5-氟尿嘧啶(5-FU)是一种干扰DNA合成的抗代谢物,已被广泛用作各种类型癌症的化疗药物。然而,耐药性的发展极大地限制了其应用。在许多类型的癌症中,ATP结合盒(ABC)转运蛋白的过表达是导致多种抗癌药物的细胞摄取减少的原因,癌症化疗的主要障碍。最近,我们获得了一种新的合成5-FU类似物,U-332[(R)-3-(4-溴苯基)-1-乙基-5-亚甲基-6-苯基二氢尿嘧啶],结合尿嘧啶骨架与外环亚甲基。U-332对HL-60细胞具有高度细胞毒性,并且在5-FU抗性亚克隆(HL-60/5FU)中显示出相似的细胞毒性,其中该类似物几乎完全消除了ATP结合盒(ABC)转运蛋白的表达,多药耐药相关蛋白1(ABCC1)。ABC转运体的表达通常与NF-κB的激活相关。这项研究的目的是确定抗性HL-60/5-FU细胞中NF-κB亚基的水平,并评估U-332抑制该细胞系中NF-κB家族成员活化的潜力。
通过MTT测定评估化合物U-332的抗增殖活性。为了揭示U-332细胞毒性的机制,NF-κB家族基因的实时定量PCR分析,c-Rel,Rela,RelB,研究了NF-κB1和NF-κB2。通过ELISA试验研究了U-332降低NF-κB成员活性的能力。
在这份报告中,使用RT-PCR和ELISA测定,NF-κB家族c-Rel的成员,Rela,RelB,NF-κB1和NF-κB2在5-FU抗性HL-60/5FU细胞中均过表达,U-332可有效降低c-Rel的活性,此细胞系中的RelA和NF-κB1亚基。
这一发现表明,c-Rel,RelA和NF-κB1亚基负责HL-60/5FU细胞对5-FU的抗性,而U-332能够逆转这种抗性。U-332可以被视为寻找不会在癌细胞中引起多药耐药性的新型候选药物的重要先导化合物。
5-Fluorouracil (5-FU) is an antimetabolite that interferes with DNA synthesis and has been widely used as a chemotherapeutic drug in various types of cancers. However, the development of drug resistance greatly limits its application. Overexpression of ATP-binding cassette (ABC) transporters in many types of cancer is responsible for the reduction of the cellular uptake of various anticancer drugs causing multidrug resistance (MDR), the major obstacle in cancer chemotherapy. Recently, we have obtained a novel synthetic 5-FU analog, U-332 [(R)-3-(4-bromophenyl)-1-ethyl-5-methylidene-6-phenyldihydrouracil], combining a uracil skeleton with an exo-cyclic methylidene group. U-332 was highly cytotoxic for HL-60 cells and showed similar cytotoxicity in the 5-FU resistant subclone (HL-60/5FU), in which this analog almost completely abolished expression of the ATP-binding cassette (ABC) transporter, multidrug resistance associate protein 1 (ABCC1). The expression of ABC transporters is usually correlated with NF-κB activation. The aim of this study was to determine the level of NF-κB subunits in the resistant HL-60/5-FU cells and to evaluate the potential of U-332 to inhibit activation of NF-κB family members in this cell line.
Anti-proliferative activity of compound U-332 was assessed by the MTT assay. In order to disclose the mechanism of U-332 cytotoxicity, quantitative real-time PCR analysis of the NF-κB family genes, c-Rel, RelA, RelB, NF-κB1, and NF-κB2, was investigated. The ability of U-332 to reduce the activity of NF-κB members was studied by ELISA test.
In this report it was demonstrated, using RT-PCR and ELISA assay, that members of the NF-κB family c-Rel, RelA, RelB, NF-κB1, and NF-κB2 were all overexpressed in the 5-FU-resistant HL-60/5FU cells and that U-332 potently reduced the activity of c-Rel, RelA and NF-κB1 subunits in this cell line.
This finding indicates that c-Rel, RelA and NF-κB1 subunits are responsible for the resistance of HL-60/5FU cells to 5-FU and that U-332 is able to reverse this resistance. U-332 can be viewed as an important lead compound in the search for novel drug candidates that would not cause multidrug resistance in cancer cells.