Reagents

Phenformin (Sigma, Santa Fe, NM, USA) and metformin (Sigma) stock solutions were prepared with phosphate buffered saline (PBS) for in vitro and in vivo studies.29 Chloroquine (CQ), MHY1485, ISRIB, and GSK2606414 were obtained from MedChemExpress (Monmouth Junction, NJ, USA) and were diluted with DMSO for in vitro studies.

In vitro cell culture

All OSCC cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). The OSCC cell lines SCC-4, SCC-25, and CAL 27 were maintained in Dulbecco’s Modified Eagle Medium basic (DMEM basic) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Beit Haemek, Israel), penicillin (100 IU/mL), and streptomycin (100 µg/mL) (Gibco). SCC-9 cells were cultured in Dulbecco’s Modified Eagle Medium: F-12 (DMEM/F-12) containing 10% FBS, 400 ng/mL hydrocortisone, penicillin (100 IU/mL), and streptomycin (100 µg/mL). All cells were cultured in a cell incubator at 37 °C in a humidified 5% CO2 atmosphere.

Normal gingival epithelial cells isolation and culture

The procedure for isolating and culturing normal gingival epithelial cells (oral keratinocytes) basically followed our previous publication.69 K-SFM (10744019, Gibco, Grand Island, NY) containing penicillin (100 IU/mL) and streptomycin (100 µg/mL) was used to culture gingival epithelial cells, and passage 3 cells were used for the experiments.

Cell counting kit-8 (CCK-8) assay

CCK-8 assays were used to analyze the numbers of viable OSCC cells or gingival epithelial cells treated with phenformin or metformin following a previously published protocol.70 Cells were seeded in 96-well plates at 5 000 cells per well and were grown overnight. Cells were cultured with the desired concentrations of phenformin or metformin as shown in the figures. At the assay time points shown in the figures, the medium was replaced with DMEM basic containing 10% CCK-8 working solution (Biosharp, Guangzhou, China). At 2 h, a multi-functional enzyme marking instrument (BMGLABTECH, Offenburg, Germany) was used to measure OD values at 450 nm.

Ethynyl-2′-deoxyuridine (EdU) staining assay

An EdU Cell Proliferation Image Kit (Abbkine, Wuhan, China) was used for EdU staining assays following the manufacturer’s instructions. SCC-9 cells and CAL 27 cells were seeded on sterile round coverslips in 96-well plates with 5 000 cells per well. The next day, OSCC cells were treated with 1 mmol/L phenformin for 48 h and were then incubated with EdU solution for 2 h in a cell incubator. After removing the medium, 0.1 mL 4% formaldehyde was added to each well and incubated for 15 min at room temperature. The cells were then washed with BSA washing solution three times and treated with PBS with 0.5% Triton X-100 for 20 min, followed by washing with BSA washing solution and staining with Click-iT reaction mixture in the dark for 30 min. After washing the cells with BSA washing solution, one drop of DAPI dye was added to each well, after which the cells were observed using a fluorescence microscope (Olympus BX53-DP80, Tokyo, Japan).

Western blot analysis

Cells were washed with ice-cold PBS and lysed with radioimmunoprecipitation assay buffer (Solarbio, Beijing, China) containing 1% phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China) and a 1% phosphatase inhibitor cocktail (Selleckchem, Houston, TX, USA) for 5 min on ice. The extracted proteins were centrifuged at 12 000 r/min for 10 min at 4 °C, followed by collecting the supernatants. The concentration of protein in each sample was measured using a BCA Protein Assay Kit (Solarbio). The supernatants were then mixed with 5 × SDS–PAGE loading buffer (Beyotime) and denatured at 100 °C for 5 min. Equal amounts of protein samples were electrophoresed in 10% or 12% sodium dodecyl sulfate-polyacrylamide gels (Beyotime) and were then electrotransferred to 0.45 µm polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were then incubated with 5% BSA blocking buffer (Solarbio) for 1 h and a primary antibody at 4 °C overnight, with GAPDH used as an intrinsic reference protein. The next day, each membrane was washed with Tris-based saline-Tween-20 (TBST) buffer (Servicebio, Wuhan, China) three times for 10 min each and incubated with corresponding secondary antibodies for 1 h at room temperature, followed by washing with TBST buffer three times again. The protein bands were visualized with Chemiluminescent HRP Substrate (Merck Millipore) and observed using a Multi-functional Imaging System (Shenhua, Hangzhou, China).

