臺灣博碩士論文加值系統

咖啡酸苯乙酯 (caffeic acid phenethyl ester, CAPE)是一種從蜂膠中萃取出的生物活性成分。本實驗室先前研究指出，不論是細胞實驗或是動物實驗，CAPE處理均能抑制男性賀爾蒙依賴型LNCaP 104-S 前列腺癌細胞生長。其分子機轉是透過抑制Akt與c-Myc的訊息傳遞路徑，因而導致細胞週期停滯在G1階段。目前轉移性前列腺癌最主要的治療方法是賀爾蒙療法 (Androgen ablation therapy)，但是大多數接受賀爾蒙療法的前列腺癌患者一至三年後，最終進展復發成非賀爾蒙依賴型前列腺癌 (CRPC)，復發後患者平均存活時間約一至兩年。目前仍然缺乏有效的治療方法，所以極需發展出新的治療方法。
LNCaP癌細胞株是從轉移到淋巴結的人類前列腺癌細胞中所產生。將男性賀爾蒙依賴型LNAaP 104-S癌細胞持續培養在缺乏男性賀爾蒙的環境下，能獲得非男性賀爾蒙依賴型的 LNCaP 104-R1 癌細胞株， LNCaP 104-R1癌細胞能模擬非賀爾蒙依賴型前列腺病患的情況。
我們發現CAPE處理能使LNCaP 104-R1癌細胞的細胞週期停滯在G1階段，進而抑制癌細胞增生，並能抑制裸鼠腫瘤生長。Micro-Western Array (MWA) 是種高通量西方點墨微陣列，能夠同時檢測6~15種樣品的96~384支抗體表現。藉由高通量西方點墨微陣列，我們發現CAPE處理在LNCaP 104-R1癌細胞中只是輕微抑制Akt訊息傳遞，卻能顯著抑制Skp2表現量。這也許可以解釋為何LNCaP 104-R1癌細胞相較於LNAaP 104-S癌細胞對於CAPE具有較大的耐受能力。Skp2過量表現能夠阻斷CAPE的抑制效果。CAPE與PI3K抑制劑-LY294002及B-cl2抑制劑ABT-737共同處理下都具有加乘效果。我們的發現指出CAPE具有潛力能做為非賀爾蒙依賴型前列腺癌的輔助治療。

Caffeic acid phenethyl ester (CAPE) is a bioactive component derived from honeybee hive propolis. We previously showed that treatment with CAPE suppresses growth of androgen-dependent AR-positive LNCaP 104-S cells both in vitro and in vivo via inhibition of Akt and c-Myc signaling, thus causes G1 cell cycle arrest. Androgen ablation therapy is the primary treatment for metastatic prostate cancer (PCa). However, a majority of PCa patients receiving the androgen ablation therapy will ultimately develop recurrent castration-resistant prostate cancer (CRPC) within 1-3 years after treatment with a median overall survival time of 1-2 years after relapse. There is currently no effective therapy and new therapeutic targets are needed. The LNCaP cancer cell line was established from a human lymph node metastatic lesion of prostatic adenocarcinoma. We have established AR-rich androgen-independent LNCaP 104-R1 cells derived from parental androgen-dependent LNCaP 104-S cells under androgen depleted condition. LNCaP 104-R1 cells mimic CRPC in patients. CAPE treatment caused G1 cell cycle arrest in 104-R1 cells and thus suppressed cell proliferation and tumor growth in nude mice. Micro-Western Array (MWA) is an antibody-based modified reverse phase array, which allows detecting protein expression level or phosphorylation status change of 96-384 different antibodies in 6-15 samples simultaneously. Using MWA platform, we found that CAPE reduced Akt-related proteins expression slightly, but decrease Skp2 abundance significantly, which may explain its larger resistance to CAPE treatment than 104-S cells. Overexpression of Skp2 blocked suppressive effect of CAPE. Co-treatment of CAPE with PI3K inhibitor LY294002 or Bcl-2 inhibitor ABT-737 both showed synergistic suppressive effects. Our finding suggested that CAPE treatment is a potential therapy for CRPC.

