Fetal bovine serum (FBS) and RPMI-1640 medium were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cisplatinum (DDP, 100 mg, MKG2946) were purchased from Millipore-Sigma (MA, USA). FITC-Annexin V/propidium iodide (PI) apoptosis assay kit (cat. no. KGA108) was purchased from Nanjing KeyGene Biotech Co., Ltd. (Nanjing, China). Antibodies to Bax (cat. no. 50599-2), Bcl-2 (cat. no. 12789-1), caspase-3 (cat. no. 19677-1); LC3 (cat. no.14600-1-AP), p62 (cat no. 18420-1AP), Beclin1 (cat. no. 11306-1-AP), PI3K (cat. no. 60225-1), AKT (cat. no. 60203-2), p-AKT (cat. no. 66444-1), and GAPDH (cat no. 60004) were purchased from Proteintech (Chicago, IL, USA). ABCB1 (cat no. 13978), ABCC1 (cat no. 72202), ABCG2 (cat. no. 42078); Cleaved Caspase-3 (cat. no. 9664), mammalian target of rapamycin (mTOR) (cat no. 2983), and phospho-mTOR (cat no. 5536) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The enhanced chemiluminescent (ECL) substrate for horseradish peroxidase (HRP) activity was acquired from proteintech (Chicago, IL, USA). Other reagents, if not specifically mentioned, were acquired from Nest (Wuxi,China).
BBD (No.180901) was purchased from Xiamen Traditional Chinese Medicine Factory Co., Ltd. (Xiamen, China). The BBD micropowder was diluted in PBS and further diluted with RPMI-1640 medium to different concentrations (0, 0.25, 0.5, 1.0 mg/ml) prior to use.
Cell lines and culture.
The human gastric carcinoma parental cell line SGC7901 and cells resistant to DDP (SGC7901/DDP) were obtained from the BeNa Culture Collection (Beijing Be Na Chuanglian Biotechnology Research Institute, Beijing, China). SGC7901/DDP cells were cultured in RPMI-1640 containing 10% FBS, 100 µg/mL streptomycin, 100 U/mL penicillin, and 1μg DDP (maintaining drug resistance). The cells were incubated at 37˚C in a 5% CO2 atmosphere. For the follow up experiments, the cells were cultured completely in RPMI 1640 without DDP.
We used an MTT assay to validate drug resistance in SGC7901/DDP cells and evaluate the effects of BBD. Briefly, cells (1×104 cells/well) in a logarithmic growth phase were seeded in 96 well plates. The next day, we replaced the original medium with medium containing different drugs: 5-FU (0-25600 μM), DOX (0-256 μM) and DDP (0-40 μM) and various concentrations of DDP combined with 0.5 mg/mL of BBD solutions with RPMI-1640 complete medium. The cell drug resistance was validated and the reversal effect of BBD on cell viability was evaluated. After incubation with the drug for 48 h, SGC7901/DDP cells were supplemented with 100 µl culture medium containing (0.5 mg/ml) MTT, and incubated for 4 h (37˚C, 5% CO2). Cells were then suspended in 100 µl DMSO. Relative cell viability (A value) was determined using a plate reader (Multiskan FC, Thermo Fisher Scientific) at an absorbance of 570 nm. We used SPSS 21.0 to calculate IC50 based on the drug resistance index (RI) = IC50 (SGC7901/DDP) / IC50 (SGC7901) and reversal fold (RF) = IC50 (DDP/ADM/5FU) / IC50 (DDP/ADM/5FU + 0.5 mg/mL BBD) were calculated.
Doxorubicin and Rho123 staining
SGC7901/DDP cells were seeded at a density of 4.0×105 in 6-well plates and cultured overnight. When the cells reached 50%~60%, different concentrations of BBD (0, 0.25, 0.5, 1.0 mg/mL) were added and incubated for 48 h. Next, the supernatant was discarded and 1 mL PBS was added to wash the cells 3 times. We used 4% paraformaldehyde to fix the cells for 10 min, then washed the cells with PBS 3 times. Doxorubicin (5 μM) or Rho123 (5 μM) staining solution was added and incubated with the cells for 15 min, and 1 mL PBS was used to wash 3 times. The samples then were observed and photographed with an inverted fluorescence microscope (400×).
After cell intervention, 4% paraformaldehyde was added to fix the cells for 15 min. 10% DAPI staining solution (in PBS) was added to each well and cultured in the darkness for 15 mins. The cells were then washed by PBS 3 times and imaged with a fluorescence microscope (400×).
The apoptotic rate was determined by flow cytometry. The AnnexinV-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGene Biotech Co., Ltd. Nanjing, China) were used. SGC-7901/DDP cells in logarithmic growth phase were plated in six-well plates at the density of 4.0×105 cells per well and treated with different concentrations of BBD (0, 0.25, 0.5, 1.0 mg/mL) for 48 h, 1×106 cells were collected and washed twice with cold PBS. Cells were resuspended in 500 µl 1×binding buffer and then incubated for 15 min at room temperature in the dark following 5 µl Annexin V/FITC and 5 µl PI additions. For each analysis, 10,000 events were recorded.
Cyto-ID autophagy detection
SGC-7901/DDP cells were treated with different concentrations of BBD for 48h, the cells were centrifuged, then the 1×106 cells were resuspended in 0.5 ml of freshly diluted Cyto-ID green detection reagent (1μL Cyto-ID green detection reagent mixed with RPMI-1640 medium to a final volumn of 2 mL). After incubation for 30 min at 37℃ in the dark, the cells were determined by flow cytometry.
Western blot analysis
Protein expression of Bax, Bcl-2, caspase-3, cleaved-caspase-3, GAPDH, LC3, p62, Beclin1, PI3K, AKT, p-AKT mTOR and p-mTOR were examined by western blot analysis. After treated with BBD as above. . Cellular proteins of SGC-7901/DDP cells were lysed by radioimmunoassay (RIPA) buffer with inhibitor cocktail (Thermo Fisher Scientific, USA), and centrifuged at 14,000 rpm for 20 min at 4℃. The final supernatants were harvested. Protein concentrations were determined by BCA protein assay kit (bovine serum albumin). Total protein (30 µg) was electrophoresed on 10% SDS-PAGE and then transferred to a PVDF membrane. Following incubation with 5% skimmed milk for 1 h at room temperature, Primary antibodies (1:1000) were diluted with TBST solution and incubated with the membranes overnight at 4˚C. Washed 3 times (TBST, 10 min), then incubated with secondary antibodies (HRP-conjugated 1:5000) for 1 h. Finally, we used Image Lab (Bio-Rad Laboratories, Inc., Berkley, California, USA) to detect protein. Three independent experiments were performed in order to get the representative data.
All representative data were obtained from three independent experiments. Statistical analyses were performed by IBM SPSS Statistics 21. P values < 0.05 were considered to be statistically significant.