br Fig CBD reduces viability
Fig. 1. CBD reduces viability and induces apoptotic cell death of human CRC cells. (A) Normal primary cell lines (CCD-18Co and Beas2B) and various CRC HC-030031 were treated with 0, 2, 4, 6, and 8 μM of the CBD for 24 h. The cell viability was detected by WST-1 assay. (B) HCT116 (upper) and DLD-1 (lower) cells were treated with 0 or 6 μM of CBD. After 2 weeks, the cells were stained with crystal violet, and the colonies were photographed using a digital camera. (C–D) The cells were incubated with CBD at the doses (C) and defined time periods (D). The levels of Cleaved PARP, caspase-3, -8, and -9 were analyzed by Western blotting. (E) Cells stained with Annexin V and PI were studied using flow cytometry to detect the apoptosis induced by exposure to 6 μM CBD in CRC cell lines. Values are shown as mean ± standard error of mean (SEM) (n = 5). *, P < 0.05. (F) Cell apoptosis was detected with TUNEL assay, and DAPI was used as a co-stained to dye the nuclei of the HCT116 (left) and DLD-1 (right) cells. After treatment of CBD (6 μΜ), damaged DNA was visualized in bright green (TUNEL-positive cells), indicating apoptosis (Scale Bar, 10 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. CBD induces apoptosis by regulating Noxa. (A–B) The increase in Noxa protein level was confirmed at diﬀerent doses of CBD (A) and diﬀerent time periods (B). (C) Treatment with 6 μΜ CBD resulted in an increase in Noxa expression in diﬀerent time periods. *, P < 0.05. (D) Under identical conditions, Noxa expression (light green) after treatment with 6 μΜ CBD was evaluated by confocal microscopy (Scale Bar, 10 μm). (E–G) The cells were transfected with control siRNA or siNoxa and then treated with CBD (6 μΜ). Noxa protein and the apoptosis marker, Cleaved PARP, caspase-3, -8, and -9 were analyzed by western blotting (E), and the rate of apoptosis was observed by flow cytometry (F–G). *, P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. CBD induces ROS and ER stress. (A) The upregulation of ROS and Unfolded proteins were measured by microarray data. (B–C) HCT116 (upper) and DLD-1 (lower) cells were stained with (B) DCF-DA and (C) MitoSOX after with or without NAC (1 mM), ROS scavenger, and CBD (6 μΜ) to trace the progress of ROS production, by flow cytometry. (D–E) After treatment of 6 μΜ CBD, the anti-oxidant proteins (D) and ER stress-related proteins (E) were also confirmed with the western blotting. (F) One of the ER stress markers, p-IRE1α protein was increased in time periods when analyzed with western blotting.
Fig. 4. ROS and ER stress induce apoptosis by regulating the Noxa protein. The cells were treated with NAC (1 mM) or left untreated, and then treated with or without the CBD (6 μΜ). (A) Cleaved PARP, an apoptosis marker, and Noxa protein were both detected by western blotting. (B) Apoptosis was also confirmed with the flow cytometry in the same condition. The cells treated with both CBD and NAC were less sensitive to apoptosis, compared to those treated with CBD but not NAC. (C–D) HCT116 and DLD-1 cells were transfected with control siRNA or siCHOP, and then treated with CBD (6 μΜ). The cleaved PARP, CHOP, and Noxa protein were confirmed with western blotting (C) and the apoptosis was detected with flow cytometry (D). *, P < 0.05. (E) ER stress sensors, p-PERK, p-IRE1α, and CHOP were confirmed with western blotting after treaed with or without NAC (1 mM), and treated with or without CBD (6 μΜ). (F) Noxa expression after the transfection of control siRNA or siCHOP along with CBD treatment can be detected by confocal microscopy (Scale bar, 10 μm).
Fig. 5. CBD upregulates Noxa and CHOP expression, and it leads to in vivo apoptosis. (A) The cell viability was detected in two PDC cells by WST-1 assay. (B–C) The Caspase 3/7 dye was added to the media after the CBD treatment, and incubated in IncuCyte ZOOM for 3 days. The intensity of Caspase 3/7 fluorescence was then analyzed by IncuCyte Live-Cell Analysis system (B) and the graph was represented by Image J (C). (D–H) HCT116 Luc+ cells (1 × 107) were subcutaneously injected into BALB/c nude mice and were raised until the tumor size reached 150 mm3. Five mice were analyzed in each group. (n = 5) (D) The mice were captured by Image J. (E) The EtOH or CBD (10 mg/kg or 20 mg/kg) was intraperitoneally treated to the mice and the tumor size was measured once in three days and (F) the photographs of tumor were taken using a digital camera. (G) Apoptosis in tissue was detected by TUNEL assay (light green) and (H) Noxa expression (red) has increased after CBD treatment (20 mg/kg). The data were observed by confocal microscopy and nuclei were co-stained with DAPI solution (Scale Bar, 10 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)