臺灣博碩士論文加值系統

口蹄疫病毒(FMDV)是經由其表面鞘蛋白質(VP1)上具有特殊的精胺酸，甘胺酸和天門冬胺酸等胺基酸來結合到細胞表面的integrin結合子。目前尚未清楚的是在此鞘蛋白質(VP1)結合到細胞表面之後，會刺激細胞有什麼樣的細胞反應。首先，我們成功的利用重組DNA的方式，純化出可水溶性的口蹄疫病毒表面鞘蛋白質(rVP1)，並發現此重組鞘蛋白質可以結合到倉鼠腎細胞表面的integrin結合子，並且引發細胞自體計畫性死亡反應。除此之外，我們並發現將重組蛋白質rVP1處理到倉鼠腎細胞，會引起Akt蛋白質的去活化，並引發下游細胞自體計畫性死亡反應，包括GSK-3B蛋白質的去磷酸化和procaspase -3, -7和-9等蛋白質的活化。此外，進一步的研究發現經重組蛋白質rVP1處理過後會引起癌細胞的自體計畫性死亡，包含: 人類乳癌細胞株(MCF-7，T-47D，MDA-MB-231)，人類前列腺癌細胞株(22Rv1，PC-3)和人類卵巢癌細胞株(SKOV3，OVCAR-3，TOV-21G)。

目前對於具轉移情況的卵巢癌病患其治療成果仍然不佳，更進一步的研發能抑制癌細胞轉移能力的藥物是迫不及待的。我們的研究發現重組蛋白質rVP1在卵巢癌細胞作用的機制，其發現其不但具有抑制卵巢癌細胞生長的效果更能在小鼠體內成功抑制癌細胞的轉移。我們的結果發現重組蛋白質rVP1會在卵巢癌細胞引發自體計畫性死亡和減少其轉移能力。這個機制是經由活化細胞內的PTEN蛋白質和GSK-3B蛋白質，並抑制FAK，Akt和MMP-2蛋白質的活性。利用抗integrin結合子的抗體或是大量表現可持續具有活性的Akt可以大幅減低重組蛋白質rVP1的作用。並經由在小鼠原位引發卵巢癌和腹腔注射卵巢癌細胞的方法，發現重組蛋白質rVP1可以在體內抑制卵巢癌細胞的生長和轉移。經由在冷光顯像系統和免疫染色方法分析在小鼠體內的癌細胞其Akt和GSK-3B蛋白質的活性皆受到重組蛋白質rVP1調控。這些結果呈現出重組蛋白質rVP1經由負調控Akt蛋白質下游反應和減低MMP-2蛋白質活性後，具有在小鼠體內抑制卵巢癌細胞的生長和轉移的能力。

VP1, a capsid protein of Foot-and-mouth disease virus (FMDV), binds to cellular integrins through an arginine-glycine-aspartic acid motif. It is unclear, however, what kind of cellular event(s) is triggered after the binding of VP1 to the cells. First of all, we found that aqueous soluble recombinant DNA derived VP1 (rVP1) of FMDV induced apoptosis in BHK-21 cells after binding to integrins. In addition, treatment of BHK-21 cells with rVP1 resulted in deactivating Akt, and enhancing several pro-apoptotic responses, such as dephosphorylation of GSK-3β and cleavage of procaspase -3, -7 and -9. We also illustrated that the rVP1 treatment caused apoptosis of cancer cells including human breast carcinoma (MCF-7, T-47D, MDA-MB-231), human prostate cancer (22Rv1, PC-3) and human ovarian cancer (SKOV3, OVCAR-3, TOV-21G).

