2.1. Reagents and antibodies
DEX-resistant human multiple myeloma cells (MM.1R) were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were preserved in our laboratory.Then they were taken out from the liquid nitrogen and characterized. Conventional resuscitation and subculture of MM.1R cells were performed. RPMI 1640 medium, as well as fetal bovine serum (FBS) were bought from Gibco (NY, USA). EPA and DEX were purchased from Sigma; cell cycle detection kits and flow cytometry apoptosis detection kits were purchased from Nanjing KGI; anti-GRα antibodies (1: 1000), anti-Hsp90 antibody and GAPDH antibody were purchased from Abcam; protein lysate, BCA protein concentration determination kit, fluorescent secondary antibody, ECL chemiluminescence kit, CCK-8 kit were purchased from Shanghai Biyuntian Company.
2.2. Cell culture experiments
MM.1R cells were inoculated in DMEM enriched with fetal bovine serum (10%), streptomycin (100U/ml), as well as penicillin (100U/ml), and then incubated in an incubator programmed at 37°C and 5% CO2. Growth media was refreshed every 2–3 days and passaging of the cells was performed accordingly, with the logarithmic phase cells selected for downstream experiments.
2.3. Group allocation
DEX was suspended in 0.05% DMSO, and dissolved to form a stock solution at a concentration of 10mmol/l, and then diluted at 1000 times with RPMI1640 medium. The final DEX concentration was adjusted to 10µmol /L. EPA reserve solution (100mmol/L) was prepared with anhydrous ethanol. The working solution(10, 20, 50, 100µmol/L) was then prepared by diluting the stock solution with RPMl1640. In the combined dosing group, different concentrations of EPA were incubated for 12h, An additional 10µmol/L DEX was introduced and incubation was continued for 24h, and then further experiments were performed. Equal volume medium was introduced to the control group. Growth of the control cells was conducted in medium with the similar concentration of DMSO or anhydrous ethanol as the DHA-possessing medium. EPA treatments at concentrations of 10–50µM suppressed cell proliferation remarkably.
2.4. Cell proliferation evaluation by the CCK-8 assay
Cells of logarithmic growth phase were collected. Digestion of the cells was performed using 0.25% trypsin, followed by cell suspension preparation. We adjusted the cell density to 1 × 105 cells/ml. Thereafter, the cells were planted in 96-well plate (200µl/well). The experiments were performed in 5 replicate holes. The cells were grown for 24h in an incubator programmed at 37°C, 5% CO2, as well as saturated humidity parameters. After adherent growth of the cells for 24h, the drug solution was added at various concentrations to the cells. CCK-8 reagent (10 µl) was introduced to each well, thorough mixing performed, then placed in an incubator for 2h. Absorbance values (OD value) were determined at 450nm using an enzyme standard instrument. The inhibition rate of cell growth= (test OD value − blank OD value)/ (control OD value − blank OD value) × 100%. The column charts of the final value were drawn and the result was the result analyzed afterwards. The experiment was replicated thrice and results averaged.
2.5. Flow cytometry analysis of the mitochondrial membrane potential
The mitochondrial membrane potential assay kit with JC-1 was employed to evaluate the mitochondrial membrane capacity. MM.1R cells in the log growth stage were planted (1×105 cells/well) in 6-well plates (2ml per well) and then different concentrations of drugs were added. Cells were collected through centrifugation 48 hours after the treatment. Samples were then rinsed with ice-cold PBS. Staining of the cells with 500µl JC-1 working solution was carried out at 37°C for 15min and the cells collected after sedimentation. After that, 500µl preheated JC-1 assay buffer was used to re-suspend the cells, and analyzed on a flow cytometer. The assay was replicated thrice and the average result used.
2.6. Western blot analysis of GRα and Hsp90 protein expression
The cells were processed as described above. MM.1R cells treatment methods ibid. After treating the cells for 24h, cells were collected, and the protein purification conducted using the RIPA lysate. Collection of the culture supernatantwas carried out, followed by centrifugation to remove any cell debris performed.Quantitation of the total protein was conducted using the BCA™ protein assay (Pierce). Fractionation of the total proteins was done using SDS-PAGE. After that, the proteins were transfer-embedded onto PVDF membranes. The blocking of the non-specific sites was performed via 1h incubation of the protein-embedded membranes with Tris-buffered saline-Tween20 (TBST) enriched with 3% bovine serum albumin at room temperature. Thereafter, incubation with primary antibody was conducted overnight in an incubator set at 4°C. Subsequently, the samples were rinsed thrice with TBST, and then conjugation with the secondary antibodies at a 1:5000 dilution for 2h was done via incubation at room temperature. After washing, color development of the protein bands was carried out using ECL for 3-5min. The bandintensity for each protein was scanned on a scanner and analyzed using the Imagaquent 5.1 image processing software. GAPDH was employed as a reference gene. The relative quantitative analysis was performed using the Imagaquent 5.1 software. The optical density of the target protein to GAPDH ratio was computed as the relative expression levelof the target protein in the samples. This experiment was performed thrice and results are presented as averaged values.
2.7. Statistical analyses
SPSS 16.0 software was applied in statistical analyses. All data were indicated as mean ± standard deviation (SD), the two-sample independent t test was used. P < 0.05 signified statistical significance.