HepG2 and HuH7 cells were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany). Hep3B cells were purchased from DSMZ (Braunschweig, Germany). HepG2 cells were grown and subcultured in Dulbecco's modified Eagle's medium:Nutrient Mixture F-12 medium (Thermo Fisher Scientific GmbH, Dreieich, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific GmbH), 20 mM Hepes-buffer and gentamicin (0.5 ml/l). HuH7 and Hep3B cells were incubated in RPMI-1640 medium, supplemented with 10% FBS, 20 mM HEPES-buffer, 1% glutamax, and 1% penicillin/streptomycin (all Gibco; Thermo Fisher Scientific GmbH) at 37°C in a humidified incubator containing 5% CO₂.

Everolimus (Novartis Pharma AG, Basel, Switzerland) and temsirolimus (LC Laboratories, Woburn, MA, USA) were dissolved in dimethyl sulfoxide (DMSO) as a 10 mM stock solution and stored in aliquots at −20°C. Prior to use, the compounds were diluted in cell culture medium. The effects of 0.1–100 nM everolimus or temsirolimus were determined on cell growth to evaluate dose dependency. All further experiments were conducted with 1 nM everolimus or temsirolimus. Cells treated with culture medium alone (supplemented with DMSO, diluted 1:10⁵-1:10⁸) served as controls. To evaluate the toxic effects of the drugs, following a 72 h incubation at 37°C in a humidified incubator containing 5% CO₂, cell viability was determined by trypan blue staining (Gibco; Thermo Fisher Scientific GmbH). A Zeiss ID 03 light microscope (Zeiss AG, Oberkochen, Germany) was used.

Cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics GmbH, Penzberg, Germany). HepG2, Hep3B and HuH6 cells, (50 µl, 1×10⁵ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48 and 72 h incubation at 37°C MTT (0.5 mg/ml) was added for an additional 4 h. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were incubated overnight at 37°C in an atmosphere containing 5% CO₂. Absorbance was measured at 550 nm using a microplate ELISA reader. Each experiment was conducted in triplicate. After subtracting background absorbance, results were expressed as percentage difference, related to a control set to 100%.

HepG2, HuH7, or Hep3B cells, treated with 1 nM everolimus or temsirolimus, were transferred to 6-well plates at 2,000 cells/well. Following 10 days of incubation, without changing the cell culture medium (everolimus and temsirolimus remained in culture), colonies were fixed with 1% glutaraldehyde for 10 min at room temperature and counted. Colonies containing ≥50 cells were counted using a Zeiss ID 03 light microscope (Zeiss AG). Non-treated cells served as controls.

Cell cycle analysis was performed once the tumor cell cultures had grown to sub-confluency, and after 24 h of drug treatment. Tumor cell populations were stained with propidium iodide using a Cycleyest Plus DNA Reagent kit (BD Biosciences, Heidelberg, Germany) and were then subjected to flow cytometry using a FACScan flow cytometer (BD Biosciences). A total of 10,000 events were collected for each sample. Data acquisition was conducted using CellQuest version 6.0 software (BD Biosciences) and cell cycle distribution was calculated using ModFit version 3.0 software (BD Biosciences). The number of gated cells in the G₁-, S-, or G₂/M-phases was expressed as a percentage of the total cell population.

To investigate the expression levels of cell cycle-regulating proteins, tumor cell lysates (50 µg; protein concentration was quantified by Bradford assay) were loaded onto a 7% polyacrylamide gel and were electrophoresed for 90 min at 100 V. The lysis buffer consisted of Tris-NaCl, 10% Tergitol, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mM NaF, 2 mM Na₃VO₄ and 2 mM PMSF. Protein was then transferred to nitrocellulose membranes for 1 h at 100 V. After blocking with non-fat dry milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with monoclonal antibodies directed against the following cell cycle-associated proteins: Phosphorylated (p)-Akt [clone 104A282, mouse immunoglobulin (Ig)G1; dilution 1:500; cat. no. 550747; BD Biosciences], p-mTOR (clone D9C2; IgG, Ser2448; dilution 1:1,000; cat. no. 5536S) and p-Raptor (IgG, Ser792; dilution 1:1,000; cat. no. 2083S; all Cell Signaling Technology Europe, B.V., Leiden, The Netherlands). Horseradish peroxidase-conjugated goat-anti-mouse IgG (dilution, 1:5,000; cat. no. 161-0380; Bio-Rad Laboratories, Inc., Hercules, CA, USA) served as the secondary antibody (30 min incubation at room temperature). The membranes were briefly incubated at room temperature with an enhanced chemiluminescence (ECL) detection reagent (ECL™; Merck KGaA, Darmstadt, Germany; cat. no. WBKLS0100) to visualize the proteins and were then analyzed using the Fusion FX7 system (Peqlab Biotechnologie GmbH, Erlangen, Germany). β-actin (1:1,000; cat. no. A5441; Sigma-Aldrich; Merck KGaA) served as an internal control.

