Hep-2 laryngeal carcinoma cells were obtained from the American Type Culture Collection (ATCC; Bethesda, MD) and maintained in RPMI-1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 units/ml of penicillin and 100 mg/ml of streptomycin) at 37°C in a 5% (v/v) CO₂ incubator.

Pak4 small interfering RNA (siRNA) oligonucleotides were purchased from Dharmacon, Inc. (Lafayette, CO). Cells were seeded onto 60-mm plates for 24 h and transfected with siRNA or control siRNA for 48 h using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions.

Total RNA was isolated using an RNeasy Mini kit (BioMed, Beijing, China). cDNA was reverse transcribed with 1 μg of total RNA, using the Takara Reverse Transcription kit (Takara Dalian, Dalian, China). cDNA was amplified using the following primers: the sequences of the forward and reverse primers for Pak4 were 5′-GACATCAAGAGCGACTCGATCC-3′ and 5′-ATCACCATTATCCCCAGCGAC-3′, respectively. GAPDH was used as the internal control. The sequences of the forward and reverse primers for GAPDH were 5′-AGAAGGCTGGGGCTCATTTG-3′ and 5′-AGGGGCCATCCACAGTCTTC-3′, respectively. PCR was performed for 35 cycles under the following conditions: annealing at 56°C (15 sec), extension at 72°C (30 sec) and denaturing at 94°C (30 sec) using a Takara thermal cycler.

Total cell extracts were obtained using lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 μg/ml leupeptin and 1 μg/ml aprotinin. Equal amounts (90 μg) of cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes and incubated with the following specific antibodies: Pak4 antibody (Abcam PLC, Cambridge, UK) was used to identify transfection efficiency. β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used as the internal control. The reaction was followed by probing with peroxidase-coupled secondary antibodies including anti-rabbit IgG, or anti-mouse IgG antibodies at dilutions ranging from 1:1,000 to 1:2,000 (Amersham Biosciences, Needham, MA) and binding results were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

For the colony formation assay, cells (200 cells per well) were seeded in 24-well tissue culture plates. Plates were incubated for 3 weeks in a humidified incubator at 37°C. Three weeks after seeding, colonies were stained with 0.05% crystal violet containing 50% methanol and counted. The colonies were counted in 4 to 5 random fields from each of the duplicate samples by using a microscope at ×100 magnification.

Caspase activities were measured by colorimetric assay kits (KeyGen Biotech. Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. After harvesting, cells were washed in ice-cold PBS and lysed; proteins were extracted and stored at −80°C. Cell lysates (20 μl) were then added to a buffer containing a p-nitroaniline (pNA)-conjugated substrate for caspase-3 (Ac-DEVD-pNA), or -9 (LEHD-pNA) to a total of 100 μl reaction volume. Incubation was carried out at room temperature for caspase-3 and -9 followed by shaking at 500 rpm for 1 min and then incubation at room temperature for 2 h. The concentration of the released pNA in each well was measured using a plate-reading luminometer (Thermo Fisher Scientific, Beijing, China). Data were from 3 independent experiments.

The Hep-2 laryngeal carcinoma cells (3×10⁵/well) were plated and incubated overnight. The control and treated cells were trypsinized, collected in PBS and fixed on ice with 1% paraformaldehyde, followed by 70% cold ethanol. Following treatment with 10 μg/ml RNase, the cells were stained with 50 μg/ml propidium iodide (PI; KeyGen Biotech. Co., Ltd.) for 15 min at room temperature for cell cycle analysis. The stained cells were analyzed by flow cytometry. Data analysis was performed with CellQuest software (BD Biosciences, Rockville, MD).

Actively replicating cells at the beginning of each hour of the S phase were first labeled with the thymidine analog, 5-iodo-2′-deoxyuridine (IdU; 50 μM) (Sigma-Aldrich, Carlsbad, CA) for 40 min, washed 3 times with PBS and then labeled with 5-chloro-2′-deoxyuridine (CldU; 100 μM) (Sigma) for 40 min. IdU and CldU incorporated into replicating DNA were later detected with red or green fluorescent antibodies, respectively, as described below. Three independent replicate experiments were performed and analyzed for the control, mock and treated cells.

