Flavonone結構相似化合物抑制腫瘤促進作用之活性研究

Anti-tumor promotion activities of structure related flavanones

柯景懷(Ching Huai Ko)

指導教授 ： 楊玲玲 ； 陳彥州

臺北醫學大學/藥學院/生藥學研究所/碩士(2001年)

摘要

Flavonoids廣泛的存於植物界中，並且許多的研究均證實其具有多種生物活性，然而flavonoids對TPA誘發癌化的結構相關活性仍有待探討。在本實驗中選取八種相關結構的flavanone，包括flavanone，2'-OH flavanone，4'-OH flavanone，6-OH flavanone，7-OH flavanone，narigin，naringenin及taxifolin，針對12-ο-tetradecanoylphorbol 13-acetate (TPA)所誘發NIH3T3細胞之癌化，探討其抑制之情形。利用MTT及[3H] thymidine incorporation的分析結果中顯示，TPA(100 ng/ml)可誘發NIH3T3細胞較對照組多達3倍的增生，而flavanone，2'-OH flavanone，4'-OH flavanone，6-OH flavanone則可對TPA誘發之細胞增生呈現明顯的劑量依循性抑制。NIH3T3細胞在TPA的處理下，PKCa會從細胞質位移至細胞膜上（translocation），flavanone，2'-OH flavanone，4'-OH flavanone，6-OH flavanone同樣的亦抑制了此位移。許多研究中指出MAPK、ODC、c-Jun、COX-2亦參與於TPA所誘導的癌化瀑布效應及PKC活化調控，在本實驗中TPA可誘導磷酸化MAPK、ODC、c-Jun、COX-2蛋白質呈時間依循性的表現，而磷酸化MAPK和其他的蛋白質分別在1小時及6小時產生最明顯的表現，Flavanone，2'-OH flavanone，4'-OH flavanone，6-OH flavanone亦可對TPA誘導的上述蛋白質表現產生明顯的劑量依循性抑制，而抑制能力的程度分別是2'-OH flavanone> 6-OH flavanone> 4'-OH flavanone> flavanone。為了深入了解此四個化合物對TPA抑制之活性，NIH3T3細胞以TPA(100ng/ml)持續處理3個月，探討此四個化合物對TPA誘導的細胞群落增生之抑制情形，以及群落增生後細胞磷酸化MAPK、ODC、c-Jun、COX-2蛋白質的表現。而此四個化合物對TPA誘導的細胞群落增生產生明顯之抑制，同時亦明顯抑制上述蛋白質的表現。由上述實驗結果推論，flavanones可以有效抑制TPA誘導的癌化作用，而且指出結構中-OH官能基數目並非影響其活性表現的主要因素。

關鍵字

類黃酮素 ； 腫瘤促進

並列摘要

Flavonoids are well distributed in plants and have been demonstrated to perform several biological activities, however the structure-activity relationship of flavanoids on TPA induced tumor promotion is still unclear. Eight structure related flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, narigin, naringenin and taxifolin were used to study their inhibitory effects on 12-ο-tetradecanoylphorbol 13-acetate (TPA) induced tumor promotion in NIH3T3 cells. On MTT and [3H] thymidine incorporation assays, TPA (100 ng/ml) treatment induced cellular proliferation about 3-fold, compared with control group, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the most significant dose dependent inhibition on TPA induced proliferation among these compounds. Translocation of PKCa from cytosol to membrane was appeared in TPA treated NIH3T3 cells, and flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone were able to block its translocation. Previous study indicated that induction of phosphorylated MAPK, ornithine decarboxylase (ODC), c-Jun and COX-2 proteins was involved in TPA induced promotion cascade and located in the downstream of PKC activation. In this study, TPA treatment was able to induce phosphorylated MAPK, ODC, c-Jun and COX-2 proteins in a time dependent manner and the maximum inductive time period is at 1 hr for MAPK and 6 hr for others. Flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone showed the dose dependent inhibition on TPA stimulated phosphorylation and indicated gene expressions. The inhibitory order is 2'-OH flavanone> 6-OH flavanone> 4'-OH flavanone> Flavanone. In order to provide more evidences to make clear the inhibitory activities of these four flavanones on TPA induced tumor promotion, colony formation was performed in this study. Continuous TPA treatment for 3 months apparently increased the numbers of colonies in NIH3T3 cells compared with control group under a microscope observation, and cotreatment of cells with TPA and indicated flavanones under the same culture condition as that in TPA treated cells showed significant suppression on TPA induced colonies. Increase of phosphorylated MAPK, ODC, c-Jun and COX-2 was detected in long-term TPA-treated cells, and these compounds blocked their activation. In addition to firstly demonstrate the inhibitory mechanism of structure related flavanones on TPA induced tumor promotion, the results of this study also indicated that number of OH group in flavanones might not be a critical determinant in the inhibition of TPA induced tumor promotive effects.

並列關鍵字

TPA ； Tumor promotion ； Flavonoid ； Jun ； ODC ； COX ； PKC ； PGE

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