Additional file 2: Table S1. of The common stress responsive transcription factor ATF3 binds genomic sites enriched with p300 and H3K27ac for transcriptional regulation

A list of unique genes containing overlapped ATF3 and p53 peaks where both ATF3 and p53 motifs are present. (XLSX 32 kb

LXY6006 is a novel HIF-1 inhibitor derived from manassantin A.

<p>(A) The scheme for LXY6006 synthesis. Reagents and conditions: (a) BnBr, K₂CO₃, I₂, NaOH; (b) Pd (OAc)2, n-Bu4NCl, KOAc, DMF, 80°C, 4 hr, 73.2%; (c) H2, Pd-C, 25°C, 2 hr, 87.5%; (d) PS-BEMP, CH3CN, 25°C, 10 hr, 97.5%; (e) PS-brohydride, CH3OH, 25°C, 28 hr, 99.5%. (B) The scheme for LXY6006 side chain synthesis. Reagents and conditions: (a) Isopropylmagnesium chloride, N,O-dimethyl Hydroxylamine hydrochloride, THF, −20°C; (b) n-BuLi, 3,4-dimethylbromobenzene,THF, −78°C; (c) Ts₂O, Pyridine, 0°C. (C) LXY6006 inhibited HIF-1 activity more potently than manassantin A. T47D cells transfected with the HIF-1 reporter were treated with various concentrations of manassantin A and LXY6007 for 16 hr in 1%O₂ and subjected to dual luciferase activity assays. Data are shown as means ±SD from one representative experiment performed in triplicate. (D) LXY6006 inhibits VEGF protein expression in T47D cells. T47D cells were treated with LXY6006 and manassantin A, and then incubated in a hypoxia chamber (1%O₂) for 16 hr. The VEGF amount in conditioned medium was determined by ELISA. Data are shown as means ±SD from one representative experiment performed in triplicate. **<i>p</i><0.01(Student t-test), compared to the DMSO (1%O₂) group.</p

LXY6006 inhibits the growth of Taxol-resistant breast tumors.

<p>Nude mice implanted with MX-1/Taxol xenografts were treated with 60 mg/kg LXY6006, 10 mg/kg Taxol, or a combination of LXY6006 and Taxol. Tumor sizes were measured and used to calculate relative tumor volume (A). Body weights were shown in (B). Tumor were also photographed (C) and weighed (D). *<i>p</i><0.05, **<i>p</i><0.01 vs control; #<i>p</i><0.05 vs LXY6006 or Taxol.</p

LXY6006 inhibits HIF-1α nuclear accumulation.

<p>(A) Nuclear extracts from T47D cells were subjected to Western blotting for HIF-1α and HIF-1β expression. HIF-1β was constitutively expressed and thus used as a loading control. (B) T47D cells pre-treated with or without 0.1 µM of LXY6006 were incubated in hypoxia conditions for different time, and then subjected to Western blotting. (C) T47D cells pretreated with or without 0.1 µM of manassantin A or LXY6006 were exposed to hypoxia (1%O₂, 8 hr) or 1,10-phenanthroline (phen) (10 µM, 8 hr), and then lysed for detection of nuclear HIF-1α expression as in (A). (D) MDA-MB-231, MX-1 and MX-1/Taxol cells were treated with or without 0.1 µM of manassantin A or LXY6006 and lysed for HIF-1α expression as above. (E) T47D cells treated with or without 0.1 µM of LXY6006 or manassantin A were exporesed to hypoxia for 8 hr, and then fixed for immunofluorescence straining for HIF-1α expression.</p

LXY6006 induces cell cycle arrest but not apoptosis.

<p>(A, B, C) T47D Cells were treated with various concentrations of LXY6006 or 1 µM of manassantin A for 4 days and 5 days, and subjected to flow cytometry for cell cycle analysis. The percentages of cells in each cell cycle phase (G1, S, and G2/M) were determined by flow cytometry. The percentage of apoptotic cells was measured by the accumulation of cells with a sub-G1 DNA content. The percentages of polyploid cells were showed in (C). *<i>p</i><0.05, **<i>p</i><0.01 vs. DMSO (n = 3). (D) T47D cells were treated with manssantin A or LXY6006 for 5 days for Western blotting to detect cdc25c, cyclin B1 and cdc2 expression.</p

Selective inhibition of growth of cultured cancer cells by LXY6006.

<p>Selective inhibition of growth of cultured cancer cells by LXY6006.</p

The HIF-1-inhibitory activity of manassantin A derivatives.

<p>(A) Structures of manassantin A and synthesized derivatives. (B) HIF-1 inhibitory activity of manassantin A derivatives measured by a HIF-1 reporter assays.</p

LXY6006 inhibits xenograft tumor growth.

<p>Nude mice implanted with MX-1, MIA Paca-2 or H460 xenografts, were administrated o.p. with LXY6006, or injected i.p. with Gemcitibine or Taxol as indicated. Tumor sizes were measured and used to calculate relative tumor volume (A, E, I). Body weights were monitored (B, F, J). At the end of experiments, tumors were also dissected out, and photographed (C, G, K) and weighed (D, H, L). *<i>p</i><0.05, **<i>p</i><0.01 vs control.</p