For the tumor cell adhesion assay, 6-well plates were coated with collagen G (extracted from calfskin, consisting of 90% collagen type I and 10% collagen type III; diluted to 400 µg/ml in PBS; Thermo Fisher Scientific GmbH) overnight. Plastic dishes served as the background control. Plates were washed with 1% bovine serum albumin (BSA; Sigma Aldrich; Merck KGaA) in PBS to block nonspecific cell adhesion. HCC tumor cells (0.5×10⁶) were then added to each well for 60 min. Subsequently, non-adherent tumor cells were washed off, and the remaining adherent cells were fixed for 10 min at room temperature with 1% glutaraldehyde and counted under a Zeiss ID 03 light microscope (Zeiss AG). Mean cellular adhesion, defined as adherent cells_{coated well}-adherent cells_background, was calculated from five different observation fields (5×0.25 mm²).

Serum-induced chemotaxis was investigated using 6-well Transwell chambers (Greiner Bio-One GmbH, Frickenhausen, Germany) with 8-µm pores. HCC tumor cells (0.5×10⁶/ml) were placed in the upper chamber in serum-free medium. The lower chamber contained complete cell culture medium including 10% serum (Sigma Aldrich; Merck KGaA). After 20 h at 37°C, the upper surface of the Transwell membrane was gently wiped with a cotton swab to remove cells that had not migrated. Cells that had moved to the lower surface of the membrane were stained using hematoxylin and counted under a light microscope (Zeiss ID 03; Zeiss AG). Mean chemotaxis was calculated from five different observation fields (5×0.25 mm²).

HepG2, HuH7 or Hep3B cells were detached from their culture flasks by Accutase (PAA Laboratories; GE Healthcare) and washed in blocking solution (PBS, 0.5% BSA). Cells were then incubated for 60 min at 4°C with phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) directed against the following integrin subtypes: Anti-α1 (mouse IgG1, clone SR84; cat. no. 742362), anti-α2 (mouse IgG2a, clone 12 F1; cat. no. 555669), anti-α3 (mouse IgG1, clone C3 II.1; cat. no. 746157), anti-α4 (mouse IgG1, clone 9F10; cat. no. 658332), anti-α5 (mouse IgG1, clone IIA1; cat. no. 555615), anti-α6 (rat IgG2a, clone GoH3; cat. no. 555734), anti-β1 (mouse IgG1, clone MAR4; cat. no. 557332), anti-β3 (mouse IgG1, clone VI-PL2; cat. no. 555752), and anti-β4 (rat IgG2a; clone 439-9B; cat. no. 555720; all 20 µl/test; using the manufacturing dilution; all BD Biosciences). Tumor cell integrin expression was then measured using a FACScan [BD Biosciences; FL-2H (log) channel histogram analysis; 1×10⁴ cells per scan] and was expressed as mean fluorescence units. A mouse IgG1-PE (MOPC-21) or IgG2a-PE (G155-178) (BD Biosciences) was used as an isotype control.

