Cell lines and reagents
Human glioblastoma U251 and human malignant glioblastoma U87 cell lines (Shanghai Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were used. Reagents included Dulbecco’s modified Eagle’s medium (DMEM; Gibco Inc.), fetal bovine serum (FBS; Gibco Inc.), trypsin, MTT powder and transwell chamber (Corning), N-cadherin, E-cadherin, and vimentin antibodies (Santa Cruz Biotechnology), matrix metalloproteinase (MMP)-2 and MMP-9 gelatinase kits (APPLYGEN, Beijing, China), and cytoskeletal staining kit (Millipore).
Preparation of COE solution
Terpenoids from Celastrus orbiculatus (Batch No: 070510) were purchased from Professor Wang Qiang’s research group at the Traditional Chinese Medicine Institute of China Pharmaceutical University (Guangzhou, China) [7]. To extract terpenoids from Celastrus orbiculatus, the stem of Celastrus orbiculatus was cut, crushed into powder, and dried. Reflux extraction was repeated three times using 95 % ethanol. A rotary evaporator was used to recover the solvent to obtain the extract. The extract was added to diatomite, dried in a vacuum at low temperature, heated and refluxed with ethyl acetate, and filtered to obtain ethyl acetate extract. Terpenoids made up 68.3 % of the extract, and the rate of obtaining extract was approximately 2 %. Concentrated COE solution was prepared by hydrotropy of the extract using dimethyl sulfoxide and culture in serum-free medium. COE solution was filtered and sterilized at atmospheric pressure for later use.
Instruments
Instruments included a 5 % CO2 incubator (Thermo Fisher Scientific, MA, USA), automatic enzyme-mark analyzer, protein electrophoresis chamber, power transfer device (Bio-Rad Company, CA, USA), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel imaging analyzer (Bio-Rad Company), inverted fluorescence microscope, and confocal laser scanning microscope (CLSM; Olympus, Japan).
Cell culture and passage
U251 and U87 cells were cultured with DMEM in a 5 % CO2 incubator at 37 °C. Cells were observed under an inverted fluorescence microscope. All cells used in this study were in the exponential phase.
MTT assay of cell viability
DMEM containing 10 % FBS was used to prepare a single-cell suspension with a concentration of 3 × 105 cells/mL. The suspension was placed in a 96-well plate with 100 μL/well. After attachment, cells were randomly divided into the control group and COE groups. Cells in COE groups were treated with different concentrations of COE ranging between 10 and 320 μg/mL, with five wells for each concentration. After cells were cultured in a 5 % CO2 incubator at 37 °C for 24, 48, or 72 h, 15 μL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was added to each well in the dark. After 4 h, 100 μL dimethyl sulfoxide was added after the supernatant was discarded. The absorbance (A) value was read at 490 nm using a microplate reader. The experiment was repeated three times. The COE inhibition rate (%) was calculated as [1 - (A of cells in the COE group/A of cells in the control group)] × 100 %. The 50 % inhibitory concentration (IC50) was also calculated.
Cell-matrix adhesion assay
Serum-free DMEM was used to dilute type I collagen stock solution to 10 μg/mL, which was placed in a 24-well plate overnight at 4 °C. Type I collagen was blocked for 1 h using 1 % bovine serum albumin and washed three times with phosphate-buffered saline (PBS). U87 and U251 cells in COE groups were treated with 20, 40, or 80 μg/mL COE for 24 h. Cells were starved overnight in serum-free DMEM, digested, centrifuged, and resuspended at a concentration of 3 × 105 cells/mL. Cells were then plated at a concentration of 3 × 104 cells/mL in the 24-well plate, with three wells for each concentration. Cells were cultured in a 5 % CO2 incubator at 37 °C for 1 h. The culture solution was then removed from the 24-well plate, and non-adherent cells were washed away three times with PBS. The remaining cells were fixed for 30 min with 2 % paraformaldehyde, stained with cresyl violet for 15 min, and observed under an inverted microscope. The experiment was repeated three times. Cell adhesion inhibition rate (%) was calculated as (1 – number of cells in the COE group/number of cells in the control group) × 100 %.
