To evaluate the expression of the EGR1 protein, sections of paraffin-embedded tissue samples were analyzed from 55 patients who underwent diagnostic biopsy for advanced LHSCC (stage III and IV) at the Chonnam National University Hwasun Hospital (Jeonnam, Korea) between July, 2004 and February, 2009. A total of 60 patients were treated with three cycles of combined cisplatin-, 5-fluorouracil- and docetaxel-based induction chemotherapy (IC). This was followed by cisplatin-based concurrent chemoradiation therapy in 56 patients who showed a complete response (CR) or partial response (PR) after IC, and in two patients who declined total laryngectomy salvage therapy, despite having stable disease (SD) after IC, while another two patients with SD after IC underwent total laryngectomy salvage therapy. Five of the initial 60 patients were subsequently excluded from the present study due to exhaustion of the small block of paraffin-embedded tissue sample taken for the diagnostic biopsy analysis. CR was defined as no visible or palpable disease, as assessed by physical examination, laryngoscopy, and computed tomography. PR was defined as a >50% decrease in tumor size, compared with the measurement prior to treatment. SD was defined as stationary or progressive disease. Patient clinicopathological characteristics were reviewed in hospital records. Tumors were staged according to the 7th edition of the American Joint Committee on Cancer Staging Manual (9). Patient survival was measured from the initiation date of the chemotherapy to the date of death or the date of last follow-up. The present study was approved by the Institutional Review Board of Chonnam National University Hwasun Hospital.

Paraffin-embedded tissue sections were deparaffinized and rehydrated. The tissue sections were incubated with a polyclonal rabbit anti-human EGR1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and then stained using the Dako Real™ EnVision™ horseradish peroxidase (HRP)-3,3′-diaminobenzidine detection system (Agilent Technologies, Santa Clara, CA, USA). Two independent observers assessed the staining intensity of each specimen, without any knowledge of the clinical information. The staining intensity was scored as follows: 0, no staining of tumor cells; 1+, weak or comparable staining in the cytoplasm and/or nucleus compared to that of non-tumor cells; 2+, readily appreciable dark-brown staining, delineating the tumor cell cytoplasm and/ or nucleus. A score of 0 or 1+ was regarded as a low expression and 2+ as a high expression of the EGR1 protein.

The human PCI50 HNSCC cell line was provided by Dr M.W. Sung (Seoul National University, Seoul, South Korea). The cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT, USA), in a humidified atmosphere of 5% CO₂ at 37°C. Small-interfering RNA (siRNA) was used to knock down endogenous EGR1 gene expression in PCI50 cells. The cells were transfected for 48 h with EGR1-specific siRNA (Santa Cruz Biotechnology, Inc.) or negative control siRNA (Qiagen, Germantown, MD, USA) using Lipofectamine™ 2000 (Invitrogen).

The cells were irradiated with a 6 MV photon beam delivered by a linear accelerator. Water-equivalent calibration plates of 1-cm thickness were placed above and below the culture dish. The attached cells in the bottom of the culture dish were covered to a 5-mm depth by culture medium.

The cells were lysed in RIPA buffer (1 M Tris HCl, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA) with 1 mM phenylmethanesulfonyl fluoride, Halt™ phosphatase inhibitor, and Halt™ protease inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL, USA). Proteins (10–20 μg) were resolved by electrophoresis, and transferred to polyvinylidene difluoride membranes (EMD; Millipore, Billerica, MA, USA). Specific proteins were detected by sequentially probing blots with cognate primary antibodies: EGR1 (Santa Cruz Biotechnology, Inc.), cleaved caspase 3, cleaved caspase 7, cleaved poly(ADP-ribose) polymerase (PARP), the X-linked inhibitor of apoptosis protein (XIAP) (Cell Signaling Technology, Danvers, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology, Inc.). Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system with an HRP substrate (EMD), and analyzed using the LAS-4000 luminescent image analyzer (Fujifilm, Tokyo, Japan).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen), reverse transcribed and amplified using specific primers for EGR1 and GAPDH. The primer sequences used were: EGR1, 5′-TCGCCTCGATGGAGC TCCTC-3′/5′-CATGTGTGCCACTGTGACGT-3′; and GAPDH,5′-ACCACAGTCCATGCCATCAC-3′/5′-TCCACCA CCCTGTTGCTGTA-3′. For cDNA synthesis, 1 μg mRNA was incubated with 50 ng/μl oligo(dT) (Promega, Madison, WI, USA), Moloney murine leukemia virus reverse transcriptase (Invitrogen), and RNAsin (Takara Bio, Shiga, Japan). PCR amplification of cDNA was carried out using specific primers (see above) and GoTaq^® DNA polymerase (Promega). PCR products were separated by electrophoresis in a 1% agarose gel containing ethidium bromide.

