臺灣博碩士論文加值系統

肺癌是目前被認為是一種轉移性強而且預後差的癌症，許多研究都積極的尋找有效的治療方法。PG是由細菌所產生的一種紅色色素，被認為是具有潛力的抗癌藥物，不過PG抑制癌細胞增生的機制目前還有待釐清。在本實驗中，我們發現PG處理可以藉由提升p27 kip1的表現有效抑制肺腺癌細胞的細胞週期進行，而且p27 kip1蛋白量的增加不是由於p27 kip1基因的高度表現，而是因為增加了p27 kip1在細胞內的蛋白穩定性。SKP2是一種E3 ubiquitin ligase的重要組成蛋白，被認為與p27 kip1的分解有直接關聯，的確PG處理後就會抑制SKP2的表現量。透過細胞群落分析，看到穩定抑制p27 kip1或是高度表現SKP2的細胞便不會受到PG抑制細胞增生效果的影響。最後，我們發現PG會透過調控PI3K/AKT/GSK-3b訊息路徑去抑制SKP2的表現，如果持續性活化AKT或共同處理GSK-3b抑制劑，會使PG降低SKP2蛋白的程度有回復現象。綜合以上結果，證實PG會抑制PI3K/AKT/GSK-3b這條訊息傳遞路徑去負調控SKP2的表現，因此使p27 kip1的蛋白穩定性提升進而導致細胞週期停滯抑制癌細胞增生，也顯示了在PG的抗癌機制中，SKP2扮演了連結上下游的重要角色，抑制此致癌基因有潛力成為未來治療肺癌的有效策略。

Lung cancer has been viewed as an aggressive disease with few treatment options and poor survival. Therefore, novel treatment strategies are needed. The anticancer agent prodigiosin (PG) is a natural red pigment produced by microorganisms. In this study, we demonstrated PG could lead to cell cycle arrest by increasing p27Kip1 expression in human lung adenocarcinoma cells. RT-PCR analysis, promoter activity assay and cycloheximide co-treatment indicated that the increased level of p27Kip1 was not due to increased p27Kip1 gene expression, but rather resulting from increased p27Kip1 protein stability. Intriguingly, SKP2, a central E3 ubiquitin ligase responsible for p27Kip1 degradation, was found to be markedly down-regulated by PG. Further analysis indicated that PG inhibits the SKP2 mRNA expression and promoter activity. Importantly, colony formation assay demonstrated that cells with stable p27Kip1 knockdown or ectopic SKP2 expression confer resistance to PG-induced antiproliferation. Finally, we found that PG regulates PI3K/AKT/GSK-3b signaling to down-regulate SKP2. Expression of constitutively active AKT or treatment with AR-A014418 could reverse the inhibition of SKP2 caused by PG. Together, these results indicates that PG inhibits PI3K/AKT/GSK-3b signaling to reduce SKP2 expression, which contributes to the increasing p27Kip1 stabilization therefore blocks the cell cycle progression. It also demonstrates that SKP2 is an important target of PG and give a potential strategy for chemotherapy in lung cancer.

目次

壹、前言………………………………………………………………………......1

一、肺癌……………………………………………………………………….1
二、靈菌紅素(Prodigiosin)…………………………………………………...2
三、CDK抑制蛋白調控細胞週期……………………………………………5
四、Ubiquitin-proteasome pathway…………………………………………7
五、SKP2 (S-phase kinase-associated protein 2)………………………...8
六、AKT 路徑………………………………………………………………...9

貳、材料與方法…………………………………………………………........…11

一、細胞培養 (Cell culture)………………………………………………...11
二、細胞之冷凍保存與解凍……………………………………………......11
三、藥物處理……………………………………………………………......12
四、細胞存活測試(Cell viability assay)…………………………………….12
五、RNA 萃取 (Total RNA extraction)…………………………………….13
六、反轉錄 (Reverse transcription)……………………………………….13
七、半定量聚合酵素連鎖反應 (Semi-quantitative RT-PCR)………….…14
八、即時定量聚合酵素連鎖反應 (Real-time RT-PCR)…………………..15
九、蛋白萃取 (Total lysate extraction)……………………………….........16
十、蛋白濃度定量分析 (Protein quantification)…………………….........16
十一、西方墨點法(Western Blot)............................................................17
十二、病毒的製備與感染 (Virus production and infection)....................19
十三、流式細胞儀分析 (Flow cytometry)...............................................21
十四、細胞群落行成能力檢測法 (Colony formation assay)……………..22
十五、蛋白半衰期測定(Cycloheximide chase assay)……………………22
十六、報導基因試驗 (Reporter gene assay)………………………….....22
十七、額外藥品列表……………………………………………………...…23

