2011-05-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643519摘要：Integrin 是細胞膜上負責調控細胞－細胞間和細胞－基質間互動的重要受體，integrin 是由α和β兩個子體所構成的異構雙圓體。在體內有二十種以上的integrin 表現，且Integrin參與了許多細胞的生理過程，包括增生、粘著、分化與移行。然而在不少疾病中，integrin也扮演著重要角色，包含在癌症細胞的生存和轉移，以及癌症的血管新生和視網膜血管新生相關疾病。在癌症發展中，細胞外基質被證實會參予調控癌症的生長、發育、轉移以及血管新生，而在這些細胞基質中，osteopontin 以及fibronectin 被認為是重要的癌症調節物質，而osteopontin 和vitronectin 的受體αvβ3 以及fibronectin 的受體α5β1 在抗癌新藥研發上被視為重要的標的。而目前在臨床試驗的αvβ3 或α5β1 的拮抗劑大多為小分子胜肽或者是單株抗體。然而小分子胜肽在體內的作用時間較短，而單株抗體對抗原的辨認只有一個點。在本計畫中，成功大學莊偉哲教授修改了由蛇毒分離出來的disintegrin,rhodostomin 的RGD 及其附近之amino acid 序列，產生了ARLDDL 之蛋白候選藥物，此蛋白擁有六個雙硫鍵，穩定度高。且經由基因修改後，對αvβ3 的選擇性非常高，對血小板之αIIbβ3 之親合力降低1000 倍，因而可以避免出血之副作用。最近我們更將此蛋白以30kD 之polyethyleneglycol 成功進行聯結，形成約40kD 之PEG-ARLDDL，以提高其在體內的停留時間並降低其在體內可能產生的免疫反應。我們最近的前驅實驗中，進一步證實了αvβ3 integrin 在癌細胞存活的重要性，我們在攝護腺PC-3 癌細胞中發現到osteopontin 會透過αvβ3 integrin 經由增加癌細胞的drugtransporter 之表現，而促使癌細胞加速排出化療藥物，經由抑制αvβ3 integrin 則可以對抗osteopontin 所誘發的抗藥性，在動物實驗中也發現到抑制αvβ3 integrin 或將osteopontinknockdown 可以促進化療藥物對癌細胞的毒殺作用。本計畫將進一步探討osteopontin 對不同種類癌細胞之各種drug transporter 表現之調節作用，尋找出αvβ3integrin 拮抗劑除了治療骨轉移之腫瘤外，還可以適用於那些種類之腫瘤與不同種類之化療藥物合併治療，此研發有助於減少腫瘤之抗藥性。我們也要針對αvβ3 以及α5β1發展同時抑制二種integrins 的拮抗劑，並將其進行pegylation，以應用於不同癌症之治療。共同主持人莊教授將負責蛋白藥物衍生物之合成及變異，而本計畫將負責藥效的測定及藥理的研究。本三年計畫我們將合成的蛋白藥物做下列藥理作用的探討：1. 癌細胞表面integrin 表現種類將與該癌細胞對何種integrin 之拮抗劑敏感度有關，因此我們將一系列探討αvβ3 及α5β1 integrin 在不同種類癌細胞之表現分佈，如A549 肺癌細胞; MDA-231, MCF-7 and 4-T1 乳癌細胞; PC-3, LNCaP cells andDU145 前列腺癌細胞; SKOV3 卵巢癌細胞; U87, A172, and C6 glioma 癌細胞;HepG2 and Hep3B 肝癌細胞; MG-63 骨癌細胞; melanoma 黑色素瘤細胞等。(第一年)2. 腫瘤形成過程常會面臨缺氧狀態，我們將探討在缺氧狀態下，不同種類癌細胞osteopontin 的表現調節有何差異。(第一年)3. 探討osteopontin 在不同種類癌細胞對於各種drug transporter 之調節作用並探討其作用機轉，下列drug transporter 將被探討：: MDR1, MDR2, MRP3, MRP4, MRP5,MRP6, ABCG2. (第一年)4. 癌細胞對於葡萄糖之需求增加，我們將探討osteopontin 在不同種類癌細胞對於各種glucose transporter 之調節作用並探討其作用機轉，探討之drug transporter 如下：GLUT1, GLUT2, GLUT3, GLUT4. (第一年)5. 探討PEG-ARLDDL 與不同種類之抗癌藥物對於不同種類癌細胞是否有協同之細胞毒殺作用。(第一年)6. 使用異種癌細胞皮下移植動物模式來探討PEG-ARLDDL 對於不同種類癌細胞(攝護腺癌、乳癌、肺癌、卵巢癌、肝癌以及神經膠質瘤)之抗癌作用。(第一、二年)7. 在癌症動物實驗中探討PEG-ARLDDL 結合化療藥物對抗不同種類癌細胞之協同作用。(第一、二年)8. 發展二代integrin 拮抗藥物，同時抑制αvβ3 及α5β1 二種integrins。(第二、三年)9. 使用matrigel 移植所誘發的血管新生以及高壓氧所誘發之視網膜血管新生模式來比較PEG-ARLDDL 以及PEG-dual integrin inhibitor 之抗血管新生作用。(第二、三年)10. 使用異種癌細胞皮下移植動物模式來比較PEG-ARLDDL 以及PEG-dual integrininhibitor 對於不同種類癌細胞(攝護腺癌、乳癌、肺癌、卵巢癌、肝癌以及神經膠質瘤)之抗癌作用。(第二、三年)由此計畫之執行，將能有助於抗癌藥物全新領域之開發。<br> Abstract: Integrins are heterodimeric cell surface receptors which mediate cell-cell and cell-matrixinteraction. There are about 20 subtypes in the integrin superfamily and integrins arecomposed with α and β subunits. They are involved in physiological cell function includingcell growth, adhesion, differentiation and migration. However, integrins also play animportant role in many diseases including the cell growth and migration of cancer cells andthe angiogenesis in cancer or age-related macular degeneration. In the process of cancer,several extracellular matrices are indicated to regulate the proliferation, growth, migration andangiogenesis of tumor. Osteopontin and fibronectin are considered as important cancerregulators. The antagonists against osteopontin and vitronectin receptor, αvβ3 or thefibronectin receptor, α5β1 are developed by many pharmaceutical companies and somemonoclonal antibodies are undergoing clinical trials. However, small molecular peptides haveshort duration in live animals and monoclonal antibody has only one binding site with target.We have collaborated with professorWoei-Jer Chuang (National Cheng-Kung University) todevelop a selective αvβ3 antagonist –a protein derived from the genetic modification of RGDdomain and its neighborhood in snake venom Rhodostomin. The most promising candidate isARLDDL. Compared with monoclonal antibody, this protein drug has 3 binding sites withαvβ3integrin and has much higher affinity with target integrin. The binding of ARLDDL toαIIbβ3of platelet is reduced to 1000 fold so that the bleeding side effect is markedly reduced.In