Heat shock protein 27 downstream of P38-PI3K/Akt signaling antagonizes melatonin-induced apoptosis of SGC-7901 gastric cancer cells

Cell culture

Human gastric cancer cell line SGC-7901 was obtained from the Cell Biology Institute
of Chinese Academy of Sciences (Shanghai, China). SGC-7901 cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM, high glucose) (Hyclone, Thermo Scientific,
Waltham, MA, USA) supplemented with 10 % (v/v) fetal bovine serum (FBS) (Hyclone)
and antibiotics (100 U/mL streptomycin and 100 ?g/mL penicillin) (Invitrogen, USA)
in a humidified incubator at 37 °C with 5 % CO
₂
. Cells were grown on coverslips for fluorescence staining and on plastic dishes for
protein extraction.

Treatment and transfection

Melatonin (Sigma, St Louis, MO, USA) was dissolved in ethanol and cells were treated
with melatonin for the indicated times and doses. In experiments to determine the
effects of inhibitors, LY294002 (Sigma) and SB203580 (Beyotime, Nantong, China) on
cell growth inhibition and apoptosis, cells were treated with these kinase inhibitors
for 30 min prior to melatonin treatment.

The sequences of small interfering RNA (siRNA) for HSP27 was 5?-UGAGAGACUGCCGCCAAGUAA-3?,
the sequence of control siRNA was 5?-UUCUCCGAACGUGUCACGUTT-3? (GenePharma Co., Shanghai,
China). Cells were transfected with control siRNA or HSP27 siRNA with Lipofectamine
2000, according to the manufacturer’s instruction.

Immunoblotting analysis

Subconfluent cells were washed with PBS, and lysed with RIPA lysis buffer (150 mmol/L
NaCl, 50 mmol/L Tris–HCl (pH 7.4), 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 %
SDS) with 1 mM sodium orthovanadate, 1 mM PMSF, and 1 % cocktail of protease inhibitors
(Sigma). The lysates were clarified by centrifugation at 12,000g for 20 min at 4 °C and separated by SDS-PAGE followed by transfer onto nitrocellulose
membranes. The following antibodies were used: rabbit anti-P38 antibody, rabbit anti-P-P38
antibody, rabbit anti-P-Akt antibody, mouse anti-HSP27 antibody and rabbit anti-P-HSP27
antibody (Cell Signaling, Danvers, MA, USA), rabbit anti-Akt antibody (Bioworld, Louis
Park, USA), rabbit anti-GAPDH antibody (Santa Cruz, CA, USA). Protein bands were detected
by incubating with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz)
and visualized with ECL reagent (Millipore, Billerica, MA, USA). Digital images of
immunoblots were obtained with a Chemidoc XRS and analyzed using the image analysis
program Quantity One (Bio-Rad, Hercules, CA, USA).

Hoechst staining

Hoechst 33258 dyes (Beyotime) are cell permeable nucleic acid stains, which are useful
for the recognition of DNA damage and cell apoptosis by monitoring the emission spectral
shifts of the dyes. Cells were stained with Hoechst 33258 (5 ?g/mL) in PBS for 30 min
at room temperature, and then washed to remove unbound dye. Observation and photography
was performed in a fluorescence microscope (Olympus BX 51, Tokyo, Japan).

Flow cytometry analysis

Cells were trypsinized and resuspended in 1× binding buffer, double-stained with Annexin
V-FITC and propidium iodide (Beyotime) at room temperature for 15 min in darkness.
Subsequently, the stained cells were analyzed by flow cytometry for apoptotic analysis
according to the manufacturer’s protocol.

Cell viability assay

Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay as described previously 18]. In brief, SGC-7901 cells were seeded at a density of 5 × 10
³
cells per well into 96-well plate and treated with melatonin for the indicated times
and doses. After culture, cells were washed, MTT was added and the plate was incubated
in the dark for 4 h, followed by measurement at 490 nm using a microplate absorbance
reader (Bio-Tek, Elx800, USA). The percent cell viability was calculated as the absorbance
of melatonin treated sample/control sample absorbance ×100 %.

Statistical analysis

Data were analyzed by Image J and statistical analyses were carried out using the
SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA). Student’s t test was used to analyze differences between two groups. Statistical significance
was considered when P  0.05.