參、結果……………………………………………........................................24

一、Prodigiosin處理導致肺腺癌細胞週期停滯並增加p21及p27的蛋白表現量.………………………………………………………………….24
二、Prodigiosin透過後轉譯層次提升p27的表現量…………………….25
三、Prodigiosin藉由提升p27的蛋白穩定度調控p27的表現………....25
四、Prodigiosin抑制SKP2的表現使p27的穩定性增加……………….26
五、Prodigiosin透過轉錄層次降低SKP2的表現…..………………...…26
六、Prodigiosin所引發抑制SKP2及促進p27的表現是導致抑制細胞增生的主要原因.....................................................................................27
七、Prodigiosin藉由PI3K-AKT訊息路徑使SKP2的表現被抑制.......…28
八、抑制GSK-3b減緩了Prodigiosin抑制SKP2的效力………….........29
九、E2F1不參予在Prodigiosin所造成的SKP2調控作用……………...30

肆、討論………………………………………………………………………….32

一、Prodigiosin抑制細胞週期進行………………………………….........32
二、Prodigiosin抑制SKP2使ubiquitin-proteasome pathway無法作用於p27……………………………………………………………………...33
三、Prodigiosin調控AKT/GSK-3b路徑使SKP2轉錄活性下降.…........34
四、轉錄因子在Prodigiosin調控SKP2的作用中所扮演的角色……….36

伍、圖表………………………………………………………………………….38

一、Prodigiosin處理導致人類肺腺癌細胞生長週期停滯於G1期..........38
二、Prodigiosin使停滯於G1期的細胞增加及進入S與G2/M期的細胞減少……………………………………………………………………….. 39
三、Prodigiosin對人類肺腺癌細胞株的細胞毒性………………………..40
四、Prodigiosin 增加人類肺腺癌細胞株p21及p27蛋白表現量……....41
五、Prodigiosin增加人類肺腺癌細胞株內的p21 mRNA表現量但不影響p27 mRNA的表現量…………………………………………………..42
六、Prodigiosin增加人類肺腺癌細胞內p21的轉錄活性…………........ 43
七、Prodigiosin不影響人類肺腺癌細胞內p27的轉錄活性………........ 44
八、Prodigiosin不影響人類肺腺癌細胞株p27 promoter活性……........45
九、Prodigiosin處理提升p27蛋白穩定度………………………………. 46
十、Prodigiosin 抑制人類肺腺癌細胞株SKP2蛋白表現量………..…...48
十一、Prodigiosin降低人類肺腺癌細胞株的SKP2 mRNA表現量.........49
十二、Prodigiosin降低人類肺腺癌細胞株SKP2 promoter活性............51
十三、Prodigiosin 不透過蛋白降解路徑降低SKP2表現量………........52
十四、p27 knockdown對於prodigiosin 引起細胞生長停滯的影響…....53
十五、SKP2 overexpression對於prodigiosin 引起細胞生長停滯的影響.................................................................................................55
十六、Prodigiosin 隨著濃度增加抑制AKT的活化程度…………..……. 57
十七、AKT持續活化對於prodigiosin降低SKP2表現量的影響…........58
十八、Prodigiosin 隨著濃度增加使AKT下游的GSK-3b活性提升........59
十九、Prodigiosin 隨著時間增加抑制AKT的活化程度…………..……. 60
二十、抑制GSK-3b 對於prodigiosin降低SKP2表現量的影響…....... 61
二十一、Prodigiosin 抑制人類肺腺癌細胞株E2F1蛋白表現量…...….... 63
二十二、Prodigiosin 抑制肺腺癌細胞株E2F1 mRNA表現量…...……...64
二十三、Prodigiosin降低肺腺癌細胞株E2F1所引發之轉錄活性..........66
二十四、E2F1不參與Prodigiosin所引起的SKP2表現量抑制現象…...67

陸、參考文獻..............................................................................................68

附錄一………………………………………………………………………........77
附錄二………………………………………………………………………........78
附錄三…………………………………………………………………………....79
附錄四………………………………………………………………………...….80
附錄五…………………………………………………………………………....81
附錄六…………………………………………………………………………....82

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