified in the mitochondrial fraction. Together, this study suggests that p38 MAPK activation is necessary for the increased mitochondrial Bcl-2, which may involve Ser70 phosphorylation, thereby supporting CL3 lung cancer cells to against apoptosis following doxorubicin.

Contents
誌謝詞........................................................................................................................I
中文摘要...................................................................................................................II
Abstract....................................................................................................................III
Abbreviations...........................................................................................................IV
Chapter 1. Literature Review and Research Aims.................................................1
1. Cancer..........................................................................................................1
1.1 Lung cancer............................................................................................1
1.2 NSCLC chemotherapy...........................................................................3
2. Doxorubicin.................................................................................................4
2.1 Roles of doxorubicin in chemotherapy..........................................5
2.2 Limitation of doxorubicin..............................................................6
3. Mitogen-activated protein kinase signaling.....................................................6
3.1 Activation and roles of the MAPK cascade...........................................7 3.2 p38 MAPK signaling.............................................................................8
3.3 p38 MAPK signaling and cancer...........................................................9
3.4 MAPK signal in chemotherapy............................................................10
4. The Bcl-2 family.............................................................................................11
4.1 Bcl-2 and cancer...................................................................................12
4.2 The roles of MAPK in regulating Bcl-2...............................................13
5. Research aims.................................................................................................14
Chapter 2. Materials and Methods.........................................................................16
1. Cell culture.................................................................................................16
2. Drug treatment............................................................................................16
3. Viability assay ............................................................................................17
4. Small interfering RNA, and transfection....................................................17
5. Flow cytometry............................................................................................18
6. Sub-cellular fractionation............................................................................18
7. Cell lysate preparation.................................................................................19
8. Antibodies and immunoblotting..................................................................20
9. Statistical analysis.......................................................................................20
Chapter 3. Results .....................................................................................................21
1. Cytotoxicity and the activation of p38 MAPK signaling caused by doxorubicin in CL3 cells...............................................................................21
2. Inhibition of the p38 MAPK activity using SB202190 augments the doxorubicin-induced cytotoxicity and apoptosis in CL3 cells......................22
3. Depletion of p38α/β MAPK using specific siRNA enhances the doxorubicin-induced apoptosis in CL3 cells.................................................23
4. The p38 MAPK activity prevents the activation of caspase-3 in doxorubicin-treated CL3 cells.......................................................................24
5. Inhibition of p38 MAPK activity decreases the doxorubicin-induced mitochondrial Bcl-2 protein level in CL3 cells.............................................24
Chapter 4. Discussion................................................................................................27
Conclusion..................................................................................................................32
References...................................................................................................................33
Figures.........................................................................................................................42
Supplement Figure.....................................................................................................50
Appendix.....................................................................................................................51

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