Cell culture

The human cell lines Huh7 and HepG2 were used and grown in RPMI 1640 medium (RPMI Gibco™; Thermo Fisher Scientific, Waltham, Massachusetts, USA), supplemented with 10% fetal bovine serum (FBS), in a humidified incubator with 5% CO2 at 37 °C. The human cell line Hep3B was grown in Minimum Essential Medium Eagle (Sigma-Aldrich, St. Louis, Missouri, USA), supplemented with 10% FBS, in a humidified incubator with 5% CO2 at 37 °C.

THLE-2 human normal liver epithelial cell line was purchased from the American Type Culture Collection (ATCC) and cultured in BEGM (Bronchial Epithelial Cell Growth Medium BulletKit™; Lonza, Basel, Switzerland). The kit includes 500 ml basal medium and separate frozen additives from which we discarded the gentamycin/amphotericin (GA) and epinephrine and from which we added an extra 5 ng/ml EGF, 70 ng/ml phosphoethanolamine and 10% FBS. The cell culture flasks used for the THLE-2 cell line were precoated with a mixture of 0.01 mg/ml fibronectin, 0.03 mg/ml bovine collagen type I, and 0.01 mg/ml bovine serum albumin dissolved in BEGM medium.

MTT

Cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay (Thermo Fisher Scientific, Waltham, Massachusetts, USA). According to the manufacturer’s instructions, cells were plated at a density of 8 × 104/well in 96‑well plates with 1.25 µM to 160 µM of tigecycline and cell viability was assessed at different time points. The absorbance of each well was measured at a wavelength of 570 nm with a background wavelength of 670 nm using a VersaMax microplate reader (Molecular devices Instruments, San Jose, California, USA). Empty wells served as blank controls. The cell viability was calculated as a comparative percentage of the values obtained from untreated cells. As an additional control we used the solvent DMSO, which was necessary for tigecycline, and we used constant amounts of DMSO with increasing concentrations of tigecycline to avoid confounding. The standard curves were obtained according to nonlinear regression in GraphPad Prism 7.04 software and then the half maximum inhibitory concentrations (IC50) of tigecycline on different cells were calculated.

Crystal violet assay

Cells were plated at a density of 8 × 104/well in 96‑well plates with 1.25 µM to 160 µM of tigecycline and cell viability was assessed at different time points. The cells were washed with phosphate buffered saline (PBS) to ensure that all medium was removed. 50 µl of 4% paraformaldehyde (PFA) were added and an incubation for 15 min at room temperature followed. PFA was discarded and plates were allowed to air dry for 15 to 20 min. Then, cells were stained with 50 µl of crystal violet (CV) solution for 15 min. After discarding the CV, the cells were washed with H2O and it was allowed to dry overnight at room temperature. The next day, the dye was dissolved in 50 µl of 33% acetic acid. The absorbance of the developed color was measured at a wavelength of 570 nm with a background wavelength of 670 nm using VersaMax microplate reader (Molecular devices Instruments, San Jose, California, USA). Empty wells served as blank controls. The cell viability was calculated as a comparative percentage to the values obtained from untreated cells. As an additional control we used the solvent DMSO, which was carrier solution for tigecycline.

Cell viability assay

Cell viability was assessed by quantifying the amount of ATP using the CellTiter-Glo 2.0 Cell Viability Assay (Promega, Walldorf, Germany) according to the manufacturer’s instructions. The cells were seeded into 6-well plates (Corning, New York, USA) for a final confluency of 85%, followed by treatment. After trypsinization, 100 µl of the cell suspension were transferred into black 96-well f-bottom plates (Greiner Bio-One, Frickenhausen, Germany). Subsequently, 100 µl of CellTiter-Glo reagent was added to each well and the cells were incubated at room temperature in the dark for 5 min. The luminescence signal was quantified with FilterMax F3 microplate reader (Molecular Devices Instruments, San Jose, California, USA) and evaluated through SoftMax Pro 7.1.2 GxP (Molecular Devices Instruments, San Jose, California, USA).

Sphere formation assay

The human cell lines Huh7 and HepG2 were cultured in suspension in sphere formation assay (SFA) medium which consisted of serum-free DMEM/F12 supplemented with B27 (1:50, Gibco™; Thermo Fisher Scientific, Waltham, Massachusetts, USA), 10 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) (ImmunoTools, Friesoythe, Germany) and 1% methylcellulose. To form HCC spheres, 1000 suspended cells were cultured per well in ultra-low attachment plates (Corning, New York, USA). After 8 days of treatment, the formed spheres were inspected under the microscope and pictures were taken with 40 × magnification. The number of clones and their size were then analyzed by ImageJ software (open-source software).