謝誌………………………………………………………………………………………i
ABSTRACT,………………………………………………………………….…………ii
中文摘要………………………………………………………………………………….iv
ABBREVIATION………………………………………………………………………vi
CHAPTER
1. INTRODUCTION …………………………..………………………………………...1
1.1 Prostate cancer………………………………………………………..……………1
1.2 Caffeic acid phenethyl ester……………………………………………….………4
1.3 Phosphatidylinositol 3-kinase (PI3K) /Akt pathway……………………..………5
1.4 The cell cycle………………………………………………………………………5
1.5 S-phase kinase–associated protein 2 (Skp2) ……………………………………...7
1.6 B-cell lymphoma 2 (Bcl-2) and Bcl-2 inhibitor ABT-737…………………….…8
1.7 The aims of the study………………………..……………………………….8
1.8 The Flowchart of the study…………………………………………...………...10
2. MATERIALS and METHODS…………………………………………………….…11
2.1 Reagents……………………………………………………………………….…11
2.2 Cell Culture………………………………………………………………………12
2.3 The Preparation and Process of CAPE………………………………………...…13
2.4 Cell Viability Assay………………………………………...…………………….14
2.5 Cell Proliferation Assay……………………………………...…………………...15
2.6 Soft Agar Colony Formation Assay………………………………...……………15
2.7 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)………………..16
2.8 Micro-Western Array……………..………………………. ……………………..17
2.9 Western Blotting Analysis……………………..…………………….………....18
2.10 Protein Overexpression……………………..……………………….………...19
2.11 Flow Cytometry assay…...……………………………..……………………...20
2.12 Tumor Xenografts………………...…………………………………………...21
2.13 Statistics…………………………...…………………………………………...22
3. RESULTS………………………………...…………………………………………..23
3.1 CAPE treatment suppresses the survival and proliferation of LNCaP 104-R1 human prostate cancer cells ……...………………………………………………23
3.2 CAPE treatment suppresses soft agar colony formation of LNCaP 104R1 cells. . …………………………………………………………………………….24
3.3 CAPE treatment disturbs cell cycle progression in LNCaP 104R1 cells………...24
3.4 CAPE causes a reduction in abundance and activity of cell cycle promoting proteins. ………………………………………………………………………….25
3.5 CAPE treatment downregulates mRNA level expression. …………………...….27
3.6 Overexpression of Akt1 or Skp2 in LNCaP 104-R1 cells rescues inhibition cause by CAPE treatment. ……………….......................................................................27
3.7 Combined treatment of CAPE with LY294002 shows synergistic and antagonistic inhibition on proliferation of LNCaP 104-R1 cell. ...............................................28
3.8 Combined treatment of CAPE with Bcl-2 inhibitor ABT-737 shows synergistic and antagonistic inhibition on proliferation of LNCaP 104-R1 cell. ....................29
3.9 CAPE treatment retards growth of prostate cancer xenografts in nude mice. ....30
4. DISCUSSION ……………………...………………………………………………..32
5. REFERENCES………………………………………….…………………………..36
6. FIGURES and FIGURE LEGENDS………………………………….……………...42
Figure 1…………………………….………………………………….……………...42
Figure 2…………………………….………………………………….……………...43
Figure 3…………………………….………………………………….……………...44
Figure 4…………………………….………………………………….……………...45
Figure 5…………………………….………………………………….……………...46
Figure 6…………………………….………………………………….……………...47
Figure 7…………………………….………………………………….……………...48
Figure 8…………………………….………………………………….……………...49
Figure 9…………………………….………………………………….……………...50
Figure 10………………………….………………………………..….……………...56
Figure 11………………………….………………………………..….……………...57
Figure 12………………………….………………………………..….……………...58
Figure 13………………………….………………………………..….……………...59
Figure 14………………………….………………………………..….……………...60
Figure 15………………………….………………………………..….……………...61
Figure 16………………………….………………………………..….……………...62
Figure 17………………………….………………………………..….……………...63
Figure 18……………………….……………………………………………………..64

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