Cisplatin was purchased from Nippon Kayaku (Tokyo, Japan). A Cell Counting kit (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan), and all other chemicals were obtained from Sigma Chemical (Tokyo, Japan).

In this study, the following antibodies were used: anti-β-actin monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA; A5441, used at 1:2,000), cyclin D1 (Thermo Fisher Scientific, Waltham, MA, USA; RB-9041, used at 1:1,000), Cdk6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-177, used at 1:500), Cdk4 (Cell Signaling Technology, Danvers, MA, USA; #2906, used at 1:1,000), horseradish peroxidase (HRP)-linked anti-mouse and anti-rabbit IgG secondary antibodies (GE Healthcare UK, Buckinghamshire, UK; used at 1:2,000).

The human hepatocellular carcinoma cell lines HLE, HLF, HuH7, Li-7, Hep3B and HepG2 were obtained from the Japanese Cancer Research Resources Bank and were passaged in our laboratory. The cell lines were authenticated by the cell bank using short tandem repeat PCR. Cells were grown in Minimum Essential Medium (MEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (533–69545; Wako) and penicillin-streptomycin (100 mg/l; Invitrogen) in a humidified atmosphere of 5% CO₂ at 37°C.

Cell proliferation assays were conducted using the CCK-8 according to the manufacturer’s instructions. Cells from each cell line (0.5×10⁴) were seeded into a 96-well plate and cultured in 100 μl of MEM supplemented with 10% FBS. After 24 h, the seeded cells were treated with 0, 1, 3 or 10 μg/ml of cisplatin added to the culture medium. At the indicated time points, the medium was changed to 100 μl of MEM with CCK-8 reagent (10 μl of CCK-8 and 90 μl of MEM). Absorbance was measured for each well at a wavelength of 450 nm using an auto-microplate reader.

The lysates were collected according to the methods described in our previous studies (20). All steps were performed at 4°C. Protein concentrations were measured using a dye-binding protein assay based on the Bradford method (21).

Samples were electrophoresed using 7.5 to 10% SDS-PAGE (22) after which the proteins were transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies after blocking and were then incubated with HRP-conjugated secondary antibodies (23). Immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Perkin Elmer Co.) on X-ray film.

The detection of caspase-cleaved cytokeratin 18 (CK18) was performed using an M30 Apoptosense ELISA kit which was purchased from Peviva AB (Bromma, Sweden). HuH7 cells (0.5×10⁴) were seeded into a 96-well plate and cultured in 100 μl of MEM supplemented with 10% FBS. After 24 h, the seeded cells were washed once with PBS and treated with 1 μg/ml of cisplatin added to the culture medium. At the indicated time points, 10 μl of PRO-PREP™ protein extraction solution was added to each well. The plates were shaken on a rotary shaker for 5 min at room temperature in order to lyse the cells. Next, 25 μl of M30 Standard (A–G), M30 Control Low, M30 Control High or samples was pipetted into the appropriate wells. A total of 75 μl of the diluted M30 HRP Conjugate solution was also added to each well. The cells were incubated at room temperature for 4 h. The incubation solution was discarded, and the plate was washed five times with 250 μl of diluted washing solution. Next, 200 μl of TMB substrate was added to each well. The cells were incubated in the dark at room temperature for 20 min. Then, 50 μl of stop solution was added to each well. After 5 min, the absorbance was measured at a wavelength of 450 nm using an auto-microplate reader.

The RayBio Human Phospho Array kit (catalog no. ARY 001) was purchased from RayBiotech, Inc (Norcross, GA, USA). An array to detect phosphorylated receptor tyrosine kinase (p-RTK) was conducted according to the manufacturer’s instructions. Briefly, p-RTK array membranes were blocked with 5% bovine serum albumin (BSA)/TBS (0.01 mol/l Tris-HCl, pH 7.6) for 1 h. The membranes were then incubated with 1.5 ml of lysate prepared from cell lines after normalization with equal amounts of protein. After extensive washing with TBS containing 0.1% v/v Tween-20 (3 washes for 10 min each), the membranes were then incubated with an anti-phospho-tyrosine-HRP detection antibody for 2 h at room temperature. The unbound HRP antibody was washed away with TBS with 0.1% Tween-20. Finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin Elmer Co.). The density of the immunoreactive band obtained on the p-RTK array was analyzed by densitometric scanning (Tlc scanner, Shimizu Co., Ltd.).

The RayBio Human Angiogenesis Antibody Array 1 kit (catalog no. AAH-ANG-1) was purchased from RayBiotech, Inc. The assay for the antibody array was performed according to the manufacturer’s instructions. Briefly, the angiogenesis antibody membranes were blocked with blocking buffer for 30 min. The membranes were then incubated with 1 ml of lysate prepared from the cell lines after normalization with equal amounts of protein. After extensive washing with TBS with 0.1% v/v Tween-20 (3 washes for 5 min each) and with TBS alone (2 washes for 5 min each) to remove unbound lysate, the membranes were then incubated with biotin-conjugated antibodies for 1 h at room temperature. After washing the membranes with TBS with 0.1% Tween-20 and with TBS alone, the membranes were incubated with HRP-conjugated streptavidin for 2 h at room temperature. After washing these membranes, finally, each array membrane was exposed to X-ray film using a chemiluminescence detection system (Perkin Elmer Co.). The density of the immunoreactive band obtained on this array was analyzed by densitometric scanning (Tlc scanner, Shimizu Co., Ltd.).

