Cell culture

The human leukemia monocytic cell line THP-1 was cultured in RPMI 1640 medium (Thermo Fisher Scientific), while the oral premalignant cell line DOK was maintained in DMEM/F12 medium (Thermo Fisher Scientific). All cell lines were maintained in their basic cell culture medium supplemented with 10% fetal bovine serum (Biological Industries), 1% glutamine (Merck), and 1 × penicillin/streptomycin/amphotericin B solution (100 × ; Merck). The cells were placed in a humidified incubator with 5% CO2 at 37 °C.

Peripheral blood mononuclear cell isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy donors by density gradient centrifugation using Histopaque (Sigma). The donors had agreed and signed an informed consent form approved by the Institutional Review Board (IRB Approval no. KMUHIRB-F(I)-20180069) of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan). Briefly, blood samples were diluted with the same amount of phosphate buffered saline (PBS) and subjected to centrifugation at 400 × g for 40 min at 25 °C. The white layer representing PBMCs was aspirated out gently and transferred into a 15 mL sterile centrifuge tube. After the addition of an equal amount of PBS, the tube was centrifuged at 400 × g for 10 min, and the supernatant containing platelets was discarded. The cell pellet was gently mixed with RPMI 1640 complete medium (Thermo Fisher Scientific) and incubated in a culture plate for 3 h. At the indicated time points, the supernatant containing lymphocytes was aspirated, and the remaining monocytes were allowed to settle onto the culture plate and maintained in RPMI 1640 complete medium supplemented with 10% fetal bovine serum (Biological Industries), 1% glutamine (Merck), and 1 × penicillin/streptomycin/amphotericin B solution (Merck). The cells were placed in a humidified incubator with 5% CO2 at 37 °C for further experiments.

Conditioned medium culture and drug treatment

THP-1 cells were seeded in 6 cm dishes at a density of 2 × 106, primed with 50 nM PMA for 6 h and refreshed with 5 mL culture medium. The cells were then treated with ANE (0.4 mg/mL for THP-1 cells and DOK cells; 0.1 µg/ml for PBMCs) or arecoline (0.1 mM and 0.3 mM) for 48 h. For functional studies, the CREB inhibitor 666–15 (80 nM, TOCRIS), the CREB inhibitor KG-501 (10 μM, MedchemExpress) [25, 26] which is a gift from lab of Prof. Long-Sen Chang [27], the VCAM-1 inhibitor δ-tocotrienol (20 µM, Cayman) was added to the culture media 30 min before adding ANE. Conditioned media were collected at 48 h after ANE treatment.

XTT cell viability assay

Cells were incubated for designated periods, and then cell viability/cell proliferation was analyzed using the XTT assay (Sigma‒Aldrich, St. Louis, MO). In brief, the media were replaced with 150 µl XTT solution (50 µg XTT and 0.4 µg phenazine methosulfate in 150 µl cell culture medium) and incubated at 37 °C for 2 h, followed by measuring OD at 470 nm with subtraction of the background at OD 660 nm.

Annexin V staining

To detect apoptotic cells after ANE or arecoline (FL-31593-250MG, Sigma‒Aldrich, St. Louis, MO) treatment, a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA) was applied. THP-1 cells were seeded on 6 cm plates at a density of 1 × 106 cells per plate, and cells were collected after ANE or arecoline treatment for 48 h. After washing three times with precooled 1 × PBS, the cells were stained with Annexin V working solution (5 µL of Annexin V-FITC reagent and 5 µL of propidium iodide (PI) solution in 100 µL of 1 × Annexin V binding buffer) for 15 min at room temperature. Cells were analyzed with a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA). The fluorescence signals of Annexin V-FITC and PI were evaluated by FL-1 and FL-3 channels, respectively. The data were analyzed with MFA32 software (Beckman Coulter, Brea, CA).

Flow cytometric assay of surface markers

THP-1 cells were primed with 50 nM PMA for 6 h, followed by ANE treatment for 48 h. After washing three times with flow cytometer buffer, the cells were fixed with 4% paraformaldehyde for 30 min and then stained with CD163 (ab182422, Abcam, Cambridge, UK), CD206 (bs4727R, Bioss, MA), or HLA-DR (ab175085, Abcam, Cambridge, UK) antibodies for 30 min. The fluorescence intensity was evaluated with a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA), and data were analyzed with FlowJo software version 10 (Tree Star Inc., San Carlos, CA).

Phospho-kinase array

A human phospho-kinase antibody array (ARY003C, R&D Systems) was applied to discover the potential signaling pathways mediating the effects of ANE in THP-1 cells. The specific sites of phosphorylation in 43 kinases were determined by using THP-1 cell lysates according to the manufacturer’s instructions.