The following primary and secondary antibodies were used: PARP Antibody (Cell Signaling Technology (CST), Cat.#9542, Boston, MA, USA), Caspase-3 Antibody (CST, Cat.#9662), Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (CST, Cat.#9664), Atg7 (D12B11) Rabbit mAb (CST, Cat.#8558), SQSTM1/p62 (D1Q5S) Rabbit mAb (CST, Cat.#39749), Beclin-1 (D40C5) Rabbit mAb (CST, Cat.#3495), LC3A/B Antibody (CST, Cat.#4108), Lamp1 antibody (CST, Cat.#9091), Recombinant Anti-REDD-1/DDIT4 antibody (Abcam, Cat.ab191871, Cambridge, UK), FAM129A Polyclonal antibody (Proteintech, Cat.No.21333-1-AP, Chicago, IL, USA), mTOR (7C10) Rabbit mAb (CST, Cat.#2983), Phospho-mTOR (Ser2448) (D9C2) XP Rabbit mAb (CST, Cat.#5536), p70 S6 Kinase (49D7) Rabbit mAb (CST, Cat.#2708), Phospho-p70 S6 Kinase (Thr389) Antibody (CST, Cat.#9205), 4E-BP1 (53H11) Rabbit mAb (CST, Cat.#9644), Phospho-4E-BP1 (Thr70) Antibody (CST, Cat.#9455), AMPKɑ (D5A2) Rabbit mAb (CST, Cat.#5831), Phospho-AMPKɑ (Thr172) (40H9) Rabbit mAb (CST, Cat.#2535), Atg5 Antibody (CST, Cat.#2630), ATF-3 (E9J4N) Rabbit mAb (CST, Cat.#18665), ATF-4 (D4B8) Rabbit mAb (CST, Cat.#11815), BiP (C50B12) Rabbit mAb (CST, Cat.#3177), CHOP (L63F7) Mouse mAb (CST, Cat.#2895), PERK (D11A8) Rabbit mAb (CST, Cat.#5683), Phospho-PERK/EIF2AK3 (Ser719) Polyclonal antibody (Proteintech, Cat.No.29546-1-AP), eIF2α (D7D3) XP® Rabbit mAb (CST, Cat.#5324), Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (CST, Cat.#3398), GAPDH (D16H11) XP Rabbit mAb (CST, Cat.#5174), Goat anti-Rabbit IgG HRP (HUABIO, Cat.HA1001, Hangzhou, China) and Goat anti-Mouse IgG Antibody (H&L)[HRP] (GenScript, Cat.No.A00160, Piscataway, NJ, USA).

Virus infection

AdV5-CMV-GFP-LC3 and GFP-RFP-LC3 were purchased from WZ Biosciences (Jinan, China) and viral infections were performed with the protocol described previously.62 In total, 2 × 105 CAL 27 cells per well were plated in 6-well plates. The next day, the desired amount of virus (MOI = 100) was added to the growth medium without antibiotics. At 12 h, the cells were washed with PBS two times and maintained in growth medium for 24 h. The old medium was then replaced with serum-free medium or growth medium with or without phenformin according to the grouping. After treatment for 6 h, images were observed using a fluorescence microscope (Olympus BX53-DP80).

RNA extraction and real-time quantitative reverse transcription PCR (qRT-PCR) assay

A SteadyPure Universal RNA Extraction Kit (Accurate Biology, Changsha, China) was used to extract the total RNAs of OSCC cells. The concentration of each total RNA was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). In total, 1 000 ng of each total RNA was reverse transcribed to complementary DNA using an Evo M-MLV RT Kit for qRCR (Accurate Biology). A SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology) was used to perform PCR reactions with a LightCycler96 Instrument (Roche Diagnostics, Basel, Switzerland) following the manufacturer’s instructions and the PCR program was carried out at 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and at 60 °C for 30 s and ended with an elongation step for 15 s at 72 °C. Relative mRNA expression levels were normalized to the mRNA expression level of GAPDH. The primers of all genes tested are shown in Table S1.