Total RNA was extracted from the samples derived from the cancer cell lines using a miRNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA samples typically showed A_260/280 ratios between 1.9 and 2.1, using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

After measurement of the RNA with an RNA 6000 Nano kit (Agilent Technologies), the samples were labeled using a miRCURY Hy3/Hy5 power labeling kit and were hybridized onto a human miRNA Oligo chip (version 19.0; Toray Industries, Tokyo, Japan). Scanning was conducted with the 3D-Gene Scanner 3000 (Toray Industries), and 3D-Gene extraction version 1.2 software (Toray Industries) was used to read the raw intensity of the image. To determine the change in miRNA expression between the cisplatin-treated samples and the control samples, the raw data were analyzed via GeneSpringGX version 10.0 (Agilent Technologies). Samples were first normalized relative to the 28S RNA, and then the baseline was corrected to the median of all samples.

Replicate data were consolidated into two groups: those from the cisplatin-treated cells and those from the control cells were organized by the hierarchical clustering and ANOVA functions in GeneSpring software. Hierarchical clustering was performed with the clustering function (condition tree) and Euclidean correlation as a distance metric. Two-way ANOVA analysis and asymptotic p-value computation without any error correction of the samples were conducted to determine the miRNAs that varied most prominently across the different groups. The p-value cutoff was set to 0.05. Only changes >50% in at least one of the time points for each sample were considered significant. All of the analyzed data were scaled by global normalization. The statistical significance of the differentially expressed miRNAs was analyzed by Student’s t-test.

All analyses were conducted using the computer-assisted JMP8.0 (SAS Institute, Cary, NC, USA). A paired analysis between the groups was conducted using Student’s t-test. A p-value of 0.05 indicated a significant difference between the groups.

To evaluate the effect on the growth activity of human HCC cell lines by cisplatin in vitro, we examined the effect of cisplatin on the proliferation of six human HCC cell lines: HLE, HLF, HuH7, Li-7, Hep3B and HepG2. To understand the direct relationship between the decrease in cell viability and the inhibition of cell proliferation, we followed the course of proliferation over three days after the addition of cisplatin. Cells were grown in culture medium and treated with 0, 1, 3 or 10 μg/ml of cisplatin. As shown in Fig. 1, cisplatin led to a strong dose- and time-dependent inhibition of cell proliferation in the human HCC cell lines HLE, HLF, HuH7, Li-7, Hep3B and HepG2. These results show that cisplatin inhibits the proliferation of human HCC cells.

After the antitumor effects of cisplatin in human HCC cell lines were established, we next used a phosphorylated-RTK array system to identify the ‘key’ RTKs that are responsible for these antitumor effects. With an antibody array (Fig. 2A), we simultaneously screened the expression of 42 different RTKs in HuH7 cells with or without cisplatin treatment. The results showed that the expression of insulin receptor (Insulin R) (Fig. 2B) was reduced by the treatment with cisplatin.

The density of the Insulin R obtained from the membrane array was analyzed by a Kodak Image Station (Eastman Kodak, Rochester, NY, USA). The densitometric ratio of the Insulin R spots of the cisplatin-treated cell lines to the Insulin R spots of the untreated cell lines was reduced to 31.2% (Fig. 2C).

We used an angiogenesis antibody array system to identify the ‘key’ angiogenesis-related proteins responsible for the antitumor effects of cisplatin. By using this antibody array (Fig. 3A), we simultaneously screened the expression of 20 different angiogenesis markers in the human HCC cell line HuH7 with or without cisplatin treatment. None of the markers of angiogenesis were changed by treatment with cisplatin as detected by the protein array (Fig. 3B).

To determine whether cisplatin affects the cell cycle in HuH7 cells, western blot analysis was used to examine the expression of various cell cycle-related molecules in HuH7 cells with and without cisplatin treatment. Cells were treated with 1 μg/ml of cisplatin or were left untreated for 24–72 h. The most remarkable change was the loss of cyclin D1, a key protein that has been implicated in the G0/G1 transition (Fig. 4). We then studied the expression of other cell cycle-related proteins (Cdk4 and Cdk6) that have also been implicated in the G0/G1 transition. Cdk4 and Cdk6, the catalytic subunits of cyclin D1, were not changed after the addition of cisplatin to the culture medium. The amount of β-actin (an internal control for protein loading) was almost the same in each lane in sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 4).

In order to establish that cisplatin induces apoptosis in HuH7 cells, we used the M30 Apoptosense method which specifically measures caspase-cleaved cytokeratin 18 in apoptotic cells. The activity in this assay is inhibited by a pan-caspase inhibitor. The M30 Apoptosense method is a useful screening tool as it measures the accumulation of the apoptotic product in cell cultures, which allows for an integrative determination of apoptosis until the cells are harvested. Cisplatin induced strong expression of caspase-cleaved cytokeratin-18 in HuH7 cells after 48 h of treatment (Fig. 5).

Using a custom micro-array platform, we analyzed the expression levels of 2019 human miRNAs in HuH7 cells with or without cisplatin treatment in vitro. As shown in Fig. 6, Tables I and II, when the expression of miRNAs was examined in HuH7 cells treated with 1 μg/ml of cisplatin in vitro and in those not treated with cisplatin, 36 miRNAs were significantly upregulated (Table I) in HuH7 cells after 24 h of cisplatin treatment, while 10 miRNAs were downregulated (Table II) out of the 2019 total miRNAs. Unsupervised hierarchical clustering analysis, with Pearson’s correlation, showed that HuH7 cells treated with cisplatin clustered together and separately from the untreated cells (Fig. 6).