Cytokine array

THP-1 cells were treated with ANE (0.4 mg/ml) for 48 h, and the conditioned media were applied to the cytokine array membranes (Proteome Profiler Human XL Cytokine Array Kit, ARY002B, R&D systems). The spot signals were developed according to the manufacturer’s instructions.

Cell secretome

To evaluate the cell secretome, THP-1 cells were treated with ANE for 48 h, and the conditioned media were filtered and frozen at -80 °C until further analysis. The concentration of the secretome was quantified using the appropriate DuoSet® Immunoassay Development kits (R&D systems) according to the manufacturer’s instructions.

Analysis of ANE components by LC/MS

ANE components were analyzed using a TSQ Quantum Ultra mass spectrometer (Thermo Scientific, Waltham, MA). A C18 column with 150 × 0.5 mm i.d., 5 μm (Agilent, Santa Clara, CA) was used to separate the components. The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B), with a flow rate of 15 μL/min. The injection volume was 3 µL with a column oven at 35 °C. The mass spectrometer with an electrospray ionization source was set in both negative and positive ionization modes. The capillary temperature was maintained at 250 °C, the source voltage and spray voltage were set at 3500 V, and the collision gas (Ar) pressure was set at 1.4 mTorr. The scan width was set at 0.4 Da with a scan time of 0.10 s.

Transwell migration assay

DOK cells were treated with the indicated conditioned medium (CM) for 48 h, and cells were harvested and seeded in 8 μM pore size transwell inserts at a density of 5 × 104 in 100 μL serum-free medium. The insert was carefully placed into a single well of a 24-well plate containing 500 μL serum-containing medium and incubated for 72 h. After incubation, the cells remaining on the upper chamber were removed using cotton swabs, while the cells that migrated into the lower chamber were stained with crystal violet solution for 15 min. The percentage of cell migration was calculated using ImageJ software.

Western blotting

Immunoblotting was performed as described previously [28]. SDS‒PAGE was performed, and proteins were transferred to PVDF membranes for further analysis. The chemiluminescence signal was captured by a Gel DocTM XR + Gel documentation system (Bio-Rad, Hercules, CA). The following primary antibodies were used: recombinant anti-human CD163 (ab182422, Abcam, Cambridge, UK), rabbit anti-human VCAM-1 (#13,662, Cell Signaling Technology, Massachusetts), rabbit anti-human p-CREB (GTX61045, Genetex, Irvine, CA), rabbit anti-human CREB (GTX112846, Genetex, Irvine, CA), rabbit anti-human EGFR (phosphor Tyr1086, GTX133599, Genetex, Irvine, CA), rabbit anti-human EGFR (GTX100448, Genetex, Irvine, CA), recombinant anti-human phospho-c-Jun (S63, MAB8930, R&D Systems, MN), mouse anti-human c-Jun (sc-166544, Santa Cruz, Texas), rabbit anti-human N-Cadherin (#4061, Cell Signaling Technology, Massachusetts), rabbit anti-human vimentin (GTX100619, Genetex, Irvine, CA), rabbit anti-human claudin-1 (#4933, Cell Signaling Technology, Massachusetts), rabbit anti-human ZO-1 (GTX108627, Genetex, Irvine, CA), mouse anti-human beta-actin (A5441, Sigma-Aldrich, St. Louis, MO). The secondary antibodies used in this study were goat anti-mouse IgG antibody (GTX213111-01, GeneTex, Irvine, CA), goat anti-rabbit IgG antibody (GTX213110-01, GeneTex, Irvine, CA), and rabbit anti-goat IgG antibody (GTX228416-01, GeneTex, Irvine, CA).

Co-immunoprecipitation (Co-IP)

A co-IP assay was performed with a Pierce Crosslink Magnetic IP/Co-IP kit (88,805, Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, 5 µg of primary antibodies were covalently cross-linked with 25 µl of protein A/G magnetic beads. Protein extraction was performed using Pierce IP lysis/wash buffer and protease and phosphatase inhibitor cocktail. A portion of each sample was used as input, while equal amounts (0.5 mg) of each protein extract were incubated with protein A/G magnetic beads cross-linked with primary antibodies overnight at 4 °C. The beads were then washed three times with 1 × modified coupling buffer to remove unbound proteins, and the bound proteins were then eluted from the antibody-crosslinking beads by a low-pH elution buffer. The elute was mixed with neutralization buffer to neutralize the low pH. Immunoprecipitates were analyzed using Western blotting. The primary antibodies used in this experiment included IGTα4 (8840, Cell Signaling Technology, Massachusetts) and ubiquitin (GTX19247, GeneTex, Irvine, CA).