Slides were treated with 70% ethanol, washed in PBS, denatured in 2.5 M HCl for 30 min and permeabilized in 0.25% Triton X-100 for 5 min and blocked with 1% bovine serum albumin. The slides were incubated at room temperature with the following antibodies: i) 1:500 mouse anti-bromodeoxyuridine (detects IdU) (Sigma); ii) 1:1,000 Alexafluor 488-conjugated anti-mouse (Sigma); iii) 1:2,000 rat anti-bromodeoxyuridine (detects CldU) (Santa Cruz Biotechnology, Inc.); and iv) 1:1,000 Alexafluor 633-conjugated anti-rat antibodies (Invitrogen Life Technologies).

After counterstaining with DAPI (1 μg/ml) (KeyGen Biotech. Co., Ltd.), photographic images were taken using an Olympus CX71 fluorescence microscope (Olympus, Tokyo, Japan).

All in vivo experiments were approved by the Ethics Committee of China Medical University. Human tumors were induced by a subcutaneous (s.c.) injection of 5×10⁶ Hep-2 cells into the dorsal flank region of nu/nu mice (CAnN.Cg-Foxn1^nu/Crl; Vital River, Beijing, China). When tumor volumes had reached a mean volume of 50±5 mm³, the animals were randomized into 3 groups (n=15 per group). All mice received 2 injections, 72 h apart (days 1 and 4). Injections were administered by mixing siRNA (20 μg) with Lipofectamine 2000 (30 μl) and PBS in a total volume of 100 μl. Tumors were measured with calipers every 5 days and tumor volumes were calculated (tumor volume = length × width² × 0.52) (15).

An additional 180 mice were used to establish xenografts to observe survival time. For these experiments, mice with xenograft tumors were treated as described above. Survival was monitored until the experiments were terminated due to heavy tumor burden.

Immunohistochemical staining was performed on 4-μm sections obtained from formalin-fixed, paraffin-embedded blocks. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min. Antigen retrieval was carried out in citrate buffer (10 mM, pH 6.0) for 30 min at 95°C in a microwave oven (16). Sections were incubated with primary antibody at 4°C overnight. Cell cycle checkpoint-regulated proteins were probed with: anti-ataxia telangiectasia mutated (ATM), anti-p53, anti-checkpoint kinase (Chk)1, anti-Chk2 (Santa Cruz Biotechnology, Inc.), anti-phospho-S345-Chk1 and anti-phospho-T68-Chk2 antibodies (Cell Signaling Technology, Danvers, MA). Cell cycle-regulated proteins were probed with: anti-Cyclin A (Santa Cruz Biotechnology, Inc.) and anti-cyclin-dependent kinase 2 (CDK2) antibodies (Abcam PLC). The sections were then incubated with a biotinylated secondary antibody and exposed to a streptavidin complex (HRP). Positive reactions were visualized with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma), followed by counterstaining with hematoxylin. Sections treated without primary antibodies were used as negative controls.

Data were analyzed using GraphPad Prism 5 software. Statistical analysis was performed using a one-tailed Student’s t-test (unilateral and unpaired). Kaplan-Meier survival plots were generated and comparisons between survival curves were made using log-rank statistical analysis. P-values <0.05 were considered to indicate statistically significant differences.

The levels of Pak4 in siRNA-treated Hep-2, untreated and mock-treated cells were detected using western blot and RT-PCR assays. In the western blot assays, the levels of Pak4 protein were higher in the untreated Hep-2 and mock-treated Hep-2 cells compared with the siRNA-treated Hep-2 cells (Fig. 1, upper panel). To determine the correlation between the levels of Pak4 and the transcription of Pak4, RT-PCR analysis of Pak4 was performed. In these assays, the levels of Pak4 mRNA were higher in the untreated Hep-2 cells compared to the treated ones and were consistent with the levels of Pak4 detected by western blot analysis (Fig. 1, lower panel).

