臺灣大學: 生化科學研究所陳義雄; 梁啟銘彭瑞銘Peng, Jei-MingJei-MingPeng2013-03-212018-07-062013-03-212018-07-062011http://ntur.lib.ntu.edu.tw//handle/246246/250971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.37296704 bytesapplication/pdfen-US化療藥物自發性死忙轉移結合子卵巢癌chemotherapeutic agentapoptosismetastasisintegrinovarian cancer[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/250971/1/ntu-100-D93b46008-1.pdf