Measurement of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the CLARIOstar system

The basal oxidative phosphorylation (determined by OCR) and glycolysis (determined by ECAR) in human monocytic THP-1 cells and oral premalignant DOK cells were measured using a CLARIOstar Plus plate reader (BMG LABTECH, Germany). To analyze OCR, the cells were seeded in 96-well culture plates (black wall with clear flat bottom) at a density of 8 × 104 cells/well in 200 µl media and cultured overnight. The media were refreshed, and after the addition of the extracellular O2 consumption reagent (ab197243, Abcam, Cambridge, UK), the wells were promptly sealed with prewarmed mineral oil. The extracellular O2 consumption signal was measured in the CLARIOstar Plus plate reader at 1.5 min intervals for 120 min at Ex/Em = 360/650 nm. To measure the ECAR rate, the cells were seeded in 96-well plates (black wall with clear flat bottom) at a density of 8 × 104 cells/well in 200 µl media and cultured overnight. After purging CO2 in a CO2-free incubator at 37 °C with 95% humidity for 3 h, the media were replaced with respiration buffer containing glycolysis assay reagent (ab197244, Abcam, Cambridge, UK). The glycolysis signal (lifetime signal) was measured in the CLARIOstar Plus plate reader at 1.5 min intervals for over 120 min at Ex/Em = 380/615 nm.

Analysis of mitochondrial function by Agilent Seahorse XF analyzer

To analyze the key parameters of functional mitochondria, an Agilent Seahorse XF analyzer (Agilent Technologies., Wilmington, DE) was applied following the detailed procedure provided by the manufacturer. There were four modulators (Seahorse XF Cell Mito Stress Test Kit) for studying the complexes of the electron transport chain (ETC) in mitochondria, namely, oligomycin, FCCP (carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone), rotenone and antimycin (all reagents from Sigma‒Aldrich), which were injected into the built-in injection ports of XF sensor cartridges. Oligomycin, an ATP synthase (ETC complex V) inhibitor, was injected first to slow electron flow through the ETC and then decrease mitochondrial respiration (OCR) and cellular ATP production. Then, FCCP, an uncoupling agent that provides an uninhibited electron flow via the ETC to consume oxygen, was injected. Finally, the complex I inhibitor rotenone and the complex III inhibitor antimycin A were injected.

Patient specimen collection

All normal oral mucosa, OPMD, and OSCC tissue specimens were collected from Kaohsiung Medical University Hospital (Kaohsiung, Taiwan) after approval by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (Approval no. KMUH-IRB-20130300, KMUHIRB-E(I)-20210343, KMUHIRB-E(I)-20190009 and KMUHIRB-F(I)-20220016, and KMUHIRB-F(I)-20180069).

Hamster buccal pouch model

The hamster buccal pouch model has been widely used in vivo to study oral lesions, including in our previous reports [29, 30]. There were two experimental groups in this study, including (1) double-distilled water (solvent for ANE) alone and (2) ANE. In brief, the buccal pouch region of hamsters was painted daily with ddH2O or ANE. At the indicated time points, the hamsters were sacrificed to collect buccal tissue, which was divided equally into 4 parts for further analysis.

Hematoxylin and eosin (H&E) staining

The tissue sections were immersed in hematoxylin for 5 min and then washed with double-distilled water for 15 min. After quickly dipping in 1% acid alcohol, the tissue sections were washed with double-distilled water for 15 min, immersed in 70% ethanol for 3 min, and then immersed in eosin for 1 min. Next, the tissue sections were washed with double-distilled water for 15 min and dehydrated by immersion in 95% alcohol twice followed by 100% twice, with 2 min for each step. The sections were immersed in xylene for 5 min twice and then mounted with Clearium Mounting Media (3,801,100, Leica, CA).

Immunohistochemistry

For immunohistochemistry (IHC), oral tissue slides were baked, dewaxed and stained with avidin–biotin complexes following our previous procedures [28,29,30]. Immunohistochemical staining was performed using a BOND-MAX IHC staining machine (Leica Microsystems). When the staining process was completed, the percentage of positively stained cells on the tissue slide was calculated as one of the following categories: 0 (0–4%), 1 (5–24%), 2 (25–49%), 3 (50–74%), and 4 (75–100%). The intensity of staining was scored as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The score was evaluated independently by two experts under the same imaging state.

Immunofluorescence (IF)

In addition to conventional immunohistochemistry for oral tissue sections with single staining, multifluorescence immunohistochemistry was carried out according to previously described procedures [31, 32], and the percentage of cells positively stained with multifluorescence was calculated as [(number of multifluorescent stained cells)/(number of all cells)] × 100% from five random fields.

Statistical analysis

All statistical analyses were performed using JMP version 14.0 for Windows (SAS Institute, Cary, NC, USA). The differences among groups were analyzed using either Student’s t test or ANOVA. For in vitro studies, data are presented as the mean ± SD from three independent experiments. The results were considered statistically significant if the p value was less than 0.05.