addition, ARLDDL is successfully pegylated with 30kD polyethyleneglycol(PEG-ARLDDL) with the similar affinity to αvβ3integrin. PEG-ARLDDL has longer halflife and the immunogenicity is reduced in vivo.Our preliminary results show that osteopontin can upregulate drug transporter in prostatePC-3 cancer cells. Inhibition of αvβ3 integrin can antagonize osteopontin-induced drugresistance. In animal models, knockdown of osteopontin or inhibition of αvβ3 integrin couldenhance the actions of cytotoxic drugs. In this project we will further investigate which kindsof tumor cells are more sensitive to disintegrin drugs. In addition, whether the antitumoractivity is enhanced by the combination of PEG-ARLDDL and different kinds of cytotoxicdrugs will also be evaluated. Since many kinds of tumor cells also over-express α5β1integrin,we will further develop the second generations of pegylated disintegrin for the dual inhibitionof αvβ3and α5β1integrins.In this three-year project, the following experiments will be conducted using these proteindrugs:1. The types of integrin distributed in cancer cells determine the sensitivity to disintegrin.Wewill examine the distribution of αvβ3and α5β1integrins in various kinds of cancer cellline, including A549 lung cancer; MDA-231, MCF-7 and 4-T1 breast cancer; PC-3,LNCaP cells and DU145 prostate cancer; SKOV3 ovary cancer; U87, A172, and C6glioma cells; HepG2 and Hep3B hepatoma cells; MG-63 osteosarcoma cells; melanomacells etc. (Year-1)2. Tumor growth will have the hypoxic environment.We will examine which kinds of tumorcells upregulate osteopontin expression in hypoxic condition. (Year-1)3. We will examine the regulation of drug transporters in various kinds of tumor cells byosteopontin. The mechanisms regarding how osteopontin induces drug resistance in tumorcells will also be investigated. The following drug transporters will be examined: MDR1,MDR2, MRP3, MRP4, MRP5, MRP6, ABCG2. (Year-1)4. Accelerated glycolysis is one of the biochemical characteristics of cancer cells and inmany cases the glucose transporter gene is upregulated in malignancy. We will examinethe effect and the mechanism of glucose transporters regulation by osteopontin in variouskinds of tumor cells. The following glucose transporters will be examined: GLUT1,GLUT2, GLUT3, GLUT4. (Year-1)5. To examine whether different cytotoxic drugs exert synergistic effect or reduction of drugresistance in causing apoptosis of cancer cells when combination with PEG-ARLDDL.(Year-1)6. To evaluate the anti-tumor efficacy of PEG-ARLDDL in different kinds of tumor cell lineby using xenograft animal models. The following tumor cell lines will be used: A549 lungcancer; MDA-231, MCF-7 and 4-T1 breast cancer; PC-3, LNCaP cells and DU145prostate cancer; SKOV3 ovary cancer; U87, A172, and C6 glioma cells; HepG2 andHep3B hepatoma cells; MG-63 osteosarcoma cells; melanoma cells etc. (Year 1~2)7. To evaluate whether the drug resistance is reduced by PEG-ARLDDL in xenograft models(subcutaneous injection of different cancer cell lines) when cytotoxic drugs is combinedwith integrin antagonist. (Year 1~2)8. To develop the second generations of disintegrin drug for dual inhibition of αvβ3andα5β1integrins. (Year 2~3)9. To compare the inhibition of angiogenesis between PEG-ARLDDL and dual inhibitor forαvβ3and α5β1integrins by using matrigel model in B6 mice or hyperoxia/normoxiamodel in neonatal ICR mice. (Year 2~3)10. To compare the inhibition of tumor growth between PEG-ARLDDL and dual inhibitor forαvβ3and α5β1integrins by using xenograft model (using lung cancer, breast cancer,prostate cancer, ovary cancer, glioma cells, hepatoma cells, osteosarcoma cells ormelanoma cells etc.). (Year 2~3)From this project, we may develop new method for the inhibition of drug resistance or thetreatment of different kinds of cancer.造骨蛋白抗藥性p-glycoprotein醣蛋白攝護腺癌組合蛋白osteopontindrug resistancep-glycoproteinPC-3αvβ3 integrinchemotherapyARLDDL