Serine protease inhibitor (SERPIN) B1 promotes oral cancer cell motility and is over-expressed in invasive oral squamous cell carcinoma
Introduction
Serine protease inhibitors (SERPINs) are the largest and most broadly distributed superfamily of protease inhibitors.1, 2 Over 1500 members of serpins to date have been identified in humans, plants, bacteria, archaea and poxviruses.1 Phylogenic analyses of this superfamily divide the eukaryotic serpins into 16 clades, among which human serpins segregate evolutionarily into nine separate clades (A-I).1 Several serpin structures have been determined, leading to the discovery that serpins share conserved tertiary structure and use a unique conformational rearrangement for their inhibitory activity.3, 4 While most serpins inhibit serine proteases, cross-class inhibitors targeting cysteine protease, caspase 1,5 and papain-like cysteine proteases6 have also been identified.
Human serpins play an important role in diverse biological functions, the majority of which are involved in proteolytic cascades of blood clotting and anti-inflammatory responses that require the protease-inhibiting activity.7 However, numerous serpin members can perform non-inhibitory functions such as hormone transporters (SERPINA6 and SERPINA7),8 molecular chaperons (Hsp47)9 and blood pressure regulator (SERPINA8).10 Due to the complexity of the inhibition mechanism, serpins are vulnerable to mutations that promote misfolding and polymerization, which may result in conformational diseases or “serpinopathies”. Indeed, compelling evidence has shown that serpins are closely linked to human diseases. For example, deficiency of SERPINA111 and SERPINC112 causes emphysema and thrombosis, respectively. On the other hand, accumulation of SERPINA1 polymers in the endoplasmic reticulum results in cirrhosis.11 Moreover, SERPINB3 (squamous cell carcinoma antigen1, SCCA1) and SERPINB4 (squamous cell carcinoma antigen2, SCCA2) have been used as diagnostic markers for advanced squamous cell carcinomas,13 while SERPINB5 (maspin), a non-inhibitory serpin, has been implicated in suppression of metastasis in breast and prostate cancers.14, 15
Lymph node metastasis accounts for poor diagnosis and survival in oral squamous cell carcinoma (OSCC), the most common malignant tumor of the oral cavity.16 Several recent studies have been focused on the identification of biomarkers for OSCC using global screening methods. For example, some previous reports have revealed the differences in gene expression profiles between metastatic and nonmetastatic primary tumors by cDNA microarray-based analysis.17, 18, 19 On the other hand, many differentially expressed proteins in OSCC have also been identified by proteomic approach.20, 21, 22, 23 Although the candidate genes validated by these studies may have provided clinical implications for oral cancer, the majority of which await further evidence to demonstrate the link between the biological functions of these candidates and malignant phenotypes.
Thus, in the present study, we sought to investigate the molecular mechanism behind oral cancer cell motility by comparing the proteomes of two oral cancer cell lines with distinct migration potentials. After protein identification by LC/MS/MS, SERPINB1, a known neutrophil protease inhibitor, was found up-regulated in a high-motility oral cancer cell line, CAL-27. Endogenous protein level of SERPINB1 was correlated with the migration ability in five different oral cancer cell lines. In addition, ectopic expression of SERPINB1 significantly enhanced migration of four different oral cancer cell lines. More importantly, SERPINB1 was over-expressed in fresh cancer tissues and paraffin-embedded OSCC tissue sections of high invasiveness. These results have suggested a possible biological role of SERPINB1 in oral cancer metastasis.
Section snippets
Cell culture and plasmid DNA transfection
Oral cancer cells, Ca9-22, SAS and SCC-9 were maintained in Dulbecco’s modified Eagle medium supplemented with nutrient mixture F-12 (DMEM/F12) while FaDu, HSC-3 and CAL-27 in DMEM (Invitrogen Technologies, Carlsbad, CA), and 10% FBS. pCMV6-XL5-SERPINB1 (Origene) was introduced into SAS, Ca9-22, CAL-27 or HSC-3 cells by using FuGENE 6 transfection reagent (Roche Applied Science). 36-48 h after transfection, cells were harvested for subsequent experiments.
Migration assay
Oral cancer cells in serum-free culture
Comparison of cell migration ability among six oral cancer cell lines
To study protein expression associated with oral cancer cells of high metastatic potential, we first attempted to determine the migration ability of six oral cancer cell lines, SAS, FaDu, SCC-9, Ca9-22, HSC-3 and CAL-27, using transwell-based migration assay. Equivalent seeding numbers of the cells were allowed to migrate for 24 h, and the migrated cells on the bottom side of the transwell inserts were scored. We found that CAL-27 showed the largest, while SAS the least, number of migrated cells
Discussion
SERPINB1, the major inhibitor of human neutrophil granule proteases, has been functionally linked to elimination of phagocytosed pathogens.27 Thus, impaired function of SERPINB1 is associated with the infiltration of activated neutrophils and subsequent protease release, resulting in airway inflammation and damage of lung tissues.28 Moreover, a rat model of lung injury suggests that SERPINB1 can protect against the destructive effects of neutrophil proteases in inflammatory lung disorders.29
Conflict of interest statement
None declared.
Acknowledgments
The authors would like to thank Dr. Yi-Hsin Yang for statistical assistance. This work was supported by Grant NSC 95-2311-B-037-004-MY2 from National Science Council and by Kaohsiung Medical University (Grant KMU-EM-97-1.1ab and Grant KMUH96-6G03).
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These two authors contributed equally to this work.