The human HCC cell line SMMC-7721 was purchased from the Soochow University Cell Banks (Suzhou, China). Lenalidomide (Natco Pharma Limited, Hyderabad, India) and thalidomide (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck Millipore) to prepare 10 and 40 mM stock solutions. Cells were stained with Annexin V-Fluorescein Isothiocyanate (FITC) following the manufacturer's instructions (Annexin V-FITC Apoptosis kit; Beijing BLKW Biotechnology Co., Ltd., Beijing, China) and analyzed for apoptosis by FACS using CellQuest software version 7.0 (BD Bioscience, Franklin Lakes, NJ, USA). Flag-tagged caspase-3 was purchased Bio-Box Biotech (Beijing, China). VEGF enzyme-linked immunosorbent assay (ELISA) analysis was performed with a commercial VEGF ELISA kit (cat. no. EH010-56; BLKW Biotechnology, Co., Ltd.) following the manufacturer's protocol. The antibodies for caspase-3 (cat. no. 9664P) and VEGF (cat. no. 2478S) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH antibody (cat. no. AB22131) was obtained from Bioworld Technology, Inc. (St. Louis Park, MN, USA) The Cell Counting Kit 8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).

SMMC-7721 cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (10%; Gibco; Thermo Fisher Scientific, Inc.). The SMMC-7721 human HCC cell line was maintained at 37°C in a humidified atmosphere with 5% CO2.

CCK-8 assay was used to evaluate the relative cell viability. Briefly, cells were plated in 96-well plates at a density of 5,000 cells/well in the media. The cells were pretreated with compounds at 6.25, 12.5, 25, 50, 100 and 200 µg/ml in a final concentration of 0.25% DMSO in triplicate at 37°C in a humidfied incubator at 5% CO2 for 48 h. Subsequently, CCK-8 reagent (100 µl/ml medium) was applied and incubated with cells at 37°C, 5% CO2 for 1 h. Cells were then incubated for an additional 4 h and the optical density (OD) was measured at 450 nm using a VersaMax Microtiter Plate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA). Relative cell viability was calculated with the following formula: Relative cell viability (%) = OD(treatment group)/OD(control group) × 100%. The experiment was performed in triplicate.

Cells (5×105) were treated with 100 µg/ml or 200 µg/ml drugs (lenalidomide and thalidomide) for 48 h then washed once with Annexin-V wash buffer (BD Biosciences). Cells were incubated with Annexin-V binding protein (5 µl) and propidium iodide (PI; 10 µl) (BD Biosciences) for 10 min. Cells were diluted with 500 µl wash buffer and analyzed by a FACSCalibur flow cytometer using CellQuest software. Furthermore, cells (5×105) were treated with the indicated treatments for 48 h, the supernatant was removed, 1 ml 70% cold ethanol was added along the six hole plate edge, then cells were fixed for 15 min. The cells were then washed with cold phosphate-buffered saline and PI (0.5 ml) was added prior to observation under the microscope.

Cells (5×105) were treated with 100 µg/ml or 200 µg/ml of the drugs (lenalidomide and thalidomide) for 48 h. Lysates (50 µl/2×106 cells) were added, then the cells were reprecipitated, placed in an ice bath for 30 min, during which they were oscillated 3–4 times (10 sec each time), then the crude cytosol was obtained as the supernatant as a result of centrifugation at 6,140 × g for 20 min at 4°C. Cell lysates (50 µl) were then extracted and mixed with Ac-DEVD-pNA (Sigma-Aldrich; Merck Millipore), incubated for 4 h at 37°C, and then the OD was measured using a microplate reader. Caspase-3 activation was determined by the rate of OD induced and OD control. All experiments were performed in triplicates.

Cells (5×105) were treated with lenalidomide and thalidomide at the indicated doses for 48 h. ELISA analysis was performed as previously described (13). VEGF ELISA was performed using 200 µl culture supernatant in duplicates using the Quantikine VEGF ELISA kit (BLKW Biotechnology, Co., Ltd.) according to the manufacturer's instructions. Briefly, 200 µl culture supernatants was added to the wells and they were incubated for 2 h at 37°C. The plate was washed with 400 µl wash buffer twice and 200 µl VEGF conjugate was added followed by incubation for 2 h. Subsequent to the addition of substrate and stop solution, the optical density was determined using a microplate reader (RT-21000; Rayto Life and Analytical Sciences Co., Ltd., Shenzhen, China) at 450 nm with wavelength correction of 570 nm.

To determine the level of VEGF proteins, lenalidomide and thalidomide-treated cell lysates were prepared as described. A total of 20 µg proteins was analyzed by western blot analysis. The PVDF membranes with the transferred proteins were incubated with primary antibodies (cleaved caspase-3, 1:500; VEGF, 1:1,000; GAPDH, 1:10,000) at 4°C overnight and horseradish peroxidase-conjugated secondary antibodies (1:10,000; cat. no. 7074P2; Cell Signaling Technology, Inc.) at room temperature for 2 h. The signal was developed by the enhanced chemiluminescence reagent (EMD Millipore, Billerica, MA, USA) and visualized by FluorChem FC2 Imaging System (Alpha Innotech, San Leandro, CA, USA).

Data are expressed as the mean ± standard deviation. Data analysis was performed using the SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). Difference between groups were assessed with Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The anti-proliferative rate was detected by CCK-8, results are expressed as the mean ± standard deviation. Treatment of cells with lenalidomide and thalidomide in different concentrations for 48 h led to a dose-dependent induction of the inhibition of cell proliferation. The anti-proliferative effects of lenalidomide were identified to be more potent than that of thalidomide in the 25, 50, 100 and 200 µg/ml groups (P<0.01; Fig. 1A and Table I).

Cells (5×105) were treated with lenalidomide and thalidomide at the indicated doses for 48 h. Typical apoptotic morphological alterations were observed using fluorescence microscopy, and included cell nucleus shrinkage, chromatin condensation and the appearance of apoptotic bodies when treated with different doses of the drugs tested (Fig. 2). Lenalidomide was observed to exhibit an increased effect of inducing cell apoptosis than thalidomide at the same concentration (Fig. 2). Caspase-3 activity of samples was detected using a microplate reader, and the OD was analyzed at 405 nm. Activity of caspase-3 was upregulated with increased lenalidomide and thalidomide concentrations. Caspase-3 activity in the lenalidomide groups was greater than in the thalidomide groups at the same concentrations, and this difference was significant (16.69±1.54 vs. 13.37±1.59; 24.31±2.24 vs. 20.75±1.75; P<0.01, P<0.05; Fig. 1B, Table II). Hepatoma SMMC-7721 cell apoptosis was examined using the Annexin-V staining-based FACS assay following lenalidomide and thalidomide treatment for 48 h. The rate of apoptosis is presented in Fig. 3. Lenalidomide has a higher rate of induced cell apoptosis than thalidomide of the same concentration (P<0.01; Fig. 3), and the effect was observed to be greater with the increase of the lenalidomide concentration (Fig. 4).

The data were presented as the mean ± standard deviation. In the current study, it was observed that lenalidomide can significantly inhibit VEGF expression of SMMC-7721 cells in vitro (Fig. 4) and is more potent than thalidomide in the 100 µg/ml groups (0.3760±0.01813; 0.4985±0.01097; P<0.05). No significant differences were observed between the 200 µg/ml groups (0.2255±0.01921; 0.2460±0.01192; P>0.05; Fig. 1C, Table III).