The human pancreatic cancer cell lines Panc-1 and Bxpc3 were purchased from the American Type Culture Collection. Panc-1 and Bxpc3 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C. Panc-1 is a human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin; Bxpc3 is a human pancreatic cancer cell line used in the study of pancreatic adenocarcinomas. We had repeated the experiment using the Bxpc3 cell line. SP1 knockdown cell lines were established by transfecting the cells with short hairpin RNA (shRNA) lentiviral plasmids. The lentiviral shRNA plasmid targeting SP1 (shSP1) was purchased from Vigene Biosciences, and the control sequence (shCtrl) from Sigma-Aldrich (Merck KGaA).

For transduction, a total of 5×10⁴ cells/well were seeded in 6-well plates. The shSP1 or shCtrl lentivirus was added to the cells in the presence of 5 µl polybrene (Sigma-Aldrich; Merck KGaA). After 96 h, the transduced cells were selected with 1 µg/ml puromycin. Subsequently, the selected cells were treated with different concentrations of propofol according to our previous study (10). In order to avoid the possible adverse effects of lipid emulsion, pure propofol was obtained from Sigma-Aldrich (Merck KGaA).

Cell viability was assessed using MTT assays. Cells were seeded in 96-well plates (6×10³ cells/well) and treated with propofol for 48 h. MTT solution (Sigma-Aldrich; Merck KGaA) was added for 4 h at 37°C. The precipitate was then dissolved in 200 µl DMSO. The absorbance was measured at 490 nm using a Multiskan Spectrum plate reader (Thermo Fisher Scientific, Inc.).

In a 6-well plate, Panc-1 and Bxpc3 cells (1×10⁶ cells/well) were incubated in DMEM overnight in order to create a cell monolayer. A scratch was made in the middle of the well using a pipette tip (7 mm) and the debris was washed away prior to the addition of new medium to the wells. Using an optical microscope (magnification, ×400), the cells were imaged, and the initial area of the scratch in the field of view was determined as the length multiplied by the width. A total of three fields of view were examined. The plate was incubated at 37°C for 24 h, after which the same field of view was imaged and the scratch area was measured again using the same methodology. The final area of the scratch wound was divided by the initial area to determine the percentage wound remaining of the initial area covered by migrating cells over the 24-h culture period.

Cells were incubated with the indicated doses of propofol for 24 h, and then washed with cold PBS. Subsequently, the cells were fixed using cold 75% ethanol overnight at 4°C, washed with cold PBS and stained with propidium iodide (PI) for 30 min at 37°C. After staining, the cells were analyzed using flow cytometry (BD FACSDiva™; BD Biosciences).

Total RNA was isolated from cell lines using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.) was used to measure RNA concentration. RT reactions were performed using the PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.) and qPCR was performed using FastStart Universal SYBR-Green Master (Roche Diagnostics GmbH) and the Step One Plus Real-time PCR system (Applied Biosystems). According to the manufacturer's protocol, the thermocycling conditions were as follows: i) Initial denaturation, 95°C for 30 sec; ii) amplification, 95°C for 5 sec, 60°C for 20 sec, 40 cycles; and iii) dissociation curve, 95°C for 60 sec, 55°C for 30 sec, 95°C for 30 sec. The 2^−ΔΔCq method was used to determine the relative mRNA expression levels, which were normalized to those of β-actin (14). All experiments were performed independently three times and set up in triplicate. The following primer sequences were used: i) ADAM8 forward, 5′-ACAATGCAGAGTTCCAGATGC-3′ and reverse, 5′-GGACCACACGGAAGTTGAGTT-3′; ii) SP1 forward, 5′-CGGAATTCATGAGCGACCAAGATCACTCCATG-3′ and reverse, 5′-CGGAATTCTTGGACCCATGCTACCTTGCATCC-3′; and iii) GAPDH forward, 5′-GTCAGTGGTGGACCTGACCT-3′ and reverse, 5′-TGGTGCTCAGTTTAGCCCAGG-3′.

Cells were lysed using RIPA buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) to extract total protein. The extracted protein (50 µg) was separated using SDS-PAGE on 10% gels, and then transferred to PVDF membranes. The membranes were incubated with antibodies against ADAM8 (cat. no. ab255608; dilution 1:1,000; Abcam), SP1 (cat. no. ab231778; dilution 1:1,000; Abcam) and β-actin (cat. no. ab8226; dilution 1:1,000; Abcam) overnight at 4°C, then with goat anti-rabbit HRP-conjugated antibody (cat. no. ab181662; dilution 1:2,000; Abcam). The protein bands were visualized using the Odyssey system (LI-COR Biosciences).

Lentiviral vectors for SP1 knockdown (shSP1) were purchased from Vigene Biosciences (cat. no. P100029). A total of 50×10⁴ cells/well were seeded in 6-well plates. The shSP1 or shCtrl virus (cat. no. TL506569V) was added to the cells in the presence of 5 µl polybrene (Sigma-Aldrich; Merck KGaA) to increase the efficiency of infection. After 96 h, the transduced cells were selected using 1 µg/ml puromycin for 2 months. The selected cells were then treated with different concentrations of propofol, the transfection efficiencies in all cell lines are over 80%. The following primer sequences were used: i) SP1-shRNA1, 5′-GCAAGTTCTGACAGGACTACCTTCAAGAGAGGTAGTCCTGTCAGAACTTGCTTTTTT-3′; ii) SP1-shRNA2, 5′-GCAACATCATTGCTGCTATGCTTCAAGAGAGCATAGCAGCAATGATGTTGCTTTTTT-3′; iii) SP1-shRNA3, 5′-GCAGACCTTTACAACTCAAGCTTCAAGAGAGCTTGAGTTGTAAAGGTCTGCTTTTTT-3′.

