Cells and cell culture
Eca-109/VCR cell line was obtained from theLife Science Centre of Hebei North University (Zhangjiakou, China). The cells were maintained at 37°C,5% CO2and saturated humidity, in RPMI-1640 medium(corning, China)supplementedwith 10% fetal bovine serum (FBS) (Gibco,USA), 100 U/ml of penicillin and 100 µg/ml of streptomycin. Cells inmid-logarithmic growth were used for experiments. When the density of the cell colonies reached ~90% confluence, the cells were detached with 0.25% trypsin (Amresco, Solon, OH, USA) and transferred to fresh flasks at a ratio of 1:2. VCR(V0129, TCIDevelopment Co.,Ltd, Shanghai, China) with a final concentration of 2 µg/mL was added to maintain the resistance of Eca-109/VCR cells. It was discontinued 1 week before the start of the experiment to maintain cell resistance.
Cell Proliferation Inhibition Assay
The MTT assay was used to assess the cytotoxic activity of matrine. The trypsin digested Eca-109/VCR cells were collectedand counted under microscope. Then cells(5x104 cells/well) were plated in 96‑well culture plates and allowed to adhere overnight at room temperature, following which the cells were treated with various concentrations of matrine (0, 0.25, 0.5, 0.75, 1.0, 1.5 or 2.0 mg/mL). The cells were subsequently incubated for 24 and 48 h then they were washed twice with PBS and treated with 20 µL of sterile MTT dye (5 mg/mL, Qilu Pharmaceutical Co., Ltd, China) for 4 h at 37°C. The cells were washed with PBS and then solubilized with 150 µL of dimethyl sulfoxide (DMSO). The 96-well plates were shaken until the formazan crystals dissolved completely. Then the absorbance value was measured on a microplate reader (SpectraMax M2, Molecular Devices, USA) at 490 nm.The rate of cell inhibition was calculated using equation: the inhibition rate (%) = [1-A490(test)/A490(blank)]×100%]. Each experiment was conducted in triplicate and included three replicates.
The Sensitivity Of Cells To Vcr By Mtt Assay
Eca-109 and Eca-109/VCR cells at logarithmic growth stage were used for the experiment. Cells(5x104 cells/well) were plated in 96‑well culture plates and allowed to adhere overnight at room temperature, following which the cells were treated with drug. Eca-109 cells were treated with different concentrations of VCR (0, 0.02, 0.04, 0.08, 0.16, 0.32 µg/ mL). Four wells were set up in each group, and the final volume of each well was 200µL. Eca-109/VCR control group and Eca-109/VCR+ matrine (1.0mg/mL matrine treated for 24h) group were set up in Eca-109/VCR cell lines. The two groups were treated with different concentrations of VCR (0.5, 1, 1.5, 2, 3 µg/mL) for 24h, and four wells were set in each group, the final volume of each well was 200 µL. The other experimental steps were the same as the above cell proliferation inhibition assay. Then 50% inhibition concentration (IC50), resistant index, and reversal index were calculated to evaluate MDR reversal effect. The inhibitory concentration 50% (IC50) was determined by a non-linear regression analysis of the concentration-response curve using the Hill equation. Resistant index = IC50 (Eca-109/VCR) / IC50(Eca-109). Reversal index = IC50 (Eca-109/VCR + VCR ) / IC50 (Eca-109/VCR + VCR + matrine).
Eca-109/VCR cells were divided into four groups: control group, matrine (1.0 mg/mL) group, VCR (2 µg/mL) group and matrine (1.0 mg/mL)+VCR (2 µg/mL) group. Cells of each group were plated onto chamber slides at 5x104 cells per chamber for 24 h. Then, they were fixed with 4% paraformaldehyde for 30 min at room temperature (RT) and subsequently permeabilized with 0.25% Triton X-100 for 20 min at RT. The slides were then treated with 0.5% bovine serum albumin (BSA; Beyotime Institute of Biotechnology, Jiangsu, China) in 0.1% Tween-20 for 30 min at RT, primary antibodies P-gp (cat. no ab170903, dilution, 1:1000; Boaosen Biotechnology Co., Ltd, Beijing, China) was added and incubated on the slides overnight at 4˚C. Secondary antibodies rhodamine-labeled affinity purified antibody (Anti-rabbit IgG (H+L) Ab, 5230-0332, dilution, 1:500; Seracare, USA) in the dark at RT for 60 min and washed with PBS three times. Finally, the cover slip was sealed with Fluorescent Mounting Media (S2100, Beijing Solarbio Technology Co., Ltd. Beijing, China). Negative controls were also employed to offset the disturbance of the primary or secondary antibody. The results were observed and recorded by fluorescence microscopy (Leica TCS-ST2 Instrument, Japan). The projected cell area was evaluated using ImageJ software. The assays were performed in triplicate in three independent experiments.
