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研究生:洪佳鈺
研究生(外文):Chia-Yu Hung
論文名稱:S6K1促進癌細胞之雄性素受器功能及生長能力
論文名稱(外文):Roles of S6K1 in promoting the function of androgen receptors for the proliferation of cancer cells
指導教授:袁大鈞
指導教授(外文):Ta-Chun Yuan
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
論文頁數:40
中文關鍵詞:雄性素接受器S6K1S6攝護腺癌口腔癌
外文關鍵詞:androgen receptorS6K1S6prostate canceroral squamous cell carcinoma
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雄性素接受器 (androgen receptor;AR) 是癌症中被廣泛研究的荷爾蒙受器蛋白。在本實驗室過去的研究顯示,口腔癌細胞中也會表現活化態的 AR,但活化機制並不清楚。S6K1 (ribosomal S6 kinase 1) 為一 serine/threonine kinase,主要功能是促進核醣體 RPS6 合成蛋白質。本研究主要是探討 S6K1 是否藉由促進 AR 磷酸化及活化,進而導致口腔癌細胞增生,同時也分析S6K1調控AR活性及細胞生長能力之機制是否也存在於攝護腺癌細胞。在比較正常攝護腺細胞PZ-HPV-7,攝護腺癌細胞LNCaP及四株口腔癌細胞(SCC4,SCC25,OECM-1及SAS)發現S6K1及下游分子RPS6之表現量及磷酸化程度與AR之表現量及活性有關。以雄性素DHT刺激LNCaP與OECM-1細胞發現S6K1與RPS6之表現量與磷酸化皆增加。以AR抑制劑Bicalutamide處理LNCaP與SAS 細胞發現S6K1與RPS6磷酸化也受到抑制,降解(knockdown) LNCaP 與 OECM-1細胞之AR表現,導致S6K1與RPS6之表現量及磷酸化程度受到抑制,而大量表現AR在SCC4細胞會促進S6K1與RPS6之表現量與磷酸化明顯增加。為進一步確定S6K1對AR活性的調控作用,將LNCaP及SCC4細胞過度表現S6K1蛋白後發現,AR Ser-81磷酸化程度及生長能力皆增加,另以RNAi技術降解LNCaP與OECM-1細胞中S6K1的表現,發現其AR Ser-81磷酸化的程度也會跟著下降,同時會導致細胞生長受到抑制。接著大量表現S6K1之LNCaP細胞處理Bicalutamide發現S6K1與AR磷酸化程度明顯受到抑制,LNCaP之PSA也受到抑制。另以大量表現S6K1之SCC25細胞處理 Bicalutamide進行細胞生長分析發現細胞隨著藥物而使生長受到抑制。為證明S6K1對細胞生長之促進作用是藉由對AR之活化,進一步探討S6K1對AR磷酸化的促進作用是與S6K1 Thr-389磷酸化或是酵素活性有關,在LNCaP與 SAS細胞處理S6K1抑制劑PF-4708671抑制S6K1的酵素活性,但促進S6K1 Thr-389的磷酸化程度。結果顯示5 μM PF-4708671處理之LNCaP及SAS細胞會抑制RPS6磷酸化,但促進S6K1及AR之磷酸化程度。本研究證明S6K1與AR訊息傳遞路徑間存在交互調控之作用而S6K1藉由本身Thr-389磷酸化調控攝護腺與口腔癌細胞之AR活性,並推測可作為治療攝護腺癌與口腔癌之有效分子標靶。
Androgen receptor is a transcription factor that has been widely studied in hormone-regulated cancers. In our previous studies, were found that oral squamous cell carcinomas (OSCC) cells, similar to prostate cancer (PCa) cells, expressed functional AR proteins. However, the underlying regulatory mechanism of AR activation is unclear. In this study, we investigated the role of S6K1 in regulating the AR activity and the proliferation of PCa and OSCC cells. Ribosomal S6 kinase 1 (S6K1) is a serine/threonine kinase that can promote the function of 40S ribosomal S6 (RPS6), a downstream of S6K1, to synthesize proteins. By examining normal and malignant prostate cell lines (PZ-HPV-7 and LNCaP) and four OSCC cell lines (SCC4, SCC25, OECM-1 and SAS), we found that the expression and activity of S6K1 were positively correlated with the expression and phosphorylation of RPS6 and AR. Treatment of LNCaP and OECM-1 cells with dihydrotestosterone led to increase expression and phosphorylation of S6K1 and RPS6, whereas treating LNCaP and SAS cells with an AR antagonist bicalutamide caused the decreased phosphorylation of S6K1 and RPS6. Knocking down AR expression in LNCaP and OECM-1 cells by shRNAs led to decrease in the levels of S6K1 and RPS6 phosphorylation, while overexpressing AR in SCC4 cells promoted the expression and phosphorylation of S6K1 and RPS6. Furthermore, overexpression of S6K1 in LNCaP and SCC4 cells resulted in increased AR phosphorylation or cell growth rate. In contrast, knocking down S6K1 expression in LNCaP and OECM-1 cells by shRNAs led to decreased AR phosphorylation and inhibited cell growth. Importantly, treatment with bicalutamide in S6K1-overexpressed LNCaP or SCC25 could abolish S6K1-mediated effects on AR activation and growth promotion. We further determined whether S6K1-promoted AR phosphorylation is mediated by its Thr-389 phosphorylation or the kinase activity. LNCaP and SAS cells were treated with PF-4708671, a S6K1 inhibitor, which can inhibit S6K1 kinase activity but promote its Thr-389 phosphorylation. We found that treatment with 5 μM PF-4708671 in LNCaP and SAS cells caused the increase of S6K1 Thr-389 and AR phosphorylation, but led to the decrease in RPS6 phosphorylation. In summary, our data collectively suggest the crosstalk between S6K1 and AR signaling, and S6K1 may via its Thr-389 phosphorylation regulate the AR activity and the stimulate growth of PCa and OSCC cells.
