Fig 1.
The structure pf austrobailignan-1 from the leaves of K. henryi Dummer.
(A) The image of K. henryi Dummer (adopted from Herbanum, Academic Sinica, http://digiarch.sinica.edu.tw/content/repository/resource_content.jsp?oid=3819630). (B) The structure of austrobailignan-1 was established via 1H- and 13C-NMR. 13C-NMR assignments were based on HETCOR and long-range HECTOR spectral results.
Fig 2.
Austrobailignan-1 induced G2/M arrest and apoptosis.
(A) A549 and H1299 cells were treated with various doses (0, 1, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h. Cell number was measured by a Trypan-blue dye exclusion method. Data are expressed as mean ± S.D. from 3 independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. control). (B) Cells were treated with varied doses (0, 3, 10, 30 and 100 nM) of austrobailignan-1 for 24 and 48 h, and then stained with propidium iodide, and flow cytometry was performed to examine the cell cycle distribution. (C) Cells were treated without or with 100 nM austrobailignan-1 for 48 h,a TUNEL assay was then performed to detect apoptotic cells (green) and the nuclear DNA was stained with DAPI (blue). The stained cells were investigated by fluorescence microscopy. Magnification x 400; scale bar, 50 μm.
Fig 3.
Austrobailignan-1 inhibited topoisomerase 1 activity and induced a DNA damage signaling pathway.
(A) Inhibition of DNA topoisomerase 1 activity. pHOT-1 DNA plasmid was incubated with various concentrations of austrobailignan-1 (0, 10, 30, and 100 nM) and topoisomerase 1 at 37°C for 30 min. The reaction products were separated by 1% agarose gel and stained by ethidium bromide. The fluorescence image was recorded by microphotography. Camptothecin (CPT) was used as a positive control. S. C. DNA: super coiled DNA, Relax DNA: unwind closed circular DNA. (B) DNA damage response. A549 and H1299 cells were treated without or with 30, 100 nM austrobailignan-1 for 24 h, and DNA damage on per cell basis was examined by a comet assay. Representative comet images from the cells exposed to austrobailignan-1 at various concentrations are shown (upper panel). The degree of DNA damage was scored by tail moment (% DNA in tail x tail length) from at least 100 cells in each treatment group (lower panel). Data are mean ± SD for three independent experiments. ** p < 0.01, *** p < 0.001. (C) Activation of ATM signaling pathway by austrobailignan-1. A549 and H1299 cells were treated with various concentrations of austrobailignan-1 for 24 h, the expressed levels of phosphorylated ATM, Chk1, Chk2, H2AX, and p53 proteins were investigated by Western blot analysis. β-actin was used as an internal loading control.
Fig 4.
Regulation of cell-cycle regulatory proteins by austrobailignan-1.
(A) A549 cells were treated with 0, 3, 10, 30 and 100 nM of austrobailignan-1 for 24. After treatment, cell extract was collected and analyzed by Western blot. (B) H1299 cells were treated with 0, 10, 30, 100 nM austrobailignan-1 for 24 h, the levels of p21Waf1/Cip1, p27Kip1, and Cdc25C were detected by Western blot. β-Actin was used as a loading control.
Fig 5.
Induction of mitochondrial apoptotic pathway by austrobailignan-1.
(A) A549 cells were treated with 100 nM of austrobailignan-1 for indicated time periods (0, 12, 24, and 48 h). (B) H1299 cells were exposed to austrobailignan-1 for 48 h. The cell lysates were harvested, and the caspase activities were determined using fluorogen substrates. Data are expressed as mean ± S.D. from three independent experiments. (*P <0.05,** P <0.01, *** P < 0.001 v.s. vehicle-treated control). (C) Caspase inhibitors block austrobailignan-1-induced cell death. A549 and H1299 cells were pretreated with 50 μM indicated caspase inhibitors for 1 h, and then treated with 100 nM austrobailignan-1 for another 48 h. The cell viability was measured by a Trypan blue dye exclusion method. (D) Regulation of Bcl-2 family proteins. A549 and H1299 cells were treated with 0, 30, 100 nM austrobailignan-1 for 48 h. The levels of indicated Bcl-2 family proteins were examined by Western blot. (E) Release of cytochrome c from mitochondria to cytosol. A549 and H1299 cells were treated without or with 100 nM austrobailignan-1 for 24 and 48 h. After treatment, particulate and cytosolic fractions were isolated, the level of cytochrome c protein was analyzed by Western blot. α-tubulin and cytochrome oxidase IV were used as loading control.
Fig 6.
p53 was not necessarily required for austrobailignan-1-induced cell cycle G2/M arrest and cell death.
(A) The A549-shRNA or A549-p53shRNA cells were treated with austrobailignan-1 (0, 10, 30, and 100 nM) for 48 h. The cell cycle distribution was determined by flow cyotmetry. (B) A549-shRNA and A549-p53shRNA cells were treated without or with 100 nM austrobailignan-1 for 48 h. The levels of p53 protein were detected by Western blot (upper panel). The cell viability was determined by a trypan blue exclusion method (lower panel).
Fig 7.
Schematic representation of the anti-cancer mechanisms of austrobailignan-1 in human non-small cell lung cancer A549 and H1299 cell lines.