Erlotinib was obtained from Roche Diagnostics (Basel, Switzerland) and dissolved in DMSO (50 mmol/l stock solution). Erlotinib was stored at −40°C as frozen aliquots and the solution was thawed directly prior to the experiments. The chemical structure of erlotinib is presented in Fig. 1A.

L-02 cells, human hepatocytes, were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). L-02 cells were maintained in complete RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C under 5% CO₂ with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), containing 100 U/ml penicillin (Invitrogen; Thermo Fisher Scientific, Inc.) and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.).

The sulforhodamine B (SRB) colorimetric assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to evaluate cell proliferation. L-02 cells (3×10³ cells/well) were cultured in 96-well plates and exposed to erlotinib (0, 1.56, 3.13, 6.25, 12.50, 25.00 or 50.00 µM) for 72 h. The cells were then fixed with 10% trichloroacetic acid (Sigma-Aldrich; Merck KGaA) for 1 h at 4°C. Once the cells had been stained with SRB for 30 min at room temperature and bound SRB had been dissolved with 10 mmol/l Tris-base, a multi-well spectrophotometer was used to measure the absorbance at 510 nm. Calculation of the cell survival rate was according to the following formula: A510 treated cells/A510 control cells ×100.

L-02 cells (3×10⁴ cells/well) cultured exponentially in 6-well plates were exposed to erlotinib (0, 6.25, 12.50 or 25.00 µM) for 48 h. To study cell morphology, the cells were rinsed in PBS followed by fixation with 0.1% Triton X-100 for 15 min. Next, DAPI (2.0 µg/ml; Sigma-Aldrich; Merck KGaA) was used to stain the cells for 15 min. All steps were performed at room temperature. The morphology of cell nuclei was examined by fluorescence microscopy (magnification, ×100).

An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to measure apoptosis rate. L-02 cells (3×10⁴ cells/well) were harvested 48 h after treatment with erlotinib (0, 6.25, 12.50, 25.00 µM). L-02 cells were washed twice with cold PBS, 1×10⁶ cells/ml were resuspended in 1X binding buffer of the kit and 100 µl cell suspension was transferred to a 5-ml culture tube. In the dark, the transferred suspension was stained with 5 µl Annexin V-FITC and 5 µl PI at 25°C for 15 min. Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with BD FACSDiva Software (version 8.0.1) within 1 h.

L-02 cells were inoculated into 6-well plates (1.5×10⁴ cells/well) for 24 h at 37°C. Following treatment with erlotinib (0, 6.25, 12.50, 25.00 µM) for 48 h, L-02 cell suspension was prepared in PBS (500 µl) and stained with 2.5 µl JC-1 (20 µg/ml) for 30 min at 37°C under 5% CO₂. As a cationic dye, once in the mitochondria, JC-1 shifts its fluorescence emission from green to red, from 525±10 to 610±10 nm, as it accumulates owing to the high membrane potential. The cell suspension was sampled as 1×10⁴ cells/sample. A FACSCalibur flow cytometer was used to analyze the fluorescence emission. The fluorescence intensity ratio of red/green decreases when mitochondrial depolarization occurs.

Briefly, protein was extracted from L-02 cells in lysis buffer (50 mmol/l Tris-HCl, 150.0 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 2.0 µg/ml aprotinin, 0.02% NaN₃). Lysates were centrifuged (1×10⁴ × g) at 4°C for 15 min. Protein expression (20 µg/lane) was detected by SDS-PAGE (8–15% gel), and proteins were electroblotted onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Primary and horseradish peroxidase (HRP)-conjugated secondary antibodies (Southern Biotech, Birmingham, AL, USA) were used to probe proteins. The primary antibodies used were: B-cell lymphoma 2 (Bcl-2; cat. no. 2872) and cleaved caspase-3 (cat. no. 9961) purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA); cleaved poly (ADP-ribose) polymerase (PARP; cat. no. sc-56196), Bcl-associated X (Bax; cat. no. sc-4239) and β-actin (cat. no. sc-1615) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). HRP-labeled secondary antibodies (GAR007, GAM007, RAG007) were purchased from MultiSciences Biotech Co., Ltd. (Hangzhou, China). The primary and secondary antibodies were diluted into 1:1,000 and 1:5,000, respectively. Incubation occurred for 2 or 0.5 h at room temperature and washing for 5 min/wash for three washes. An enhanced chemiluminescence detection system (Biological Industries, Beit Haemek, Israel) was used for protein detection.

SPSS version 18.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analyses. Survival rates were presented as mean ± standard deviation and analyzed using two-way analysis of variance. P<0.05 (two-way) was considered to indicate a statistically significant difference.

Cell viability was assessed using a SRB colorimetric assay following treatment of L-02 cells with erlotinib (0, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00 µM) for 72 h. As presented in Fig. 1B and C, a dose-dependent inhibition of L-02 cell proliferation was detected in L-02 cells following exposure to erlotinib for 72 h (P<0.05). The percentage of cells that survived (compared with untreated control cells) was 70.31±4.90 and 48.64±6.83% at the lowest (P=0.03), and highest (P=0.003) concentration tested, respectively. The growth of L-02 cells following the 72-h treatment was analyzed using light microscopy. Cellular swelling, a cloudy cytoplasm, slow cell growth and impaired cell adherence were observed (Fig. 1D).

Following treatment of L-02 cells with 6.25, 12.50 and 25.00 µM erlotinib for 48 h, DAPI staining was used to detect variations in the nucleus morphology caused by apoptosis. Upon examination by fluorescence microscopy, L-02 cells treated with 12.50 µM erlotinib exhibited chromatin condensation and karyopyknosis, which are typical apoptotic phenomena. Further marked nuclear pyknosis was observed at a concentration of 25.00 µM, indicating the dose-dependent effect of erlotinib on the apoptosis of L-02 cells (Fig. 2A). Furthermore, detection of apoptotic protein expression was performed by western blot analysis. In concordance with the morphological changes, the levels of cleaved caspase-3 and cleaved PARP increased in a dose-dependent manner in treated cells, indicating the upregulation of apoptotic proteins (Fig. 2B). Subsequently, measurement by flow cytometry of the apoptotic rate of cells stained with Annexin V-FITC/PI was performed. Compared with the control, the late apoptotic rate in erlotinib-treated cells increased significantly and dose-dependently (P<0.05; Fig. 3). These results revealed that apoptosis contributes to erlotinib-induced hepatotoxicity.

During drug-induced apoptosis, mitochondrial changes are the key events. Therefore, the function of mitochondria in erlotinib-induced apoptosis was investigated. JC-1 staining and western blot analysis was used to examined the variation of ΔΨ_m, and associated protein expression in L-02 cells treated with erlotinib (0, 6.25, 12.50 and 25.00 µM) for 48 h. Using JC-1 staining, treated cells exhibited a decreased red/green fluorescence intensity ratio in line with the increasing concentration of erlotinib (P<0.05), which indicated that erlotinib treatment induced a dose-dependent loss of ΔΨ_m and mitochondrial depolarization (Fig. 4). Since the mitochondrial pathway of apoptosis is regulated by proteins of the Bcl-2 family (8), the levels of the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were analyzed. The levels of Bcl-2 protein decreased whereas those of Bax increased in L-02 cells 24 h after erlotinib treatment (6.25, 12.50, 25.00 µmol/l), in a dose-dependent manner (Fig. 5). The present study indicated that the mitochondrial pathway may be involved in apoptosis induced by erlotinib.