中文摘要……………………………...…………………………....................... I
英文摘要……………………………...…………………………....................... II
縮寫表……………………………...…………………………........................... IV
目錄……………………………...…………………………............................... VI
第一章、文獻回顧………………………………...…………………………... 1
1. 雄性素受體 …………………………………………..…………...…… 1
2. Ribosomal protein S6 kinase 1..….………………………………...…… 3
3. Dihydrotestosterone 與 Bicalutamide…………………...……...……... 5
第二章、研究目的…………………………………………...……..………… 7
第三章、實驗設計……………………………………...…………..………… 9
第四章、研究方法……………………………………...…………..………… 11
1. 細胞株之繼代培養(cell culture) …………...……….………….……… 11
2. 細胞生長能力分析………………………...…........…….……………… 11
3. 西方墨點法…………..…………………………..……………………… 11
3.1蛋白質樣本製備…………….……………….……………………… 12
3.2 蛋白質濃度測定……………………………..…...………………… 12
3.3 聚丙烯醯胺膠體電泳分析…………………….....………………… 12
3.4 轉漬………………………………………….……………………… 12
3.5 免疫呈色反應………………………………….…………………… 13
4. 質體 DNA 之製備………….……………………...…………………… 13
5. 質體之構築…………………………….…………..…………………… 14
5.1 S6K1/AR 質體構築…………………...……...…………………… 14
5.2 pCDH-S6K1/ pCDH-AR 質體建構……………...…..……….…… 14
6. 病毒製備及感染………………….…………..………………………… 15
6.1 病毒製備………………………….………………….……………… 15
6.2 病毒感染………………………….………………..…...…………… 16
7. 統計分析…………………….………………..………………………… 16
第五章、結果……………………………...……………..….………………… 17
1. mTOR/S6K1 訊息傳遞分子與AR在攝護腺細胞與口腔鱗狀上皮癌細胞中之表現量及其磷酸化程.……………………….………...… 17
2. 雄性素DHT或AR抑制劑Bicalutamide對攝護腺癌與口腔癌細胞之S6K1與RPS6之表現量及其活性影響…….………………….… 17
3. 大量表現S6K1蛋白對LNCaP與SCC4細胞之AR表現量,磷酸化程度或生長能力之影響分析…………….………………………… 18
4. 降解S6K1表現量對LNCaP與OECM-1細胞中AR表現量,磷酸化程度及細胞生長能力之影響。…………….…………....……… 18
5. AR抑制劑Bicalutamide對S6K1大量表現之LNCaP細胞中S6K1,RPS6與AR磷酸化程度,與S6K1增幅表現之SCC25 細胞生長能力之影響……………….………………………………...… 19
6. S6K1抑制劑PF-4708671對LNCaP及SAS細胞之S6K1,RPS6及AR之蛋白質表現量與磷酸化程度之影響………………….…… 19
第六章、討論…………………………………………………..………...…… 21
第七章、結論……………………………………...………………...………… 25
第八章、未來研究計畫……………...……………………………..…….…… 27
第九章、圖表…………………...………………………………..……….…… 29
圖三、mTOR/S6K1/RPS6訊息傳遞路徑與雄性素受器AR在攝護腺細胞與口腔鱗狀上皮癌細胞中之表現量及其磷酸化程度...…………… 29
圖四、AR訊息傳遞路徑對攝護腺癌細胞與口腔癌細胞中S6K1及 RPS6 之表現量與磷酸化程度…………………………...………….…… 30
圖五、大量表現 S6K1蛋白對 LNCaP與SCC4細胞之AR表現量,磷酸化程度或生長能力之影響分析………………………………....…... 31
圖六、降解 S6K1表現量對LNCaP與OECM-1細胞中AR表現量,磷酸化程度及細胞生長能力之影響……………………...…...……….… 32
圖七、AR抑制劑Bicalutamide對S6K1大量表現之LNCaP細胞中RPS6與 AR表現量,磷酸化程度,與S6K1增幅表現之SCC25細胞生長能力之影響…...………………………………………….…...…… 33
圖八、S6K1抑制劑PF-4708671對LNCaP及SAS細胞之S6K1,RPS6及AR 表現量與磷酸化程度之影響………………………...……. 34
第十章、參考文獻………………………..…………...………………………. 35
第十一章、附表……………………………………..……...…………………. 39
(一) 抗體………………….………………………...…………………...…. 39
(二) 抑制劑……………………….……………...………………...………. 39
(三) 引子序列…………………….……………...………………...………. 40

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