WT1 is known to drive its own transcription using an auto-regulatory mechanism

WT1 is known to drive its own transcription using an auto-regulatory mechanism. contains supplementary material, which is available to authorized users. (Miq.) T. Anders is a Thai medicinal herb belonging to the family of Guttiferae, and is known in Thai as Saraphi [1]. Previous phytochemical studies of have led to the isolation and structural determination of coumarins (mammea, BMS-740808 surangin, therapin, calanone, mammeanoyl, etc.) found in the root, leaf, twig, stem, bark, blossom, and seed of and [2C10]. Coumarins are well-known natural products that have been shown to have various biological activities, such as insecticidal [11], antioxidant [5, 12, 13], antibacterial [5], antifungal [14], CDKN1B anti-malarial [15], anti-HIV [16], and anticancer activities [4, 7, 10, 12, 13]. A previous study reported the isolation and structural determination of phenolic compounds from seeds, including siamensone A, surangin B, mammea E/BB (Fig.?1), and -tocotrienol [6]. Recently, compounds from your flowers of were found to exert antiproliferative actions through apoptotic cell death in leukemia cells [10]. Open in a separate windows Fig. 1 Chemical structure of Mammea E/BB The (gene expression in both main and leukemic cells [19]. In addition, Semsri et al. reported that real turmeric curcumin affected WT1 protein-promoter binding and decreased WT1 mRNA and protein levels through inhibition of the PI3K/PKC/JNK pathway in K562 cells [20]. Moreover, expression of the gene and its product has been used as biological markers for diagnosis and evaluation of the prognosis in leukemia and minimal residual disease (MRD) [18, 21]. A previous study revealed that mammea E/BB also suppressed WT1 protein expression when compared to surangin A and surangin C [22]. However, the down-regulatory mechanism was unknown. The current study therefore aimed to examine the inhibitory mechanism of mammea E/BB on gene expression, WT1 protein and mRNA stability, and cell proliferation in K562 cell collection. Methods Materials seeds were collected from Chiang Mai University or college, Amphoe Muang, Chiang Mai province, Thailand in May 2010. The herb material used in this study was recognized by Mr. James Franklin Maxwell. A voucher specimen (J.F. Maxwell, No.92-70) is deposited in the CMU herbarium, Faculty of Science, Chiang Mai University or college, Chiang Mai, Thailand. RPMI-1640, fetal bovine serum, using column chromatography, extraction, and isolation as previously explained [22]. Mammea E/BB was obtained as a pale yellowish BMS-740808 gum with []D27 ?65.7 (c?=?0.40, MeOH). The UV spectra of mammea E/BB exhibited absorption maxima bands at 337 and 265?nm; these are characteristic for coumarin [23]. The complete stereochemistry at C-1 and C-2 was assigned to be from its unfavorable optical rotation value [12]. The mammea E/BB identity was confirmed by comparison of the 1H and 13C NMR spectra data (Additional file 1: Physique S1 and Additional file 2: Physique S2) with those reported in the literature [24, 25]. Cells and cell culture conditions The K562 cell collection, a BMS-740808 model of WT1-overexpressing leukemic cells, was cultured in RPMI-1640 medium supplemented with BMS-740808 10?% fetal bovine serum, 1?mM?gene using the ChIP assay. WT1 is known to drive its own transcription using an auto-regulatory mechanism. The WT1 promoter has been found to contain one AP-1 consensus sequence, TGAGTGA, at +144 to +150. Treatment of K562 cells with 3.5?M mammea E/BB for 72?h could significantly inhibit WT1 binding to its own promoter, by up to 75?% (Fig.?7a and ?andb).b). Mammea E/BB also disrupted c-Fos/AP-1 binding to the WT1 promoter by 50?% as compared to the vehicle control by standard PCR. Open in a separate window Fig. 7 Mammea E/BB BMS-740808 treatment attenuated WT1 – DNA binding to the proximal WT1 promoter and WT1 promoter activity. a K562 cells were treated with 3.5?M mammea E/BB for 72?h and ChIPs were performed. Chromatin lysates were immunoprecipitated with antibodies to WT1, c-Fos/AP-1, Pol II (gene expression is the WT1 proximal promoter (-301?bp) [20]. The WT1 (-50 to -39) consensus binding site is included in this proximal promoter element (Fig.?7c). Transfection of this 301?bp construct, contained within the pGL3 reporter vector into K562 cells, demonstrated high luciferase activity in vehicle control treated cells and a.