2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657228摘要：背景: 有非常多的研究曾廣泛性的探討黃酮類及chalcones 衍生物，在抗癌方面的活性及相關的訊息傳遞路徑。其作用的標的，特別是-酵素標的，也曾被廣泛研究，如:protein tyrosine kinases、12-lipoxygenase 、phosphatidylinositol 3-kinase (PI3-kinase)/Akt、topoisomerases 及pim-1kinases。特別值得注意的是，有些作用標的酵素在訊息傳遞路徑中，被證明很重要，有些標的則僅是被某些黃酮類成分”作用到”，但其貢獻於抗癌活性的程度則並不如預期。在許多已發表的研究當中，大都針對個別的酵素活性大小，做化學結構及作用活性的關係來分析，甚少有同時針對酵素活性大小及抗癌活性(如抑制腫瘤細胞增生及促進細胞凋亡)的化學結構及作用活性的關係來分析，因為若同時考慮這些因素，則結果時常很不一致。因此，本計畫之特殊目的及初期結果如下：在我們的初期成果中，以pim-1 酵素為例，與apigenin、genistein 及naringenin比較起來，quercetin 及kaempferol 是很強的pim-1 的抑制劑，其IC50 值為0.10 (quercetin)、0.44(karmpferol)、10.4 (apigenin)、51.2 (genistein)及114 (naringenin) μM。然而，在肝癌細胞Hep3B及多種其它腫瘤細胞(皆含有pim-1 酵素蛋白)，其抑制腫瘤細胞增生及促進細胞凋亡的活性大小為genistein > apigenin > quercetin > kaempferol > naringenin，這些不一致的結果背後應有其重要原因，也因此，我們第一年的工作，將先針對現有的三十個天然及化學合成修飾的黃酮類及chalcones 衍生物，探討其在肝癌及多種癌細胞的抑制腫瘤細胞增生及促進細胞凋亡的活性大小，以及對於多種酵素活性的抑制作用大小，包括protein tyrosine kinase、12-lipoxygenase、PI3-kinase、topoisomerase II 及pim-1 等酵素。分析每一種酵素抑制作用與抗癌活性的關係，藉此研究出重要的酵素，並以siRNA 的技術剔除酵素蛋白，來做更證確的驗證。第二年，我們的工作同仁也會根據第一年的結果，合成最優化的結構化合物，來對於標的酵素及抗癌活性做最好的抑制。此外，根據我們初期的成果內容，微管的作用藥物(如paclitaxel)皆能顯著的增加pim-1 的蛋白表現，將pim-1 剔除，則除了會增加腫瘤細胞的凋亡外，也會大大的增加微管作用藥物的抗癌作用，這些結果顯示pim-1 為存活(pro-survival)因子，且是抗藥性蛋白。也因此，本計畫也要執行合併治療，將合併最優化衍生物及抗癌藥物，如paclitaxel、vincristine、doxorubicin、5-Fu、mitoxantrone、irinotecan、SAHA 及actinomycin D。藉此找出最好的合併內容。第三年，我們將設計出肝腫瘤同位癌動物模式(orthotopic xenograft tumor model，此模式日後也將服務國內學者)，根據第一及第二年的結果，將這些潛力化合物作用於動物模式，並且有單一及合併療法的作用方式，來研究其動物體內活性。此外，我們也將研究在動物體內作用的機轉探討及相關的訊息傳遞內容。在三年計畫結束後，我們預期將發表八篇以上的研究論文(2+3+3)，期望對生物醫學有所貢獻。<br> Abstract: Background: Flavonoids and chalcones are extensively studied to display anticancer activity throughnumerous signaling pathways. The primary targets, in particular the enzyme targets, have beenidentified being protein tyrosine kinases, 12-lipoxygenase, phosphatidylinositol 3-kinase(PI3-kinase)/Akt, topoisomerases and pim-1 kinases. It is worth noting that some of the targetproteins and kinases are in depth elucidated and are well recognized as key regulators in the signalingpathways. However, some targets seem to be “affected”by the individual flavonoid but do not soundpersuasive enough. In many published studies, the structure-activity-relationship has beenconstructed according to the target enzyme. Nevertheless, few studies concomitantly dealt with thetarget enzyme and anticancer activities (e.g., antiproliferation and apoptotic cell death). If both partsare taken into account, the difficulty may come about by inconsistent relationship. Specific goals andpreliminary data: Take pim-1 kinase activity as an example, our preliminary data showed thatquercetin and kaempferol are much more potent inhibitors of pim-1 kinase than apigenin, genisteinand naringenin. The IC50 values are 0.10, 0.44, 10.4, 51.2, and 114 μM, respectively. However, therank order of antiproliferation and apoptotic activities is genistein > apigenin > quercetin >kaempferol > naringenin in human hepatoma Hep3B cells and some other cancer cell lines that arerich in pim-1 kinases. Because of the controversy, there are several specific goals in the three-yearproposal. The first year, we already have thirty natural and chemically synthetic flavonoid andchalcone derivatives. We will determine the antiproliferation and apoptotic effect of these derivativesas well as the effect on the enzyme activities, including protein tyrosine kinase, 12-lipoxygenase,PI3-kinase, Top-II and pim-1. The correlation between every single enzyme effect and cell-basedanticancer activity will be constructed to discover critical target by the derivatives. The siRNAtechnique will be subsequently used to knockdown the enzyme and validate the proposed target. Thesecond year, our colleague will synthesize more derivatives according to the first year’s work andoptimize the structures based on the data. From the structure-optimized derivative, we will performthe holistic mechanism study of the anticancer signaling cascades. Moreover, according to ourpreliminary data, anti-tubulin agents stimulate a profound increase of pim-1 expression. Thepreliminary data showed that the knockdown of pim-1 increased intrinsically the apoptosis as well asanti-tubulin agent-induced apoptosis. The data substantiated that pim-1 kinase is a pro-survival factorand exhibits drug resistance to anti-tubulin drugs. From the data we anticipate that the combination ofpim-1 inhibitors with anti-tubulin agents may potentiate apoptosis of cancer cells. Accordingly, theapproach of combination therapy between the derivative and cancer chemotherapeutic drugs, such aspaclitaxel, vincristine, doxorubicin, 5-Fu, mitoxantrone, irinotecan, SAHA, and actinomycin D willbe carry out to determine the optimal combination. The third year, after the achievement of the firsttwo years, several potential compounds will be obtained that are specific target-oriented. We willdesign animal studies for the determination of in vivo efficacy of the compounds. An orthotopicxenograft tumor model of hepatocellular carcinoma will be created and the single as well ascombination treatment will be tested. Besides, the mechanism study associated with related signalingwill be conducted in an in vivo model. We anticipate that more than eight original papers will bepublished in the end of the project.黃酮類衍生物抗癌活性合併療法肝癌Flavonoid derivativesanticancer activitycombinatory treatmentliver cancer