Pak4 mRNA and protein levels are high in Hep-2 cells. We determined whether its downregulation would reduce the tumorigenicity of laryngeal carcinoma cells. Clonogenic assay showed that the proliferation rate of siRNA-treated Hep-2 cells was decreased compared to the untreated and mock-treated cells (Fig. 2A, p<0.05). Caspase-3 and caspase-9 activities in the treated cells was also found to be higher compared to the untreated and mock-treated cells (Fig. 2B, p<0.05). PI staining of cells revealed that Pak4-siRNA cells were arrested in the S phase (Fig. 3, p<0.05).

To further analyze the proliferation activity of Hep-2 cells after siRNA treatment, a CldU/IdU double-labeling method was applied to distinguish CldU and IdU incorporated into cellular DNA. According to the method of Bakker et al(17), we assessed the G₁/S and S/G₂ checkpoint in Hep-2 cells following siRNA treatment. Our labeling strategy is shown in Fig. 4 (upper panel). In this assay, single-labeled IdU cells (green) are cells that enter the S phase after the first labeling period of the experiment. Single-labeled CldU cells (red) are cells that are left int he S phase before the second labeling period of the experiment. Cells labeled both with CldU and IdU (yellow) are in the S phase. Non-labeled cells are not in the S phase. This method helped us to identify the percentage of cells in the G₁/S and S/G₂ transitions. As shown by our results, there was no difference in the number of Hep-2 cells and mock cells in the G₁/S transition, S phase and S/G₂ transition. Non-labeled cells were considered cells in other phases (Fig. 4, lower panel). However, we found that the number of Hep-2 cells following siRNA treatment in the S/G₂ transition was higher than that of cells in the G₁/S transition (Fig. 4, lower panel). This indicated that Hep-2 cells were obstructed in the S/G₂ transition following siRNA treatment.

The anti-tumor effect of Pak4-siRNA was analyzed in vivo using Hep-2 laryngeal carcinoma subcutaneous xenografts. When the established tumors reached 5–7 mm in diameter, siRNA was injected into the tumors. Treatment with siRNA was performed by mixing the plasmid DNA (20 μg) with Lipofectamine 2000 (30 μl) and PBS in a total volume of 100 μl per mouse and injecting the mice intratumorally twice every 3 days. As is shown in Fig. 5B, on day 15, the tumor volume of the control group was 623.1±43.3 mm³, that of the mock-treated group was 637.4±48.6 mm³ and that of the siRNA-treated group was 324.6±32.7 mm³. These results demonstrate the suppressive effect of Pak4-siRNA on tumor growth. Moreover, Pak4-siRNA showed the same effects on tumor weight (Fig. 5C). However, on day 20, tumor volume and weight showed no significant differences between the 3 groups. Subsequently, the effectiveness of siRNA on the xenografts gradually diminished after 15 days.

A significantly improved survival rate was observed in the mice treated with siRNA (Fig. 5D). The mice began to die first in the siRNA-treated group on day 9, and then in the control and mock-treated groups on days 3 and 4, respectively. At the end of this experiment, there were 10 out of 15 mice left alive in the siRNA group. However, all mice died in the other 2 groups.

To identify the mechanism of the cell cycle arrest induced by Pak4, immunohistochemical staining was performed to detect changes in cell cycle-related proteins. The results demonstrated that the downregulation of Pak4 induced a significant decrease in the levels of cyclin A (Fig. 6A). Decreased levels of CDK2 were also detected (Fig. 6A). In further experiments, we found that p53 was activated following the downregulation of Pak4 (Fig. 6A). These results indicate that the cell cycle arrest is dependent on p53 function in these cells.

The protein kinases, ATM and Chk1/Chk2, are major components of the mechanisms that oversee the control of DNA replication and genomic integrity (18). In our study, we found that the levels of Chk1 and Chk2 were not altered in the siRNA-treated group compared with the control and mock-treated groups (Fig. 6B). However, the levels of P-Chk1 and P-Chk2 were significantly increased in the siRNA-treated group (Fig. 6B). To examine the hypothesis that the ATM/Chk1/Chk2-p53 pathway is activated by the downregulation of Pak4 in the siRNA-treated group, we detected the levels of ATM, the upstream protein of Chk1 and Chk2. We found that ATM levels also increased (Fig. 6B). These data are consistent with the results of the cell cycle arrest at the S/G₂ transition.