Wound healing assay

Huh7 and HepG2 cells were seeded in 6-well plates. When the cells grew in a full monolayer, a wound was produced by a straight scratch across the cell monolayer using a 200 μl sterile tip. The cells were then washed gently with PBS and new serum-free medium was added. Pictures were taken immediately (0 h) as well as after 24 and 48 h in the same location and with the same magnification. The area of each wound was analyzed at different time points using ImageJ software (open-source software). The reduction of the wound area was then calculated and interpreted as the cell migration ratio. The migration ratio was defined as the reduction of wound area under treatment related to the reduction of wound area without treatment.

Transwell assay

Matrigel (Matrigel GFR Basement Membrane Matrix, cat. no. 354230; Corning, New York, USA) and serum-free medium were dissolved at 4 °C at a ratio of 1:3, mixed thoroughly, and added to the upper chamber of an 8.0 µm pore size transwell plate (Corning, New York, USA). 40 µl were added to each chamber so that it fully covered the bottom of the chamber. Then it was incubated in a humidified incubator with 5% CO2 at 37 °C overnight to allow the matrigel to fully solidify. A total number of 100,000 cells in 300 µl serum-free medium were seeded into the upper chamber of the transwell plate and 600 µl complete medium was added to the lower compartment. The cells were incubated for 24 h in a humidified incubator with 5% CO2 at 37 °C. After incubation for 24 h, the upper chamber was wiped twice with cotton swabs. Next, the cells were fixed with 4% paraformaldehyde for 15 to 20 min and stained with 0.5% crystal violet for another 15 to 20 min at room temperature. The numbers of invading cells in three randomly selected fields were counted under an inverted light microscope (TE2000-U Inverted Microscope, 100 × magnification; Nikon, Tokyo, Japan).

Detection of reactive oxygen speciesTwo methods were applied to examine changes in Reactive Oxygen Species (ROS)

Evaluation via luminescence: The level of ROS was quantified by luminescence measurement using the Ros-GLO™ H2O2 Assay (Promega, Walldorf, Germany) according to the manufacturer’s instructions. The cells were seeded into white 96-well plates with clear flat-bottom (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for a final confluency of 85%, followed by treatment for 48 h. Five hours prior to measurement, 20 µl of freshly prepared H2O2 Substrate Solution diluted with the Substrate Dilution Buffer at a ratio of 1:80 was added into the wells containing 80 µl of corresponding media. This was followed by adding 100 µl ROS-Glo Detection Solution prepared with Recon Buffer and Luciferin, D-Cysteine 100x, and Signal Enhancer in a ratio of 100:1:1 and incubation at room temperature for 20 min. The luminescence signal was measured with the FilterMax™ F3 microplate reader (Molecular Devices Instruments, San Jose, California, USA) and evaluated through SoftMax® Pro 7.1.2 GxP (Molecular Devices Instruments, San Jose, California, USA).

Evaluation via flow cytometry: The cells were cultured in 6-well plates (Corning, New York, USA) and incubated for 48 h at 37 °C with a final confluency of 85%. This was followed by trypsinization and subsequent transfer into 5 ml round-bottom polystyrene test tubes (Corning, New York, USA) and centrifuged at 500 rpm for 5 min. The pellet was resuspended in DPBS, followed by repetition of the washing step, and the supernatant was discarded. Determination of ROS by flow cytometry was performed using the Total Reactive Oxygen Species (ROS) Assay Kit 520 nm (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. The cells were supplied with 500 µl of DPBS prior to measurement. The events were recorded in the FITC-A channel and gated using FlowJo v10 (BD Life Sciences, Ashland, California, USA).