As prognosis for patients with metastatic ovarian cancer is generally poor, advances in treatments are needed. We have studied the effect of rVP1 on ovarian tumor growth and metastasis in vivo. We showed that rVP1 promoted the apoptosis of human ovarian cancer cells and decreased the cell invasion. This effect of rVP1 was accompanied by the activation of PTEN and GSK-3β as well as downregulation of FAK, Akt and MMP-2. Anti-integrin antibodies or overexpression of constitutively active Akt reversed the cellular effects of rVP1. Orthotopic and intraperitoneal xenograft mouse models demonstrated that rVP1 attenuated survival and metastasis of human ovarian cancer SKOV3 cell line in vivo through selective regulation of Akt and GSK-3β activity as revealed by bioluminescence imaging of mice and immunohistochemical analysis. These results indicate that negative regulation of Akt signaling and MMP-2 by rVP1 may have the potential to suppress ovarian tumor growth and metastasis in vivo.

Table of Contents i
List of Figures iv
Abbreviations ix
Abstract xi
中文摘要 xiii
Introduction 1
Materials and Methods 26
1. Materials 26
2. Site-directed mutagenesis 27
3. Expression and purification of rVP1 28
4. Cell lines and Treatments 28
5. DNA Fragmentation Assay by eletrophoresis analysis 30
6. DAPI stain for Cell Survival and Apoptosis 31
7. Cell Transfection and Plasmid Expression 31
8. Indirect Immunofluorescence 32
9. Cell viability assay 32
10. Internalization of rVP1 on SKOV3 cells 33
11. Caspase activity assay 34
12. DNA fragmentation assay 34
13. Matrigel invasion assay 35
14. Gelatin zymographic analysis 36
15. Isolation of intraperitoneal cells 36
16. Preparation of pCMV-GFP/leuciferase-lentivirus and establishment of a stable SKOV3-GL cell line 37
17. Detection of luciferase activity in vivo by bioluminescence imaging 38
18. Immunohistochemistry 39
19. Orthotopic xenograft mouse model of ovarian cancer 40
20. Intraperitoneal xenograft mouse model of ovarian cancer 41
21. Statistical analysis 42


Results 43
1. rVP1 induces apoptosis 43
2. rVP1 is more potent than cRGD and p29 in inhibitio of cell viability 44
3. rVP1 induces apoptosis by binding to integrin 44
4. rVP1 treatment deactivates Akt 46
5. rVP1 activates cleavage of procaspases 47
6. rVP1 binds to the surface of cancer cell 48
7. rVP1 induces DNA fragmentation in cancer cell 48
8. rVP1 induces cancer apoptosis disregarding the absence of caspase-3 and S1P3. 49
9. MCD can reverse rVP1 induced DNA fragmentation in cancer cell 50
10. rVP1 induces apoptosis and suppresses invasion of ovarian cancer cells in vitro 51
11. rVP1 inhibits survival of SKOV3 cells via dephosphorylation of Akt, GSK-3β and phosphorylation of PTEN 52
12. rVP1 reduces invasive capacity of SKOV3 cells via integrin and Akt/MMP-2 54
13. rVP1 reduces tumor growth and metastasis of SKOV3 cells in an orthotopic xenograft mouse model 55
14. rVP1 reduces advanced stage ovarian tumor and metastasis in an intraperitoneal xenograft mouse model 57
15. rVP1 reduces early stage ovarian tumor and metastasis in an intraperitoneal xenograft mouse model 59
16. rVP1 can not reduces SKOV3Akt-DA tumor and metastasis in an orthotopic xenograft mouse model 60
17. rVP1 suppresses invasive capacity of peritoneal adapted SKOV3 cells via integrin and Akt/MMP-2 61
18. rVP1 shows a synergistic effect with doxorubicin and cisplatin in cancer cells 62
19. MCD and anti-α5β1 antibody can not reverse doxorubicin and cisplatin induced cell death 63
20. rVP1 binds and inhibits the activity of topoisomerase II 64
21. Both paclitaxel and rVP1 reduces SKOV3 tumor and metastasis in an orthotopic xenograft mouse model 64




Discussion 66-75
Figure 76-153
Table 154-155
Reference 156-166
Appendix 167-171

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