title: Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter.
abstract: Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3'-oxime and 7-methoxyindirubin 3'-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3'-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3'-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.
authors: Departments of Biochemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Toru
authors: T
authors: Tanaka
authors: Departments of Biochemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Sachiyo
authors: S
authors: Ohashi
authors: Departments of Bio-organic Chemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Hiroaki
authors: H
authors: Saito
authors: Departments of Biochemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Takashi
authors: T
authors: Higuchi
authors: Departments of Clinical Medicine, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Keiichi
authors: K
authors: Tabata
authors: Departments of Pharmacology, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Yasuhiro
authors: Y
authors: Kosuge
authors: Departments of Clinical Medicine, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Takashi
authors: T
authors: Suzuki
authors: Departments of Bio-organic Chemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan.
authors: Shinichi
authors: S
authors: Miyairi
authors: Departments of Biochemistry, School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, Chiba 274-8555, Japan. Electronic address: kobayashi.shunsuke@nihon-u.ac.jp.
authors: Shunsuke
authors: S
authors: Kobayashi
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D061986:MCF-7 Cells; D051379:Mice; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D007211:Indoles; C027185:indirubin
keywords: Indirubin derivatives; MDR1 gene promoter; NF-Y; Tumor cell-type specificity