Fig 1.
Structures of andrographolide (ANDRO, 1) and an NBD-conjugated andrographolide derivative 2 (ANDRO-NBD).
Fig 2.
Reagents and conditions: (a) 2,2-dimethoxypropane, PPTS, acetone, 30 min, 97%; (b) (i) Ac2O, reflux for 1.5 h, (ii) AcOH/H2O (7:3), 10 min, 83% over 2 steps; (c) 5, 2,4,6-trichlorobenzoyl chloride, TEA, DMAP, THF, 30 min, -20°C, 54%.
Table 1.
Cytotoxicity of ANDRO-NBD on various human cell lines.
Fig 3.
ANDRO-NBD suppressed migration activity of melanoma cells.
B16F10 cells (3x104) were treated with vehicle (0.1% DMSO), andrographolide (ANDRO), or indicated concentrations of ANDRO-NBD and analyzed using migration assays. (A) The migrated cells were stained with 2% crystal violet and photographed under a phase-contrast microscope (100x). (B) Quantification of the migrated cells. The stained cells from five independent fields (100x) were photographed and counted. Data are presented as mean ± SD, and ** indicates significant difference versus control (p<0.01).
Fig 4.
ANDRO-NBD suppressed v-Src-mediated cellular transformation and reduced the expression of oncoproteins, v-Src and Bcr-abl.
(A) The ts-v-Src cells (105) were treated with vehicle or ANDRO-NBD for 24 h at 35°C and cell morphology was photographed under a phase-contrast microscope. (B) Vehicle- and ANDRO-NBD treated ts-v-Src cells grown at 35°C were lysed in modified RIPA buffer and analyzed with Western blot. (C) Vehicle- and ANDRO-NBD-treated K562 cells were lysed in modified RIPA buffer and analyzed with Western blot. Data are presented as mean ± SD, and * indicates significant difference versus vehicle (*p<0.05).
Fig 5.
The kinetics of ANDRO-NBD uptake by v-Src-transformed cells.
The ts-v-Src cells (2x105) grown at 35°C were treated with ANDRO-NBD (2 μM) for the indicated time periods. The images were photographed using a phase-contrast microscope with or without fluorescence illuminator.
Fig 6.
Cellular distribution of ANDRO-NBD in v-Src-transformed cells.
The ts-v-Src cells (105) grown at 35°C were co-treated with ANDRO-NBD (2 μM) and the indicated organelle tracker for 2 hours. DAPI, MitoTracker, LysoTracker and ERTracker are for staining nucleus, mitochondria, lysosome and endoplasmic reticulum (ER), respectively. The images were analyzed using a Laser confocal microscope (ZEISS LSM 700).
Fig 7.
ANDRO-NBD suppresses transcriptional activation of NF-κB and could covalently bind to known target proteins of andrographolide.
(A) The RAW 264.7/Luc-P1 cells (4x105) were treated with vehicle (0.1% DMSO) or indicated compounds, and analyzed using reporter assays. In both (B) and (C), one microgram of recombinant protein was incubated with ANDRO-NBD in PBS buffer for 1 h at 25°C, and then analyzed with 10% SDS-polyacrylamide gels. The fluorescent signals were detected using Typhoon TRIO scanner. Quantitation data from three independent experiments are presented as mean ± SD, and * indicates significant difference versus control (p<0.05).