I. Introduction

Aberrations on the short arm of chromosome 8 (8p) have been observed in various types of carcinoma including oral cancer. Loss of heterozygosity on chromosome 8p has been found in carcinomas of prostate ^[1], sporadic colorectal adenoma ^[2], breast ^[3], urinary bladder ^[4], head and neck ^[5], oropharyngeal ^[6]. These reports suggest the commonly deleted region located in chromosome 8p22 should play crucial roles in carcinogenesis in various carcinomas. Ishii et al. revealed that the expression of FEZ1 gene at chromosome 8q22, encoding a leucine-zipper protein, was down-regulated ^[7], and we previously reported the down-regulation of FEZ1/LZTS1 gene with frequent loss of heterozygosity in oral squamous cell carcinoma (OSCC)^[8]. Cathepsin B gene is known to locate at chromosome 8q22, and some studies demonstrated that cathepsin B expression is related to the progression of cancer, including oral carcinoma ^[9-11]. Thus, we hypothesized that chromosomal region 8p22 is one of important regions for oral carcinoma and altered expression of cathepsin B, locating at this region, should be involved in oral carcinomas. In this study, we performed quantitative real time quantitative RT-PCR in order to clarify the role of cathepsin B in OSCC.

II. Materials and methods

(1) Tissue specimens

Forty-seven pairs of primary OSCC samples and corresponding normal oral epithelium tissues were obtained at the time of surgery performed at Chiba University Hospital between 1998 and 2005. Profiles of OSCC patients examined in the present study were summarized in Table 1. All patients provided informed consent according to the protocol that was reviewed and approved by the institutional review board of Chiba University before any procedures were performed. Postoperative follow-up data were collected until April 2006 or until the day of the patient’s death, metastasis, or local recurrence. The median follow-up time was 2.1 years (range, 3 months to 8 years).

The resected tissues were divided into two parts: one was frozen immediately after removal of the surrounding normal tissue and stored at -80℃ until RNA extraction, and another was fixed in 10% buffered formaldehyde solution for pathologic diagnosis. Histopathologic diagnosis of each tumor specimen was performed according to the International Histological Classification of Tumors by the Department of Pathology, Chiba University Hospital. Clinicopathological staging was determined by the TNM classification of the International Union against Cancer. All OSCC samples were histologically confirmed and checked to ensure the presence of tumor in greater than 80% of specimens.

(2) mRNA extraction

Total RNA was extracted using Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instruction. All of extracted RNA was stored separately at －80℃ until use.

(3) Cathepsin B mRNA expression analysis

The expression levels of cathepsin B mRNA was examined in 47 OSCC specimens from patients with primary OSCC. Control reactions were prepared in parallel without reverse transcriptase. Before cDNA synthesis, residual genomic DNA was removed from the total RNA using DNase I treatment (DNA-free; Ambion, Austin, TX, USA). The primer sequences used to analyze cathepsin B mRNA expression were 5-ACTCCATCCCTCCCTGTGA-3 (nucleotides 227-246) and 5-CCGTAGTGCTTGTAATGTTTGTAG-3 (nucleotides 359-383). The sequences of specific primers were checked before use to avoid amplification of genomic DNA or pseudogenes by the Primer3 program (available at http:www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Amplified products were analyzed by 3% agarose gel electrophoresis to ascertain size and purity. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed with a single method using the LightCycler FastStart DNA Master SYBR Green I kit (Roche, Mannheim, Germany). To prepare the standard curve, 3 μg of total RNA from normal oral tissue was reverse-transcribed with Superscript reverse transcriptase (Life Technologies, Grand Island, NY, USA) and oligo-d (T) 12-18 primer, after which serial dilutions were made corresponding to cDNA transcribed from 300, 30, 3.0, and 0.3 ng of total RNA. Real time qRT-PCR analysis using LightCycler apparatus was carried out in a final volume of 20 μl of reaction mixture consisting of 2 μl of FirstStart DNA Master SYBR Green I mix, 3 mM MgCl2, and 0.2 μl of primers, according to the manufacturer’s instructions. The reaction mixture then was loaded into glass capillary tubes and submitted to an initial denaturation at 95℃ for 10 min, followed by 45 rounds of amplification at 95℃ (10 sec) for denaturation, 58℃ (10 sec) for annealing and 72℃ for extension, with a temperature slope of 20℃/sec, performed in the LightCycler. The transcript amount for cathepsin B was estimated from the respective standard curves and normalized to the GAPDH transcript amount determined in corresponding samples.