Dual luciferase reporter assays were performed as previously described (15). Wild-type or mutant ADAM8 (containing mutations in the putative binding site for SP1 located in 3′-untranslated region), together with a synthesized promoter mimic or vector, were co-transfected for 48 h. The transfected cells were then harvested to determine luciferase activity using a dual luciferase reporter assay system (Promega Corp.).

Panc-1 cells were lysed using RIPA buffer (50 mM Tris-HCl, pH 7.8; 150 mM NaCl; 5 mM EDTA; 0.1% Triton X-100; 0.05% NP-40). Subsequently, the lysates were incubated overnight at 4°C in an orbital shaker with 2 µg anti-ADAM8 or anti-SP1 antibody alongside a negative control containing 2 µg rabbit IgG antibody. Cell lysate without any antibody (input) was used as a positive control. After incubation, the mixture was incubated with agarose beads at 4°C for 3 h. The beads were collected and sequentially washed five times with 1 ml RIPA lysis buffer, then analyzed by western blotting using anti-ADAM8 or anti-SP1 antibodies. The intensity of the specific bands was evaluated using ImageJ software, version 1.46 (National Institutes of Health). The assays were repeated at least three times.

The data are presented as the mean ± SD of three independent experiments. SSPS 20.0 software (IBM Corp.) was used for statistical analysis. One-way ANOVA followed by Duncan's post hoc test and unpaired Student's t-tests were used to compare the experimental groups. P<0.05 was considered to indicate a statistically significant difference.

Firstly, the effect of 0 (negative control), 5, 10 and 20 µg/ml propofol on Panc-1 and Bxpc3 cell viability was evaluated using MTT assays. As shown in Fig. 1, propofol significantly suppressed the viability of Panc-1 and Bxpc3 cells when compared with the negative control. It was also observed that 10 µg/ml propofol resulted in the lowest viability. These findings indicated that propofol treatment could inhibit the viability of pancreatic cancer cells.

The role of propofol on the cell cycle progression of Panc-1 and Bxpc3 cells was examined. Panc-1 and Bxpc3 cells were treated with 0, 5, 10 and 20 µg/ml propofol, and the distribution of Panc-1 and Bxpc3 cells in different phases of the cell cycle was examined using flow cytometry. The results indicated that propofol affected the cell cycle progression of Panc-1 and Bxpc3 cells, as evidenced by a significant gradual reduction in the number of pancreatic cancer cells in the S-phase with increasing propofol concentration (Fig. 2). While the number of cells in the G1-phase appeared to be increased, this was not statistically significant. This demonstrated that cells were blocked at the S-phase, which may indicate a relative decrease in DNA synthesis and replication.

In addition to its inhibitory effects on cell viability, the potential role of propofol on the malignant behavior of Panc-1 and Bxpc3 cells was also examined. For this purpose, wound healing assays were performed. The results demonstrated that propofol treatment significantly suppressed the migration of Panc-1 and Bxpc3 cells (Fig. 3). Indeed, the wounds healed at a slower rate following treatment with higher concentrations of propofol.

To investigate the effects of propofol on ADAM8, mRNA and protein were extracted from Panc-1 and Bxpc3 cells treated with 0, 5, 10 and 20 µg/ml propofol. It was observed that propofol treatment significantly reduced the ADAM8 mRNA and protein expression levels in a dose-dependent manner (Fig. 4).

Verification of the direct interaction between SP1 and ADAM8. In the University of California Santa Cruz database, the promoter region of ADAM8 was predicted to be located at chr10:133,262,422-133,264,422 (GRCh38). The luciferase reporter vectors containing the indicated genomic fragments of the ADAM8 gene were constructed. To investigate the potential regulators involved in ADAM8 expression, potential transcription factor binding sites in the ADAM8 promoter were identified using three online software packages: PubMed (https://pmlegacy.ncbi.nlm.nih.gov/gene/101), JASPAR (http://jaspar.genereg.net/) and GeneCards (http://genecards.org) (16). Binding sites for the transcription factor SP1 and zinc finger and BTB domain containing 40 were found in the promoter region of ADAM8 (Fig. 5A). Of the two candidate transcription factors, only SP1 mimics markedly enhanced luciferase activity (Fig. 5B). Co-IP experiments were performed in order to confirm whether SP1 binds to the promoter region of ADAM8. The results indicated that SP1 and ADAM8 were detectable in the corresponding precipitated protein complexes, indicating that SP1 interacted directly with ADAM8 (Fig. 5C). This suggested that the SP1 transcription factor may be targeted by propofol.

Propofol potentially targets SP1 to regulate ADAM8. To investigate whether propofol acts through ADAM8 via SP1, Panc-1 and Bxpc3 cells were treated with 0, 5, 10 and 20 µg/ml propofol and used in a dual luciferase reporter assay. The luciferase activity was significantly inhibited, with a concentration of 10 µg/ml propofol resulting in the lowest luciferase activity (Fig. 6A and B). Additionally, Panc-1 shCtrl and three Panc-1 shSP1 cell lines (Panc-1-SP1-shRNA1, 2 and 3) were established, and the shSP1 cell line expressing the lowest mRNA and protein levels of SP1 was used in the subsequent experiments (Fig. 6C-H). Protein and mRNA were extracted from the Panc-1/Bxpc3-shSP1-shRNA and the Panc-1/Bxpc3-NC-shRNA lines to determine whether the expression of ADAM8 was regulated by propofol in the absence of SP1. The expression of ADAM8 was decreased at different concentrations of propofol in the control groups (Figs. 7A-C and 8A-C), but not in the experimental groups (Figs. 7D-F and 8D-F).