Measurements of mitochondrial respiration

To assess mitochondrial respiratory function, oxygen consumption rates (OCR) were analyzed using the Seahorse XFp Analyzer (Agilent Technologies, Santa Clara, California, USA). 12,000 cells were seeded in Seahorse 8-well mini-plates per well and cultured at 37 ℃ with 5% CO2 and treated with 10 µM to 160 µM of tigecycline for 48 h. After 48 h of treatment, media was replaced with Seahorse assay medium, cells were incubated at 37 ℃ without CO2 for 1 h and then 2 µM oligomycin and 0.5 µM rotenone/antimycin A were added sequentially to assess mitochondrial respiratory capacity. For short-term treatment of Huh7, HepG2, and THLE-2, cells were treated with tigecycline in Seahorse 8-well mini-plates for only 6 h. After 6 h of treatment, the medium was replaced with a seahorse analysis medium and then the subsequent measurements were performed. Also, mitochondrial basal respiration, ATP production, and proton leak were measured according to the manufacturer’s protocol.

Measurement of mitochondrial mass

Cells were cultured as described before, followed by trypsinization, subsequent transfer into 5 ml round-bottom polystyrene test tubes (Falcon, Corning, Wiesbaden, Germany) and centrifuged at 500 rpm for 5 min. The pellet was resuspended in DPBS, followed by repetition of the washing step, and the supernatant was discarded. To measure mitochondrial mass, we used Nonyl-Acridine Orange (NAO) (Acridine Orange 10-Nonyl Bromide) (Invitrogen, Carlsbad, California, USA). The cell pellet was fixed by drop-wise addition of ice-cold 70% ethanol with simultaneous vortexing. Ethanol was removed through two wash steps with DPBS followed by staining with 100 nm NAO diluted in DPBS, with incubation at 4 °C for 10 min in the dark. The cells were supplied with 500 µl of DPBS prior to measurement (BD LSRFortessa, BD Biosciences, San Jose, CA, USA). The events were recorded in the FITC-A channel and gated using FlowJo v10 (BD Life Sciences, Ashland, California, USA).

Western blot analysis

Huh7, HepG2, and THLE-2 cells were washed twice with cold PBS and harvested with protein lysis buffer including protease and phosphatase inhibitors (Roche, Basel, Switzerland). The protein concentrations were assessed using a BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Equal amounts of proteins (25 μg/lane) from each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% and 13% gels (Bio‑Rad Laboratories, Hercules, California, USA) and transferred to polyvinylidene difluoride membranes (Merck Group, Darmstadt, Germany). After blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature, the membranes were incubated with specific primary antibodies at 4 °C overnight. Then, after washing three times with tris‑buffered saline containing Tween‑20 (TBST), the membranes were incubated with horseradish peroxidase‑conjugated secondary antibodies for 1 h at room temperature. Proteins were visualized by chemiluminescence using enhanced chemiluminescent substrate (Bio‑Rad Laboratories, Hercules, California, USA). Immunoreactive bands were examined using the ChemiDoc Imaging System (Bio‑Rad Laboratories, Hercules, California, USA). The following antibodies were used: Rabbit Rac1/Cdc42 antibody (cat. no. 4561, dilution 1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit p44/42 MAPK (Erk1/2) (cat. no. 4695, dilution 1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit Phospho-p44/42 MAPK (Erk1/2) (cat. no. 8544, dilution 1:1000; Cell Signaling Technology, Danvers, Massachusetts, USA), Mouse OXPHOS antibody (cat. no. ab110411, dilution 1:1500; Abcam, Cambridge, United Kingdom) and Rabbit GAPDH antibody (cat. no. sc-25778, dilution 1:5000; Santa Cruz Biotechnology, Texas, USA). GAPDH was used as an internal control for each membrane.

Flow cytometric analysis

Flow cytometry and BrdU Flow kits (BD Pharmingen, San Diego, California, USA) were used to analyze the cell cycle changes and live/dead cell populations of the three used cell lines Huh7, HepG2, and THLE-2 after tigecycline treatment. All cells were seeded in 6-well plates with different numbers of cells depending on the morphological size of the cells to obtain a confluence of 80% before treatment. For cell cycle analysis, culturing was then started at the same time for all groups. The control group was left untreated for the whole time of the experiment, the 24-h group was treated in the last 24 h and the 48-h group was treated in the last 48 h of the experiment. All samples were harvested at the same time. Therefore, 20 µl of BrdU solution (1 mM BrdU in 1xDPBS) were added directly to 2 ml of culture medium and the cells were incubated for 1 h. Then, the cells were trypsinized from the wells and transferred into FACS tubes. They were centrifuged for 5 min at 200 to 300 G and the supernatant was discarded. The cells were resuspended in 100 µl of BD Cytofix/Cytoperm buffer and incubated for 30 min at room temperature. After washing with 1 ml 1xBD Perm/Wash buffer, the cells were resuspended in 100 µl BD Cytoperm Permeabilization Buffer Plus and incubated for 10 min on ice. The measurements were performed by flow cytometry immediately after staining the cells with fluorescent anti-BrdU and 7-AAD solution according to the manufacturer’s instructions for the kit.

