Xanthatin mediates G 2 /M cell cycle arrest, autophagy and apoptosis via ROS/XIAP signaling in human colon cancer cells

Xanthatin is a natural plant bicyclic sesquiterpene lactone extracted from Xanthium plants (Asteraceae). In the present study, we demonstrated for the first time that Xanthatin inhibited cell proliferation and mediated G 2 /M phase arrest in human colon cancer cells. Xanthatin also activated caspase and mediated apoptosis in these cells. Concomitantly, Xanthatin triggered cell autophagic response. We found down-regulation of X-linked inhibitor of apoptosis protein (XIAP) contribute to the induction of apoptosis and autophagy. Moreover, reactive oxygen species (ROS) production was triggered upon exposure to Xanthatin in colon cancer cells. ROS inhibitor N-acetylcysteine (NAC) significantly reversed Xanthatin-mediated XIAP down-regulation, G 2 /M phase arrest, apoptosis and autophagosome accumulation. In summary, our findings demonstrated that Xanthatin caused G 2 /M phase arrest and mediated apoptosis and autophagy through ROS/XIAP in human colon cancer cells. We provided molecular bases for developing Xanthatin as a promising antitumor candidate for colon cancer therapy.


Cell culture
HT-29 cells and DLD-1 cells were purchased from the Cell Bank of Shanghai Institute of Biochemistry and & Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were grown in DMEM or DME/F12 medium supplemented with 10 % fetal bovine serum, exponentially growing cultures were maintained in a humidified atmosphere of 37 °C and 5 % CO 2 .

Cell viability assay
Cells were seeded in 96-well plates at a density of 5000 cells in 200 μl medium per well and incubated overnight. The cells were treated with Xanthatin or 5-FU at different concentrations, or subjecting to Xanthatin co-treatment with 3-MA. MTS (5 mg/ml) was dissolved with PBS and filter sterilized, then 20 μl of the prepared solution was added to each well and cells were incubated until a purple precipitate was visible. The absorbance was measured on an ELISA reader (SpectraMax®I3, USA) at a test wavelength of 570 nm and a reference wavelength of 650 nm.

Colony formation assay
Colon cancer cells were seeded on 6-well plate at a density of 1000 cells per well and incubated overnight. Then cells were treated with Xanthatin and/or NAC at indicated concentrations. Medium was replaced after 24 h incubation. Colonies were further observed 10 days later. After being fixed with 4% paraformaldehyde for 20 min, and washed with PBS, the plates were then stained with crystal violet solution (Beyotime, Shanghai, China) and colonies were counted under microscope.

Cell cycle analysis
In brief, after treatment with Xanthatin and/or NAC at indicated concentrations, cells were harvested and fixed with 70 % ethanol at -20°C overnight. Then the cells were stained with PI/RNase staining buffer (BestBio, Shanghai, China) for 15 min and analyzed by flow cytometer (Beckman Coulter, A00-1-1102).

Hoechst 33258 staining
HT-29 cells and DLD-1 cells were treated with Xanthatin and/or NAC at indicated concentrations. Then cells were stained with Hoechst 33258 reagent (Beyotime, Shanghai, China) for 5 min. The cells were plated on the glass slide and covered with cover slip, then observed under fluorescent microscope.

Hydrogen peroxide measurement
The fluorescent dye 2'7'-dichlorfluorescein-diacetate (DCFH-DA, Beyotime) was used to measure hydrogen peroxide formation. Cells were seeded in a 6-well plate and treated with Xanthatin at different concentrations (0-40 μM). After 24 h, cells were collected and exposed to serum-free medium containing 10 mM DCFH-DA for 30 min, and then cells were washed with DMEM for three times. Fluorescent intensity was measured by the fluorescence microscopy.

Western blot analysis
After treated with Xanthatin and/or NAC at indicated concentrations, cells were lysed in Western blotting lysis buffer and 12000 g centrifugation for 10 min. The protein content of supernatant was quantified using a BCA protein assay kit (Beyotime). Equal amounts of protein were separated on SDS-polyacrylamide gels, and electroblotted onto PVDF membranes. Then the membranes were blocked in blocking buffer (TBST plus 5% skimmed milk), and incubated with primary antibodies overnight at 4°C. The membranes were washed with TBST and probed with secondary isotype specific antibodies tagged with horseradish peroxidase (Cell Signaling Technology). Bound immuno-complexes were detected using ChemiScope 3300 Mini (Shanghai, China).

Statistical analysis
The results were analyzed using one-way ANOVA followed by Tukey's range test. The data are given as the means ± SD. Significance was set at p < 0.05.