Cell culture and treatment
Human ovarian cancer cell lines ES-2 and SKOV3 obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% FBS. Cells were treated with dezocine, and DMSO was used as negative control (NC). The cDNA sequence od CRABP2 was cloned into pcDNA3.1 vector, and the blank pcDNA3.1 was used as control. The plasmids were transfected into ovarian cancer cells by Lipofectamine 2000 (Invitrogen, USA).
Cells were treated with different concentrations of dezocine (0, 2.5, 5, 10, 20, 30, 40, 50, 60, 80, 120, and 200 µg/ml) in a 96-well plate at 37 °C for 24 h, respectively. Then, Cell Counting Kit-8 (CCK-8) reagent (Beijing Solarbio Science & Technology, Beijing, China) was added in each well and incubated for 90 min. The absorbance at 450 nm was recorded with a Bio-Rad microplate reader (Bio-Rad, USA).
Cell proliferation assay
Cell proliferation was measured using CCK8 assay. Ovarian cancer cells were exposed to dezocine n a 96-well plate at 37 °C for 0, 24, 48, and 72 h, respectively. The absorbance at 450 nm was recorded according to the above steps.
Colony formation assay
Following treatment with dezocine for 24 h, cells were seeded into 35 mm-plates with 5 × 102 cells/well and cultured with DMEM at 37 °C for 1–2 week. Colonies were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. The number of colonies was counted under an optical microscope.
When the cells (5 × 105) reached a confluency of 90% in 6-well plates, they were scraped by a sterile pipette tip, and were treated with dezocine for 0, 24 h. Photographs were taken at the same wound location using an optical microscope (OLYMPUS, Japan), and the width of the wound was measured using ImageJ software (NIH, Bethesda, USA).
Transwell migration and invasion assay
Cell invasion was analyzed by Transwell chambers (8.0 µm; Millipore, MA, USA). After 24 h of treatment with dezocine or DMSO, cells (1 × 105) were inoculated into the upper chamber precoated with Matrigel (BD Bioscience, CA, USA), and the lower chamber was filled with 700 µl of medium containing 20% FBS. After incubation for 24 h, cells in the upper chamber were removed, and then the invaded cells were fixed with 4% paraformaldehyde and stained with 5% crystal violet solution for 20 min. For cell migration assays, the Matrigel precoating was not performed. The migrated and invaded cells were photographed under a light microscope (100 × magnification; Nikon, Japan).
Apoptosis was detected using the Annexin V-FITC kit (BioVision, USA) according to the manufacturer's instructions. The cells treated with dezocine or DMSO for 24 h were collected, and incubated in serum-free medium for another 24 h. Thereafter, cells were incubated with Annexin V-FITC and PI for 30 min at room temperature in the dark. Finally, the percentage of apoptotic cells was measured using a BD FACSCalibur (Beckman Coulter, CA, USA).
Western blotting analysis
RIPA Lysis Buffer (CWBIO, Beijing, China) was used to extract proteins in cells treated with dezocine for 48 h. Following the concentration was determined by BCA kit (CWBIO), the protein samples (20 µg) were separated by 10% SDS-PAGE and transferred onto PVDF members (Millipore, Billerica, MA, USA). After blocked with 5% dried skimmed milk for 1 h, the members were incubated with primary antibodies at 4 °C overnight, and then incubated with HRP-conjugated secondary antibodies at room temperature. An enhanced chemiluminescence reagents (CWBIO) was performed to visualize the blot bands. The antibodies, including anti-Bcl-2 (Cat no. 12789-1-AP), anti-Bax (Cat no. 50599-2-Ig), anti-p-Akt (Cat no. 66444-1- Ig), anti-mTOR (Cat no. 20657-1-AP), anti-GAPDH (Cat no. 10494-1-AP), and anti-CRABP2 (Cat no. 10225-1-AP) were obtained from Proteintech Group (IL, USA); anti-cleaved Caspase 3 (Cat no. 9661), anti-Akt (Cat no. 9272), anti-p-mTOR (Cat no. 5536), anti-p70s6k (Cat no. 9204), and secondary antibodies were obtained from Cell Signaling Technology (Danvers, USA).
The values were presented as Mean ± SD and statistical analyzed using GraphPad Prism software. Differences between groups was assessed using Student’s t-test or one-way ANOVA followed by Dunnett’s test. P-values less than 0.05 indicate a significant difference.