For live/dead cell staining, cells were trypsinized from the wells and transferred into FACS tubes. The cells were washed twice with 1xDPBS with centrifugation for 5 min at 200 to 300 G, and stained with 7-AAD for 10 min in the dark.

RNA isolation and real‑time PCR

The total RNA from Huh7, and HepG2 cells was extracted with the Qiagen total RNA extraction kit (Qiagen, Venlo, Netherlands) and reverse transcription was performed using the SuperScript™ IV VILO™ Master Mix kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The cDNA concentration was measured with NanoDrop (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The quantitative RT‑PCR was performed using QuantiNova™ SYBR Green PCR Kit (Qiagen, Venlo, Netherlands) in a 20 μl PCR mixture on a Bio‑Rad CFX96 Real‑Time PCR system (Bio‑Rad Laboratories, Hercules, California, USA) according to the manufacturer’s standard protocols. An initial step of 2 min at 95 °C was followed by 40 cycles of 5 s at 95 °C and 10 s at 60 °C. Each sample was performed in duplicate and negative control with sterile RNase-free H2O was. The housekeeping gene GAPDH was used to normalize the variation of cDNA. Three independent experiments were performed for each group. Relative gene expression was normalized to GAPDH and calculated using the 2‑ΔΔCq method.

RNA sequencing and analysis

Cells were seeded into 6-well plates (Corning, New York, USA), grown for 48 h in the presence and absence of 10 µM tigecycline and harvested at a confluency of 85%. RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). After RNA isolation, RNA integrity number (RIN) was measured using the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, California, USA). RNA with a RIN value > 7 was selected for mRNA sequencing (poly-A selected). The libraries were prepared using the Illumina stranded mRNAprep ligation kit (Illumina, San Diego, California, USA), following the manufacturer's instructions. After final quality control, the libraries were sequenced in a paired-end mode (2 × 100 bases) in the Novaseq6000 sequencer (Illumina, San Diego, California, USA) with a depth of ≥ 25 million reads per sample. The analysis of RNA sequencing data sets was conducted using the web-based platform, www.usegalaxy.eu.

Bioinformatic analysis

The molecular structure of tigecycline was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Information on a total of 6,876 HCC-related genes was retrieved from the GeneCards database using “hepatocellular carcinoma” as a keyword. According to a GeneCards Inferred Functionality Score (GIFtS) greater than 20, we selected the top 5.4% of genes which comprised a total of 376 genes. Similarly, a search in the Disgenet database (https://www.disgenet.org/) using “hepatocellular carcinoma” as a keyword comprised 96 relevant genes for HCC. Excluding the 43 genes contained in both databases, we obtained a total of 429 potential genes related to HCC. Potential target proteins for tigecycline and their corresponding genes were obtained from the PharmMapper database (http://www.lilab-ecust.cn/pharmmapper/) and the Comparative Toxicogenomics Database (CTD, http://ctdbase.org/). Hereby, we got a total of 34 potential target genes. These 34 potential target genes and the 429 relevant genes were intersected and we got 11 genes that are both potential targets of tigecycline and related to HCC. Furthermore, survival data for the 11 potential genes was retrieved from the Kaplan–Meier plotter (https://kmplot.com/analysis/) and data for differential gene expression were obtained from GEPIA (http://gepia.cancer-pku.cn/) and The Human Protein Atlas (https://www.proteinatlas.org/). STRING 11.5 was used to analyze the correlation of the 11 genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed to determine the pathways significantly associated with the 11 potential target genes. Functional enrichment analysis of the 11 potential target genes was performed by the R programming language.

Statistical analysis

All experiments were independently performed at least three times. The mean standard deviation (SD) was determined for each group. Statistical analyses were performed using one/two‑way analysis of variance (ANOVA) for multiple group comparisons or student's t‑test for individual comparisons. Statistical significance was considered at p < 0.05. In all statistical graphs, bar graphs represent the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, and ns means no significance compared with the control group.