Eca-109/VCR cells were divided into four groups: control group, matrine (1.0 mg/mL) group, VCR (2 µg/mL) group and matrine (1.0 mg/mL)+VCR (2 µg/mL) group. Western blot was used to detect the MDR effect of matrine on Eca-109/VCR cells. Furthermore, the PI3K/Akt/mTOR pathway plays an important role in regulating the cell cycle, apoptosis and autophagy. Therefore, we next investigated whether the effects of matrine on Eca-109/VCR cells were mediated by this pathway. Western blot methods was performed as previously described. Briefly,cellswere lysed in RIPA lysis buffer (Beyotime, China). Cell protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to PVDF membranes (Immobilon 0.45 µm, Millipore, USA), and immersed in a blocking solution containing 5% fat-free milk and 0.1% Tween-20 for 1h. After blocking, membranes were incubated with P-gp (cat. no ab170903, dilution, 1:1000;Boaosen Biotechnology Co., Ltd, Beijing, China), MRP1(cat. no ab233383, dilution, 1:1000;Boaosen Biotechnology Co., Ltd, Beijing, China), p-mTOR (cat. no ET1608-5, dilution, 1:1000;Hangzhou HuaAn Biotechnology Co.,Ltd, Hangzhou, China), p-Akt (cat. no ET1607-73, dilution, 1:1000;Hangzhou HuaAn Biotechnology Co.,Ltd, Hangzhou, China), p-p70S6K-T421/S424 (cat. no AP0502, dilution, 1:1000;ABclonal Technology, Wuhan, China), LC3 (cat. no PM036,dilution, 1:1000; Medical & Biological Laboratories Co., Ltd, Japan), p62(SQSTM1)(cat. no PM045, dilution, 1:1000;Medical & Biological LaboratoriesCo., Ltd, Japan), Beclin 1 (cat. no PD017, dilution, 1:1000; Medical & Biological LaboratoriesCo., Ltd, Japan) overnight at 4°C and then with diluted horseradish peroxidase-conjugated secondary antibody (sc-2004, dilution, 1:1000, Santa Cruz) for 2h at room temperature. After washing, the resulting bands were visualized using the standard ECL procedure, quantified by densitometry, and normalized to the corresponding β-actin (cat. no AC026, dilution, 1:1000;ABclonalTechnology, Wuhan, China)bands.
Total RNA extraction andqRT-PCR
Total RNA from Eca-109/VCR cells in different groups was isolated with TRIzol®reagent (Invitrogen, Thermo Fisher Scientific, Inc.) and were then converted to cDNA with a PrimeScript RT Reagent kit (cat. no RR036A,Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. SYBR Premix Ex Taq (cat. no RR820A, Takara Biotechnology Co., Ltd.) was used for the RT-qPCR assays to detect the expression levels of MDR1in cells. The sequences of the primers were as follows: MDR1: 5′-GAGTGTCTGTTCCGTTTCA-3′ and 5′-TGCTCCGTCCAAATCA-3′; GAPDH (glyceraldehyde-3-phosphate dehydrogenase): 5′-GTCTCCTCTGACTTCAACAGCG-3′ and 5′-ACCACCCTGTTGCTGTAGCCAA-3′. qRT-PCR was performed on the ABI 7300 system (Applied Biosystems, Foster City, CA, USA) in 30-µL reactions using the following program: 40 cycles of 95°C for 120 s, 60°C for 30 s and 72°C for 30 s. All samples were amplified in triplicate in one assay run simultaneously. Transcription levels were normalized against GAPDH, and relative gene expression was quantified using the 2−ΔΔCtmethod.
Transmission Electron Microscope (Tem) Sample Preparation Of Culture Cell
Cell suspension with concentrationof5×104cells/mL was prepared and inoculated in 50 mL culture flasks.After cultured overnight, culture medium was discarded.In the experiment group(Eca-109/VCR cells treated with 1.0 mg / mL Matrine for 48 h ), 1.5 mL cell suspension and 1.5 mL culture medium were added to reach the terminal volume of 3 mL.In the control group, same terminal volume of culture medium was added.After 24h digestion with trypsin, cells andsupernatant were collected into a centrifuge tube. Thecentrifugecondition is rotation speed 1500r/min and time 5min. The sample was washed with PBS for 3 times, and fixed at room temperature using glutaraldehyde, followed by osmic acid. After fixation with aqueous fixatives, samples are dehydrated in increasing concentrations of acetone, followed by propylene oxide. The samples were embedded, and 60 - 80 nm sections were stained with uranyl acetatefollowed by lead citrate to enhance contrast.
Data analysis was performed by SPSS 20.0. Data were described as the mean ± SD. Student’st-test, pairedt-test and ANOVA were conducted to analyse quantitative variables.AP-value ≤0.05 was considered statistically significant.