To determine whether integrin α1 impacts tumor growth, HepG2 cells were incubated for 60 min at 37°C with 10 µg/ml function-blocking anti-integrin α1 (clone FB12) mAb (Merck KGaA; cat. no. MAB1973Z). Controls were incubated with cell culture medium alone. Subsequently, tumor cell growth was analyzed using the MTT assay, as aforementioned. To evaluate whether integrin α1 acts on tumor cell motility, adhesion and migration experiments were conducted using HepG2 cells following integrin α1 suppression. To evaluate tumor cell binding to matrix proteins, 24-well plates coated with fibronectin (BD Biosciences) were used. Plastic dishes served as the background control. Plates were washed with 1% BSA in PBS to block nonspecific cell adhesion. Subsequently, 0.5×10⁶ tumor cells were added to each well and incubated for 60 min at 37°C. Non-adherent tumor cells were washed off and the remaining adherent cells were fixed with 1% glutaraldehyde for 10 min at room temperature and counted by a light microscope (Zeiss ID 03; Zeiss AG). The mean cellular adhesion rate, defined as adherent cells_{coated well}-adherent cells_background, was calculated from five different observation fields. To evaluate tumor cell migration, serum-induced chemotaxis was examined using 6-well Transwell chambers (Greiner Bio-One GmbH) with 8-µm pores preformed as described above (Tumor cell chemotaxis subsection), however, only 0.5×10⁶ HepG2 cells were applied.

Statistical analysis was performed using BiAS software version 11.06 (http://www.bias-online.de/). All experiments were performed between three and six times. Data are presented as the mean ± standard deviation. Statistical significance between groups was determined using the Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.

Ascending concentrations of everolimus or temsirolimus induced a dose-dependent significant reduction in the number of HCC cells. Everolimus exerted a growth inhibitory effect at 0.1 nM in Hep3B and HuH7 cells, where temsirolimus exerted an inhibitory effect at 1 nM in HepG2 and HuH7 cells; the most obvious effect was apparent at 100 nM compared with the untreated controls (Fig. 1). No signs of toxicity were apparent, as determined by a trypan blue exclusion assay (data not shown).

Clonogenic growth was significantly reduced when all three tumor cell lines were treated with 1 nM everolimus or temsirolimus (Fig. 2A-C). In addition, everolimus (1 nM) and temsirolimus (1 nM) modulated cell cycle progression. The number of HepG2 cells in the G₂/M- and S-phases was reduced, whereas the number of tumor cells in the G₀/G1-phase was increased compared with the controls (Fig. 2D).

Since everolimus and temsirolimus target the mTOR signaling pathway, the Akt-mTOR axis was also evaluated. p-mTOR and p-Raptor expression was suppressed following treatment of all cell lines with both compounds (Fig. 2E). However, as total protein content was not detected in the present study, the activation status of the proteins cannot be confirmed. In addition, p-Akt expression was reduced in the HepG2 and Hep3B cell lines, but was undetectable in HuH7 cells.

The integrin subtypes α1, α2, α6 and β1 were strongly expressed on HepG2 cells, whereas α3, α4, α5, β3 and β4 were not detected (Fig. 3A). Integrin expression was also detected on Hep3B and HuH7 cells; it was demonstrated that α1, α2, α6 and β1 were expressed on these cell lines (Fig. 3B). Treatment of HepG2 cells with everolimus or temsirolimus significantly elevated the expression of integrin α1 on the tumor cell surface, without acting on α2, α6 or β1 expression (Fig. 3C).

Treatment with everolimus or temsirolimus (1 nM) significantly downregulated adhesion of HepG2, HuH7 and Hep3B cells to immobilized collagen (Fig. 4A). The most prominent effects were evoked in HepG2 and Hep3B cells. Control values were 61.4±11.2 cells/mm² (HepG2), 66.8±14.2 cells/mm² (HuH7) and 79.4±14.8 cells/mm² (Hep3B). A chemotaxis assay revealed that HepG2 and Hep3B motility was enhanced following exposure to temsirolimus or everolimus exposure (Fig. 4B). However, the control values were low (HepG2: 16.2±4.2 cells/mm², Hep3B: 6.8±3.5 cells/mm²). HuH7 cells did not migrate through the filter membrane.

Since everolimus and temsirolimus induced a distinct upregulation in integrin α1 on HepG2 cells, subsequent experiments aimed to determine the functional relevance of this receptor. Suppression of integrin α1 slightly decreased HepG2 cell growth; however, this was not significant (Fig. 4C). The strongest effects were observed with respect to tumor cell adhesion, which was reduced by >40% following integrin α1 suppression (Fig. 4D). Chemotactic activity was also significantly decreased in the presence of anti-integrin α1 compared with the control group (30% decrease; Fig. 4D).