(4) Statistical analysis

Differences of the cathepsin B mRNA expression in normal tissues and OSCCs, and correlation with clinicopathological profiles were analyzed using the Mann-Whitney’s U-test. The criterion for statistical significance was P＜0.05.

III. Results

Relative mRNA expression of cathepsin B was examined by real-time qRT-PCR analysis. In 38 of 47 (80.9%) samples, mRNA expressions were greater in OSCC samples than in normal tissue samples ^{(Fig. 1)}. The median amounts of mRNA expression of cathepsin B in OSCC and normal tissue were 2.10 and 0.81, respectively. Statistically significant difference was observed between these two groups ^{(Fig. 2)}. These data suggest that cathepsin B mRNA expression was up-regulated in OSCC samples compared to normal oral tissues. We investigated the relationship between cathepsin B expression and clinicopathological profiles, and data were summarized in Table 2. Significant differences were found in clinicopathological profiles of differentiation (P＝0.035), tumor size (P＝0.006), regional neck metastasis (P＝0.009) and TNM stage classification (P＝0.001). These strong relationships suggest cathepsin B should be associated with advance of OSCC.

IV. Discussion

Various investigations concerning the aberrant alteration of chromosomes and genes have been performed in oral carcinomas ^[12-14]. Chromosome 8 is thought to be one of important regions in many different types of cancer. Moreover, suppressive effect of FEZ1; leucine-zipper protein, locating at 8p22 chromosome region was suggested in various types of carcinoma ^[7]. We previously reported allelic loss on the short arm of chromosome 8 in oral squamous cell carcinoma ^[15], and also reported down-regulation of FEZ1 gene with frequent loss of heterozygosity in oral squamous cell carcinoma ^[15]. These studies suggest that chromosome 8p22 should be crucial chromosome region for oncogenesis in oral cancer. In order to confirm the significance of this chromosome region, the expression of cathepsin B, which locates at chromosome 8q22, in OSCC carcinoma was examined in the current study. The data revealed the mRNA expression of cathepsin B was significantly up-regulated in OSCC compared to normal tissues. Our results are consistent with previous studies describing the significance of this region and all of these investigations strongly suggest that chromosome 8p22 region plays important roles in oral carcinomas.

Cathepsin B is a member of lysosomal cysteine protease of the papain family. It is known to function mainly as a protease, and it may also be involved in other physiological processes, such as processing of antigens in the immune response, hormone activation and bone turnover. In addition, cathepsin B is implicated in the pathology of chronic inflammatory diseases of airways and joints, and in cancer and pancreatitis ^[9]. In OSCC, previous studies demonstrated that increased expression of cathepsin B is closely associated with advanced tumor stage and poor histologic malignancy grade ^[11], motility and invasiveness ^[16], invasion and progression ^[17], invasion and metastasis ^[18]. The current study also demonstrated close association with mRNA expression of cathepsin B and clinicopathological profiles in OSCC. All of these facts imply the usefulness of cathepsin B as a prognostic marker.

Although little is known for functions of cathepsin B in carcinomas, Nagaraj et al. demonstrated the involvement of cathepsin B with TRAIL (tumor necrosis factor-related apoptosis inducing ligand) -induced apoptosis pathway ^[19]. This suggests the possibility for molecular therapeutic application through control of cathepsin B expression in the treatment for carcinomas. Molecular therapy approaches based on cathepsin B inhibitors should exhibit suppressive effect in invasiveness, on the other hand, the inhibition might lead enrichment of apoptosis-prone tumor cells and increase selective survival of highly malignant sub-populations of cancer cells.

In conclusion, the current study revealed over expression of cathepsin B mRNA in OSCC and it correlated clinicopathological profiles. This implies cathepsin B would play crucial roles in advance of OSCC, and the current study strongly suggests that chromosome 8p22 should be involved in OSCC. Further investigation to control the expression of cathepsin B should be necessary.