Materials
Triptolide (Tianjin Medical Co., Ltd, Tianjin, CHINA) was lysed in dimethyl sulfoxide (DMSO) as a storage solution and freshly diluted into 5 mmol·L−1 in RPMI 1640 medium before use. Cisplatin (Qilu Pharmacy Co., Ltd), Rhodanmine123 (Rh123, fanbobiochemicals Co., Ltd), 3 (4, 5 dimethylthiazol-2-yl) 2, 5 diphenyltetrazolium bromide (MTT), Doxorubicin(Dox) (ZHEJIANG HISUN PHARMACEUTICAL CO., LTD), just as well as the β‑actin mouse monoclonal antibody (1:1000) were acquired from Santa Cruz Biotechnology (Santa Cruz, USA). The P-glycoprotein, MRP1, CAP1, CAP2, and Caspase-3 antibodies (1:500) were obtained from Abcam (Abcam, Hong Kong).
Cells and cell culture
The HNE1/DDP cells were provided by the biochemical pharmacology experiment center of Bengbu Medical College (Bengbu, China). Briefly, cells were incubated in RPMI‑1640 solution, supplemented with Newborn Calf Serum (10%) and streptomycin/penicillin (100 U/mL) at 37 °C under 5% humidified CO2 atmosphere. The HNE1/DDP cells were incubated in DDP (1 μg/mL)-containing culture medium in order to maintain their DDP resistance.
Cell viability assay
The HNE1/DDP cells were added in plates with 6,000 cells a well and cultured overnight for adherence. The supernatant was discarded and replaced by the fresh medium containing TPL (0, 12.5, 25, 50, 100 nmol·L-1) or doxorubicin (0.1, 0.2, 0.4, 0.8 μmol·L-1) or TPL (12.5 nmol·L-1) and doxorubicin (0.1, 0.2, 0.4, 0.8 μmol·L-1). The cells have been cultured for 24h or 48h. Following treatment, MTT (5 mg/mL) was added into the supernatant (15 μL) and maintained in culture for additional 4 h. The supernatant removed, 150 μL DMSO was added to each well and then incubated at 37℃ for 30 minutes. Subsequently, the absorbance of all wells was detected at 570 nm with a Microplate Reader (BioTek, USA).
Apoptosis detection
HNE1/DDP cells were seeded in a 6-well plate with a density of 3 ×105 cells per well. After overnight incubation for attachment, cells were treated with different concentrations of TPL (0, 12.5, 25, and 50 nmol L−1) for 24 h. Next, cells were digested with trypsin, washed with cold PBS, and fixed with alcohol solution overnight at 4 °C. The fixed cells were washed twice with PBS, stained with a PI solution (50 mg/mL PI, 0.05% Triton X-100, and 0.1 mg/mL RNase A) for 30 mins in the dark. Finally, at least 6000 events per sample were acquired using a flow cytometer (BD, Becton, Dickinson, and Co., Piscataway, NJ).
Intracellular Dox content estimation
The cellular efflux and uptake of Dox with or without TPL were fixed using a flow cytometer (BD, USA). Briefly, HNE1/DDP cells were captured in the exponential growth phase and seeded onto 12-well plates at a density of 50,000 cells per well. Following 12 h of incubation, the wells were divided into 5 groups, each group comprising 3 wells. The five groups of cells were pretreated with TPL (0, 12.5, 25, 50 nmol·L−1, respectively) for 2 h, then treated with Dox (0.2 μmol L−1) with or without TPL (12.5, 25 and 50 nmol L−1, respectively) for 8 h. Next, the cells were washed twice with PBS and incubated with fresh RPMI-1640 for 6 h. Subsequently, the cells were collected to analyze the Dox fluorescence. Baseline controls were also provided for the Dox efflux evaluation. Non-treated cells were used to set the baseline. We detected 10,000 events from each harvested sample. The net Dox uptake was presented as the fluorescence intensity difference between the treated and non-treated cells.
Cellular Rh123 uptake and efflux estimation
The extracellular secretion of P-gp was evaluated by rhodamine 123 (Rh-123), a fluorescent P-gp substrate. Briefly, the cellular uptake and efflux of Rh123 with or without TPL were determined using a flow cytometer (BD, USA). The HNE1/DDP cells were captured and seeded onto 12-well plates at a density of 30,000 cells per well, then incubated overnight. The wells were divided into four groups and pretreated with TPL (0, 12.5, 25, 50 nmol·L−1) for 4 h, followed by the removal of the supernatant. Next, Rh123 (0.5 μmol·L−1) alone or in combination with triptolide (12.5, 25, and 50 nmol·L−1, respectively) were supplemented, and the cells were incubated for an additional 4 h, treated the same way as described for the intracellular Dox content estimation.
Intracellular ATP measurement
The cells were seeded onto 6-well dishes at a density of 2 × 105 cells per well. After being incubated overnight, the wells were divided into five groups, each group comprising three wells, and treated with TPL at different concentrations (0, 12.5, 25, 50, and 100 nmol·L−1, respectively) for 24 h. The cells were then collected and the ATP levels were determined using a Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The luminescence levels were measured using a microplate reader.
P-gp, MRP1, Caspase-3, CAP1, and CAP2 expression level evaluation in HNE1/DDP cells
The cell suspensions were seeded onto 6-well dishes at a density of 7.5×106 cells per well. After overnight incubation for attachment, the cells were treated with different concentrations of TPL (0, 12.5, 25, and 50 nmol·L−1, respectively) for 24 h. The cells were then harvested and lysed in lysis buffer (40 mmol L−1 Tris (pH = 8), 0.1% NP40, 120 mmol L−1 NaCl) on ice for 30 mins, and centrifuged for 30 mins at 12000 rpm. The protein quantity was essayed using a BCA Protein Assay kit. Equal protein quantities were electrophoresed, transferred onto polyvinylidene difluoride (PVDF) membranes, and incubated with the primary antibodies overnight at 4 °C. After washing three times with TPBS, the membranes were exposed to the secondary antibodies at room temperature for 2 h. The blots were detected using the immobilon western chemiluminescent HRP substrate (Millipore).
Data analysis
Data were analyzed by ANOVA using the SPSS 17.0 software, presented as the mean ± standard deviation, and differences of p < 0.05 or 0.01 were regarded as statistically significant.