DMAMCL and its active metabolic form micheliolide (MCL) were synthesized as previously reported . The positive probe (MCL-biotin) and the negative probe (MCL-S-biotin, NP) were synthesized as described . Gemcitabine was obtained from Med Chem. Express (CAS: 95058-81-4).
Human pancreatic cancer cell lines (PANC-1, AsPC-1, BxPC-3) were originally obtained from JIKAI GENE (NICR, 3111C0001CCC000023, 3111C0001CCC000214, 3142C0001000001478). PANC-1 cells were cultured in DMEM medium (Corning, 10-017-CVR) supplemented with 10% fetal bovine serum (BI, 04010-1A) and penicillin/streptomycin (Sigma, V900929) in a humidified incubator with 5% CO2 at 37℃. AsPC-1 cells and BxPC-3 cells were cultured in RPMI1640 medium (Corning, 10-040-CVR) supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37℃.
Cell viability assay
For cell viability assay, human pancreatic cancer cell lines were seeded in 96-well-plates (4000 cells/well). Cells were treated with varying concentrations of DMAMCL or MCL for a certain time. After 48 or 72 h, 20 μL MTT reagent (Solarbio, M8180) (5 mg/mL) was added to each well. After 4-h incubation, the liquid in the wells was replaced by 150 μL dimethyl sulfoxide (DMSO). Cell viability was determined by optical density (OD) values at 570 nm.
Colony formation assay
For colony formation assay, human pancreatic cancer cell lines were seeded in 6-well-plate (2000 cells/well). Cells were treated with varying concentrations of DMAMCL or MCL for 14 days. After that, the culture medium was discarded, and cells were fixed with ice methanol for 15 min. Fixed cells were stained with crystal violet (Solarbio, G1063) for 15 min and washed 3 times with PBS. Finally, the plates were photographed by a digital camera.
The apoptosis assay was detected by FITC coupled with Annexin-V apoptosis detection kit (Beyotime, C1062). PANC-1 cells were seeded in 6-well-plates (2×105 cells/well). After cells adhered overnight, they were treated with different concentrations of DMAMCL or MCL, with or without gemcitabine. After 48 h treatment, cells were washed 3 times with PBS and digested with trypsin without EDTA (Gibco) in EP tubes, followed by centrifugation at 1000 g for 3 min. The cell pellets were washed twice with PBS, following by adding 100 μL 1×binding buffer to suspend the cells. For cell staining, 5 μL FITC Annexin V and 5 μL of PI were added to each tube and incubated at room temperature (keep in dark) for 15 min. Finally, each tube was supplemented with 400 μL 1×binding buffer. The samples were analyzed by FACSCalibur flow cytometer (Beckman, BD LSRFortessa ), and the data was analyzed by FlowJo 7.6.1 software (10,0,0,0).
Chemo-proteomics to identify MCL-bound proteins
Pull-down experiment was carried out according to the method of literature . Briefly, BxPC-3 cells were plated on 10 cm2 cell culture dish and grown to confluence for 24 h. Cells were harvested and lysed in NP40 lysed buffer containing 1 mM PMSF and protease inhibitors. MCL-biotin (Probe) or MCL-S-biotin (NC probe) was incubated with cell lysates overnight at 4℃, then the prewashed streptavidin beads (Thermo, 20349) were added to each sample and incubated overnight at 4℃. On the second day, the beads ware washed six times with lysis buffer, and the bead-bound proteins were eluted and boiled in 2×SDS loading buffer. The bead-bound proteins were separated by SDS-PAGE and visualized by silver staining. The protein-containing band in the gel was excised, followed by in-gel digestion and analysis by LC-MS/MS.
Site-directed mutant assay
Site-directed mutant assay was constructed using QuikChange II XL site-directed mutagenesis kit (Stratagene, USA). In order to mutate the cysteine at position 132 of pETM3C-ANXA2 plasmid to glycine, primers were designed (forward, 5'-CTCATTGAGATCATCGGCTCCAGAAC-3'; reverse, 5'-CTCCTGAGAGAGTAA CTCTAGTAGC-3') and synthesized (GENEWIZ, China). According to the instructions of the site-directed mutagenesis kit, the mutated plasmid was obtained by PCR. Agarose gel electrophoresis was used to detect whether the target band of the PCR product is correct. If the target band was correct, added 1 μL of DMT enzyme to the PCR product and mixed well. The mixture was kept in a 37℃ water bath for 1 h to obtain digestion products. The digested product was purified by agarose gel DNA recovery kit (TIANGEN, DP209-03). The purified PCR product was transformed to E.coli strain DH5α, and the plasmid was extracted according to the plasmid extraction instructions, and then sent to the company for sequencing. The finally confirmed target plasmid was transformed into E. coli strain BL21 (DE3) for the expression and purification of protein.
The molecular docking experiment was performed with Schrodinger software. The ANXA2 protein structure was downloaded from the UniProt datebase (https://www.uniprot.org), and the protein was optimized through Schrodinger software to obtain the best protein model. The three-dimensional structure of MCL was prepared using Chemdraw software. LigPrep tool was used to minimize the energy of the ligand to correct the coordinates, ionization, stereochemistry, and tautomeric substitution structure to obtain the proper conformation by adding or removing hydrogen bonds. Gliding agent ligand docking was used to dock MCL to the active site of ANXA2, and calculate the binding energy. All docking calculations used Glide XP (super precision) mode.
