DHA was purchased from Energy Chemical Reagent Company (Shanghai, China). Mito-DHA5 was synthesized in our laboratory. Compounds were dissolved in dimethyl sulfoxide (DMSO). During the experiment, the concentration of DMSO did not exceed 0.1%. Dulbecco’s modified Eagle’s medium (DMEM, U.S.), fetal bovine serum (Hyclone, Logan, UT, USA), penicillin-streptomycin solution, 0.25% trypsin and phosphate buffer (Hyclone, USA) were bought from Hyclone company. MMP Detection Kit was bought from Solarbio company (Beijing, China). AnnexinV- FITC/PI Apoptosis Detection Kit was purchased from Vazyme (Nanjing, China). ROS Detection Kit, Hochest33258 Kit, Bax (AF0057) and PARP protein antibodies were bought from Beyotime company (Shanghai, China). Bcl-2 (CAS7511) protein antibody was bought from Bioworld. Caspase-3 (#9662) and Cytochrome C (Cyt-C, D18C7) antibodies were purchased from Cell Signaling Technology company.
Cell culture
Human bladder cancer cell T24 was obtained from Dr. P Guo (Xi’an Jiaotong University). The cells were grown in DMEM contained with 10% of FBS and 1% of penicillin–streptomycin at 37 °C, in a constant temperature incubator containing 5% of CO2.
MTT assay
Briefly, T24 cells were planted in 96 well plates (8.0 × 103 per well). After 12 h, treated T24 cells with different concentrations (0, 3, 10, 30 and 100 µM) of Mito-DHA5 or DHA for 24, 48 or 72 h. The medium was removed after 4 h incubation with MTT (2 mg/ml, 50 µL) at 37℃ and 150 µL DMSO was added. Finally, OD values were measured at 490 nm by using a microplate reader (Biotek).
Clonogenic assay
In brief, T24 cells were planted in 24 well plates (3 × 103 cells per well). After 12 h, T24 cells were treated with different concentrations (0, 1, 2, 4 µM) of Mito-DHA5 or DHA for about 7 days. T24 cells were fixed with 10% formaldehyde for 1 h and then 0.1% crystal violet was added to stain cells for 12 h. Finally, the OD values were measured by using an area scanning microplate reader at 550 nm.
Wound healing assay
Briefly, T24 cells were planted in 12-well-plates (5 × 105 cells per well). After the density of cells reached 90%, we created a wound on the monolayer of cell by using a 200 µL pipette tip. The floating cells were washed off by PBS for three times. Then, different concentrations (0, 1, 2, 4 µM) of Mito-DHA5 and DHA which were dissolved in serum-free medium were added. Finally, the scratch width of 0, 24, 48, 72 h were recorded and imaged by fluorescence microscope (DFC450C; Leica, Wetzlar, Germany).
Hoechst 33,258 staining
Briefly, T24 cells were planted in 24 well plates (2.0 × 105 cells per well). After 12 h, T24 cells were treated with Mito-DHA5 at 30 µM or DHA at 30 µM for 48 h. In order to investigate the effect of NAC on Mito-DHA5-induced apoptisis, pretreated T24 cells by 5 mM NAC for 2 h and incubated in presence of Mito-DHA5 (0, 10 µM) for 48 h. Then T24 cells were washed three times with PBS and stained with Hoechst 33,258 for 5 min. After three times washing with cold PBS, the morphology apoptosis of T24 cells were observed by fluorescence microscope (DFC450C; Leica, Wetzlar, Germany).
Cell apoptosis determination
In brief, T24 cells were plated in 6-well plates (5 × 105 cells per well). After 12 h, T24 cells were treated with Mito-DHA5 (0, 3, 10, 30 µM). After 48 h, cells were washed with cold PBS for three times and resuspended in binding buffer. Then 5 µL annexin V-FITC and 5 µL PI solution were added for staining and incubated at room temperature for 10 min in the dark. Finally, the apoptosis rate of T24 was tested with a flow cytometry (Becton Dickinson).
Measurement of intracellular ROS generation
Briefly, T24 cells were planted in 12-well plates (3 × 105 cells per well). After 12 h, T24 cells were treated with Mito-DHA5 (0, 3, 10 µM) or pretreated by 5 mM NAC and then cultured by Mito-DHA5 (0, 3 and 10 µM) for 12 h. Next removed the culture medium and then incubated with 10 µM DCFH-DA at 37 °C for 30 min. After that, washed with serum-free medium for three times and then photographed by a fluorescence microscopy (DFC450C; Leica, Wetzlar, Germany).
JC-1 assay
MMP was detected using JC-1 staining kit. In brief, T24 cells were plated in 12-well plates (2.0 × 105 cells per well). After 12 h, T24 cells were treated with different concentrations (0, 3, 10 and 30 µM) of Mito-DHA5 or DHA (30 µM) for 48 h. First, prepared incubated JC-1 stain working solution according to the instruction. Briefly, added 50 µL JC-1 (200X) to 8 mL super-pure water and mixed fully. Then 2 mL JC-1 staining buffer (5X) was added to it. After the completion of drug treatment, T24 cells were stained with JC-1 stain working solution (0.5 mL) at 37 °C for 20 min. Finally, washed with JC-1 staining buffer (1×) for twice and added 2 mL incubation medium. Fluorescence microscopy was used to measure MMP.
Western blotting analysis
Briefly, T24 cells were planted in 6-well-plates (5 × 105 cells per well). After 12 h, T24 cells were treated with Mito-DHA5 (0, 3, 10 and 30 µM). After 24 h, washed with cold PBS for twice and lysate was added for 30 min on ice. Then samples were cooked in boiling water for 10 min immediately and stored at -20 ℃. Western blotting experiment was carried out with regular procedure. In brief, PVDF membrane was blocked in 5% milk for 1 h after eletrophoresis finished. Then incubated in first antibody at 4℃. After 15-18 h, washed with PBST for three times (10 min per time) and then incubated in secondary antibody for 1 h at room temperature. After that, washed with PBST for three times again. Finally, developing solution was added and imaged on Chemi Doc (Bio-Rad, USA).
Data analysis
We used the GraphPad Prism (version 6.01) software to analyze our data and the results were expressed as the mean ± SD. The statistical significance of the data was analysed by T-test and was indicated as: *p<0.05, **p<0.01, ***p<0.001 and **** p<0.0001 ns: represent no significance.