The relationship between EGR1 protein expression and various clinicopathological parameters was compared using the χ² and Fisher’s exact tests. Survival curves were calculated using the Kaplan-Meier method, and comparison of curves was conducted using the log-rank test. The Statistical Package for the Social Sciences, version 21.0 (MicroCal Software Inc., Chicago, IL, USA) was used for all analyses. P<0.05 was considered to indicate a statistically significant result.

Local research ethics committee approval from the Chonnam National University Hwasun Hospital Institutional Review Board.

The patients in the present study included 54 men and one woman. The mean age was 64.4±8.0 years (mean ± standard deviation), with a range of 47–83 years. The mean follow-up period while the patient was alive was 49.9±30.17 months with a range of 8.1–121.3 months. To decrease the influence due to variable treatment strategies and evaluate whether EGR1 contributes to the radiosensitivity of LHSCC, only patients who were treated with the same chemoradiation regimen were included in the present study.

EGR1 protein expression was investigated by immunohistochemical staining of formalin-fixed, paraffin-embedded biopsy tissue obtained from 55 LHSCC patients. Immunohistochemical staining revealed the overexpression of EGR1 in LHSCC tissue (Fig. 1). Based on our criteria, a high expression of EGR1, with 2+ staining, was observed in 67.3% of patients (37/55), while a low expression of EGR1, with 1+ or no staining, was observed in 32.7% of patients (18/55).

Patient data, and the correlation between EGR1 expression and various clinicopathological factors in LHSCC, are shown in Table I. A high EGR1 expression was significantly associated with improved survival with laryngeal preservation (p=0.04). However, no significant correlation was found between EGR1 expression and other clinicopathological factors, including age, gender, tumor location, tumor stage, T stage (tumor invasion), N stage (lymph-node metastasis), chemotherapy sensitivity, tumor response following chemoradiation therapy and disease-specific survival (p>0.05).

For the 55 patients with stage III or IV LHSCC that were enrolled in the present study, the 3-/5-year disease-specific survival rate was 60/55%, and the 3-/5-year disease-specific survival rate with larynx preservation was 52/49%. The 5-year disease-specific survival rate with larynx preservation was 59% in patients with high EGR1 expression vs. 30% in patients with low EGR1 expression, however, analysis of Kaplan-Meier curves for survival with larynx preservation, using the log-rank test, did not demonstrate a significant difference between these two groups of patients (p=0.15) (Fig. 2).

To determine whether radiation in upregulation of the EGR1, total RNA and whole-cell protein extracts were prepared from PCI50 HNSCC cells at different time intervals following exposure to a 5 Gy dose of ionizing radiation, and analyzed by RT-PCR and western blotting, respectively. As shown in Fig. 3, EGR1 mRNA expression was induced at 30–60 min post-irradiation and EGR1 protein expression was induced at 60–120 min post-irradiation.

To evaluate the impact of EGR1 on radiation-induced apoptosis, we used siRNA to inhibit endogenous EGR1 gene expression. EGR1 mRNA and EGR1 protein expression were reduced by EGR1-specific siRNA, as compared to the negative control siRNA, in transfected PCI50 cells (Fig. 4A). The levels of the cleaved caspase 7, cleaved PARP and XIAP, were increased in the negative control siRNA-transfected PCI50 cells at 6 h after a 5 Gy irradiation dose, as compared to the levels at 0 h (Fig. 4B). By contrast, the levels of cleaved caspase 3, cleaved caspase 7, and cleaved PARP were decreased, while the level of XIAP was increased, in EGR1-specific siRNA-transfected PCI50 cells, as compared to the negative control cells, at all time points (Fig. 4B). These findings suggested that EGR1 knockdown inhibits radiation-induced apoptosis.