Cell migration and invasion assays
For the wound-healing assay, U87 and U251 cells were cultured in DMEM at a concentration of 5 × 105 cells/mL until cell confluence reached 90 %. Micropipette tips were used to make linear scratches, and the exfoliated cells were washed off three times with PBS. The remaining cells were starved overnight with serum-free medium to exclude the effect of proliferation on migration. Cells in COE groups were treated with 20, 40, or 80 μg/mL COE and cultured for another 24 h before images were taken. The experiment was repeated three times. The degree of wound healing (%), calculated as (scratch width of the control group - scratch width of the COE group)/scratch width of the control group × 100 %, was used to measure the migration capacity of cells.
In the transwell invasion assay, matrigel (1:8) was diluted with serum-free DMEM, and the basement membrane of the upper chamber of the transwell was coated. The solution was kept at 37 °C for 1 h to transform the matrigel aggregate into gel. Cells were prepared at a concentration of 5 × 105 cells/mL in serum-free DMEM. Two hundred μL was added to the upper chamber of the transwell, and 600 μL culture medium containing 20 % FBS was added to the lower chamber. Cells in the COE group were treated with 20, 40, or 80 μg/mL COE, with three wells for each concentration. Cells at each concentration were cultured in a 24-well plate in a 5 % CO2 incubator at 37 °C for 24 h. The culture medium in each well was then discarded, and the chamber was washed twice with PBS. Cells that did not migrate were physically cleared from the upper chamber with cotton swabs. The cells that migrated were fixed with methanol for 15 min, stained with 0.1 % cresyl violet, washed three times with PBS, and air-dried. The chamber was inverted on a microslide and observed under a microscope. Five fields (200× magnification) were randomly selected for counting the number of migrated cells, and images were taken. In the transwell migration assay, matrigel was not used, otherwise the procedure was the same as that used in the invasion assay.
E-cadherin, N-cadherin, and vimentin expression
U87 and U251 cells in COE groups were treated with 20, 40, or 80 μg/mL COE. After 24 h, total cellular protein was extracted. After bicinchoninic acid assay, SDS-PAGE was performed. Separated protein was transferred to a polyvinylidene fluoride membrane, kept at room temperature for 2 h with 5 % skim milk, and incubated at 4 °C overnight with the primary antibody (1:1000). The corresponding secondary antibody (1:1000) was added after membrane washing, and protein was incubated at room temperature for 2 h. ECL detection reagent was used to develop and detect specific protein bands.
Gelatin zymography for detecting MMP-2 and MMP-9 activity
Supernatant was collected and cultured. After mixing with buffer solution at 1:1, 8 % SDS-PAGE containing 1 % gelatin was added, and electrophoresis was performed for 2 h. The gel was washed with 2.5 % Triton X-100 twice at room temperature for 30 min and transferred to substrate buffer (10 mmol/L Tris Base, 40 mmol/L Tris-Cl, 0.2 mol/L NaCl, 5 mmol/L CaCl2, 0.02 % Brij 35, pH 7.6). After reaction overnight at 37 °C and fixation for 2 h, Coomassie blue staining was performed for 20 min. Eluent destaining was performed until clear digestion bands were observed. Image analysis was performed on the electrophoresis results.
Immunofluorescence and CLSM for detecting cytoskeletal organization
Cytoskeletal organization was visualized by using the actin cytoskeletal and focal adhesion staining kit (FAK100;Millipore,Billerica,MA). U87 and U251 cells were placed in a 6-well plate at a concentration of 5 × 105 cells/mL. When cell confluence reached 50 %, cells were treated with 80 μg/mL COE for 24 h and then washed twice with PBS. Cells were fixed immediately with 4 % paraformaldehyde for 15 min at room temperature, and then permeabilized in 0.1 M PBS containing 0.2 % Triton X-100 for 5 min. After being blocked with 5 % bovine serum albumin, the cells were immune-labeled with anti-vinculin (1:200) at 37 °C for 1 h. Then, cells were incubated with FITC-anti-mouse (1:200) and treated with 0.5 μmol/L phalloidin-tetramethylrhodamine isothiocyanate (TRITC), placed in the chamber’s cassette for 45 min, then washed twice with PBS, stained with 6-diamidino-2-phenylindole (DAPI) for 5 min, and mounted with anti-fade fluorescence mounting medium. The structure of actin microfilaments and vinculin were evaluated by using confocal microscope (Olympus, Japan). All images were obtained under the same conditions of excitation and registration.
Statistical analysis
Each experiment was repeated at least three times. Data were analyzed by one-way analysis of variance using SPSS 16.0 (SPSS Inc., Chicago, IL). Data are shown as mean ± standard deviation. P-values < 0.05 and P-values < 0.01 means difference were considered statistically significant.
