Chen X, Hasuma T, Yano Y, Yoshimata T, Morishima Y, Wang Y, Otani S
Department of Biochemistry, Osaka City University Medical School, Japan.
Anticancer Res. 1997 Jul-Aug;17(4A):2555-64.
Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) in order to extend their biological activity. Previous studies have indicate an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group, and that using inhibitors of the mevalonate pathway could block the transforming properties of ras oncogene. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, we partially purified farnesyl protein transferase (FPTase) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro from porcine kidney epithelial-like LLC-PK 1 cells, human lung adenocarcinoma A549 cells and human pancreatic cancer MIA PaCa-2 cells. This FPTase activity requires a divalent cation, such as Mg2+ or Zn2+ ions, sulfhydryl protecting agent, DTT, as well as certain pH which is linear with time and with enzyme concentration, and is present in many mammalian normal cell and tumor cell lines. Sequentially, we observed the effects of the monoterpene compound, d-limonene; curcumin derivatives, CD-I and CD-II; polyphenol compound, gallotannin; Salvia miltiorrhiza derivative, SMD; and retinoid acid derivative, RAD on FPTase activity. We found that curcumin derivatives and gallotannin had a strong inhibition on FPTase besides d-limonene, while gallotannin was the strongest among synthetic and natural compounds tested. Salivia miltiorrhiza and retinoid acid derivatives had no influence on FPTase activity. Our results suggest that compounds containing polyphenol hydroxyl may be a new source of FPTase inhibitors. The experiment also showed that availability of an in vitro farnesyl protein transferase assay could be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.
Ras蛋白必须在靠近羧基末端的保守半胱氨酸残基（哺乳动物Ras p21蛋白中的Cys-186）处进行异戊二烯化修饰，以延长其生物学活性。先前的研究表明，甲羟戊酸途径中的一种中间体，很可能是法尼基焦磷酸，是这种异戊二烯基团的供体，并且使用甲羟戊酸途径的抑制剂可以阻断ras癌基因的转化特性。不幸的是，甲羟戊酸是哺乳动物细胞必需的各种终产物的前体，如多萜醇、泛醌、血红素A和胆固醇。在本研究中，我们从猪肾上皮样LLC-PK 1细胞、人肺腺癌A549细胞和人胰腺癌MIA PaCa-2细胞中部分纯化了能够在体外催化未加工的Ras p21蛋白法尼基化的法尼基蛋白转移酶（FPTase）。这种FPTase活性需要二价阳离子，如Mg2+或Zn2+离子、巯基保护剂DTT以及特定的pH值，其与时间和酶浓度呈线性关系，并且存在于许多哺乳动物正常细胞和肿瘤细胞系中。随后，我们观察了单萜化合物d-柠檬烯、姜黄素衍生物CD-I和CD-II、多酚化合物没食子单宁、丹参衍生物SMD以及视黄酸衍生物RAD对FPTase活性的影响。我们发现，除了d-柠檬烯外，姜黄素衍生物和没食子单宁对FPTase有强烈的抑制作用，而在测试的合成和天然化合物中没食子单宁的抑制作用最强。丹参和视黄酸衍生物对FPTase活性没有影响。我们的结果表明，含有多酚羟基的化合物可能是FPTase抑制剂的新来源。该实验还表明，体外法尼基蛋白转移酶测定法可用于筛选不会干扰甲羟戊酸途径其他方面的ras癌基因功能的潜在抑制剂。