Expression and purification of recombinant ANXA2
The pETM3C-ANXA2 plasmid was transformed into BL21 competent cells, coated on a kana-resistant solid medium plate, and placed in a 37℃ incubator for 12-14 h. Monoclonal strains were picked into 5 mL of LB medium with kana resistance and shaken overnight at 37℃ and 180 rpm constant temperature shaker. The bacterial liquid obtained above was transferred to 250 mL of LB medium with kana resistance, and expanded for 4 h at 37℃ and 180 rpm constant temperature shaker. The bacterial solution was added to 25 μL IPTG (1 mmol/L), and it was induced for 20 h at 18℃ and 180 rpm constant temperature oscillator. The bacterial solution was collected, centrifuged at 4000 rpm, 4℃ for 30 min, and the supernatant was discarded. The pellet was resuspended in an appropriate volume of 1× bacteriolytic buffer and stored at -80℃ for the next step of purification. The bacterial pellet was ultrasonically disrupted for 15 min, centrifuged at 4000 rpm, 4℃ for 30 min, and the supernatant was collected. The supernatant was added to a nickel column equilibrated with 10 mM imidazole in advance, and repeated 2 times. Contaminated proteins were eluted with 30 mM imidazole until the collection solution detected by Coomassie Brilliant Blue G250 was not blue. The target protein was eluted with 250 mM imidazole and collected into 1.5 mL centrifuge tubes on ice until the collection solution detected by Coomassie Brilliant Blue G250 was not blue. 20 μL of eluate were drawn from each tube, and 5 μL of 5×loading buffer was added to the suction fluid, mixed, and boiled to obtain protein samples. The protein purity was detected by SDS-PAGE and Coomassie Brilliant Blue G250 copying method. The high-purity protein collection solution was used to remove imidazole by ultrafiltration, and then the protein solution was obtained by adding an appropriate volume of physiological saline.
The same procedure was used to express and purify the pETM3C-ANXA2-C132G mutant.
293T cells were seeded in a 6-well plate (3.5×105 cells/well) and cultured overnight in a humidified incubator with 5% CO2 at 37℃.The medium was changed to serum-free medium (1 mL/well) before cell transfection. According to the instructions of JIKAI GENE, the lentiviral packaging systems corresponding to the three plasmids of ANXA2-RNAi (16427-1), ANXA2-RNAi (16423-1) and ANXA2-RNAi (16424-1) plasmids were obtained, and the system volume of each is 50 μL. The lentivirus packaging system was added to 293T cells and incubated in a humidified incubator with 5% CO2 at 37℃ for 6 h. After 6 h, the mediums in the cells were replaced with new complete mediums (2 mL/well). After the cells were cultured in a humidified incubator with 5% CO2 at 37℃ for 48 h or 72 h, shRNA virus was obtained, and the shRNA virus was centrifuged to take the supernatant for use or stored at -80℃. For lentiviral transduction, AsPC-1 cells were cultured in serum-free medium containing lentivirus for 6 h, then replaced by new complete medium for 24 h. For generating stable cell lines, infected cells were selected with 1 μg/mL puromycin (Sigma, 540411) for 72 h. Western blot experiments verified which plasmid has the best knockdown effect and used it for the next knockdown experiment.
Western blot analysis
Cell lines were lysed in NP40 lysis buffer with protease inhibitors and the total protein concentration was quantified by BCA assay (Thermo Fisher Scientific, 23227). The normalized samples were analyzed by SDS-PAGE and western blot using standard protocols and the following primary antibodies: anti-ANXA2 (1:2000 dilution, Immunoway, YT0236), anti-AKT (1:2000 dilution, Cell Signaling, 9272S), anti-pAKT (S473) (1:1000 dilution, Cell Signaling, 4060S), anti-α-tublin (1:5000 dilution, TEL, KM9007), anti-GAPDH (1:5000 dilution, Cell Signaling, 2118S), goat anti-rabbit HRP-IgG (1:5000 dilution, Solarbio, SE134), goat anti-mouse HRP-IgG (1:5000 dilution, Solarbio, SE131).
Synergy index calculation
Human pancreatic cancer cell lines were seeded in 96-well plates (4000 cells/well). The cells were treated with DMAMCL, gemcitabine and DMAMCL combined gemcitabine (concentration ratio 1:3) for 72 h, respectively. After 72 h, 20 μL MTT reagent (Solarbio, M8180) (5 mg/mL) was added to each well. After 4 h incubation, the liquid in the wells was replaced by 150 μL dimethyl sulfoxide (DMSO). Cell viability was determined by optical density (OD) values at 570 nm and the inhibition rate of each treatment was calculated. The inhibition effect and drug combination index of DMAMCL and gemcitabine was calculated by the Compusyn software (Dr. Dorothy Chou) using constant drug concentration ratio.
Results are representative examples of three individual experiments. Statistical analysis and graphical presentation were using GraphPad Prism 5.0. P values were determined by a two-tailed Student’s t test. All histogram data was presented as mean ± SEM. *, P< 0.05 and **, P<0.01.