Flow cytometry (FACS) analysis

The effect of phenformin on the apoptosis of OSCC cells was measured using an Annexin V, FITC Apoptosis Detection Kit (Dojindo, Kumamoto Prefecture, Japan) as described previously.71 Similarly, OSCC cells were plated in 6-well plates with 2 × 105 cells per well. After incubation with phenformin for 24 h, those cells were collected and resuspended in ice-cold PBS. Apoptosis of the control group (Con) cells was induced by repeated freeze (−20 °C for 20 min)-thawing (37 °C for 3 min) three times. After centrifugation, 1 × 105 cells from each group were resuspended in 500 μL 1 × binding buffer containing 5 μL Annexin V-FITC conjugate and/or 5 μL PI solution according to the grouping and was incubated in the dark for 15 min on ice. Apoptosis rates were analyzed using a flow cytometer (FACS Calibur, BD Biosciences, Franklin Lakes, NJ, USA).

In vivo tumor xenograft assay

Eight-week-old female nude/nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co. (Beijing, China) and were randomly divided into two groups of three mice each: a control group (Con, PBS) and an experimental group (Phen, 150 mg/kg/day phenformin). Procedures of tumor xenografts were as described previously.72 In brief, 1 × 106 OSCC cells suspended in 100 μL DMEM-opti (Gibco) were injected subcutaneously into four sites in the dorsal skin of each nude mouse. At 1 week, the nude mice in the Phen group were fed with phenformin dissolved in PBS twice a day for 2 weeks, while mice in the control group were fed with an equal amount of PBS as a control. At 2 weeks, all mice were euthanized and weighed, and their tumors were collected, weighed, and photographed for analysis.

Immunohistochemical (IHC) staining and immunofluorescence (IF) staining

IHC and IF staining were performed as previously described.62 The tumor tissues were cut into 5-micron sections. Sections on slides were deparaffinized by immersion in dewaxing solution, anhydrous ethanol, and different concentrations of alcohol, in that order and were subsequently immersed in EDTA antigen repair solution (Solarbio) for antigen repair. After blocking endogenous peroxidase in the sections using a 3% hydrogen peroxide solution, 3% BSA (Solarbio) was added to cover the tissue sections evenly at room temperature. The blocking solution was gently shaken off and the sections were incubated with each primary antibody in a humidified box at 4 °C overnight. After washing three times with PBS for 5 min, the sections were incubated with the corresponding secondary antibody and washed three times with PBS again. For IHC staining, freshly prepared DAB solution was added dropwise to the sections and hematoxylin was used to stain cell nuclei, followed by dehydration. For IF staining, sections were incubated with DAPI for 5 min in the dark to stain nuclei. An immunofluorescence microscope (Olympus BX53-DP80) was used for analysis of staining.

The following primary and secondary antibodies were used: Anti-Ki67 antibody (Abcam, Cat.ab16667), Anti-LC3B antibody (Abcam, Cat. ab232940), SQSTM1/p62 (D6M5X) Rabbit mAb (CST, Cat.#23214), Anti-Beclin 1 antibody (Abcam, Cat.ab62557), Recombinant Anti-REDD-1/DDIT4 antibody (Abcam, Cat.ab191871), FAM129A Polyclonal antibody (Proteintech, Cat.No.21333-1-AP), and DyLight 594 goat anti-rabbit IgG (H + L) (Multisciences, Cat.GAR007, Hangzhou, China).

RNA-seq analysis

Total RNAs from CAL 27 and SCC-9 OSCC cells treated with phenformin or PBS (Control) for 12 h were collected and purified using a SteadyPure Universal RNA Extraction Kit. Purified RNAs were sent to Beijing Genomics Institute (BGI, Beijing, China) for RNA sequencing and further analysis. DEGs were screened with | log2 Fold Change | > 1.5 and q value < 0.05. Volcano plots, KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Network Diagrams and heat maps were plotted based on DEGs.

siRNA transfection

siRNA transfections were performed as described previously.72 Briefly, CAL 27 cells were plated in 6-well plates at 3 × 105 cells per well. The next day, those CAL 27 cells were transfected with the specific siRNA or the scramble siRNA (Control) diluted in DMEM-opti containing Lipofectamine 3000 (Thermo Fisher Scientific). After 24 h, the transfected cells were treated with phenformin or with PBS (Control) for the desired times as indicated in the figures. The cells were then collected for qRT-PCR or Western blot analysis. The oligo sequences of siRNAs are listed in Table S2.

Statistical analyses

All experiments were replicated three times. Data summarization included the mean ± standard deviation of at least three scoring results and statistical analysis was performed using GraphPad Prism 7. Student’s t test was used to compare two groups, one-way or two-way analysis of variance (ANOVA) was used to compare three or more groups. “*” indicates a significant difference between groups.