br stored at C Dimethylthiazol
stored at −20 °C. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenylte-trazo-lium bromide (MTT), propidium iodide (PI), pifithrin-α (PFT-α) were obtained from Sigma-Aldrich (Poole, UK). DMEM medium, RPMI 1640 medium, and fetal bovine serum were obtained from Invitrogen (Carlsbad, CA, USA). Monoclonal Oxaliplatin against GAPDH, PARP, p21, cleaved PARP, caspase-3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cyclin B, cyclin A, p53 were from Bioworld (Minneapolis, MN, USA).
2.2. Cell lines and culture conditions
Human colorectal cell lines HCT116, HCT15, LOVO and SW480 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM or RPMI 1640 with 10% fetal bovine serum, 100 U/ml peni-cillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 atmosphere.
2.3. Cell viability assay
The cell viability was measured by the MTT assay and expressed as the concentration inhibiting 50% of cells growth (IC50). Firstly, cells (1 × 104) were plated into each well of a 96-well plate and treated with 8-AHN at concentrations from 0 to 80 μM at a ratio of 1:2 for 48 h. The 0 μM 8-AHN group added DMSO and was used as negative control group. After treatment, the cells were washed twice with phosphate-
Fig. 1. 8-AHN inhibited cell proliferation.
(A) Chemical structure and abbreviation of 8 compounds isolated from the root of Toddalia asiatica.
(B) Eﬀects of 8 compounds on HCT15 cells were shown as the IC50 values.
(C) Cells were treated with various concentrations of 8-AHN for 48 h, and cell viability was assessed by MTT assay. Data were presented as means ± S.D. from three independent experiments.
Fig. 2. 8-AHN induced cell cycle arrest.
(A) After release from a double thymidine block, HCT116 cells were treated with 5 μM 8-AHN at various times (0 h, 24 h, and 48 h). PI prior to FACS analysis for DNA content was analyzed by flow cytometry (left) and quantitatively analyzed (right).
(B) HCT116 or LOVO cells were treated with 8-AHN for 24 h. Cyclin A, cyclin B and cyclin D protein expression levels were checked by Western blot analysis (left) and quantitatively analyzed by Image J (right). (C) Cells were treated with 8-AHN (0, 4, 8 μM) for 24 h and the RNA expression of cyclin B and cyclin A was determined by RT-PCR. Data were presented as means ± S.D. from three independent experiments.
buﬀered saline (PBS), and 100 μl of 0.25 mg/ml MTT in culture medium was added to each well. The plate was incubated at 37 °C for 4 h. Then, the culture medium was removed, and DMSO (150 μl) was added to each well to dissolve the dark blue crystal. The absorbance was mea-sured at 490 nm using a microplate reader (Spectra MAX 340; Molecular Devices, Sunnyvale, CA, USA).
2.4. Cell cycle analysis
Control and treated cells were collected into flow cytometry tubes and centrifuged at 300×g for 5 min to obtain cell pellets. The cells were washed with PBS and fixed with 70% ethanol (−20 °C ice-cold) for night at 4 °C. Fixed cells were washed with PBS and incubated with
RNase A (0.1 mg/ml) for 30 min followed by incubation with propi-dium iodide (50 μg/ml) for 15 min at room temperature. Cell cycle analysis was performed with a Coulter Epics XL flow cytometry system (Beckman–Coulter, Miami, FL, USA). In each analysis, 10,000 events were recorded. The percentages of cells at G0/G1, S, and G2/M were calculated using EXPO32 ADC analysis software (Beckman–Coulter).
2.5. Cell synchronization analysis
HCT116 cells were synchronized by double-thymidine block. Exponentially growing cells were incubated for 12 h at 37 °C in medium containing 2.5 mM thymidine, followed by incubation in thymidine-free medium for 12 h. Synchronization of cells at the G1/S boundary