Analysis of tissue and salivary nicotinamide N-methyltransferase in oral squamous cell carcinoma: basis for the development of a noninvasive diagnostic test for early-stage disease

Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer worldwide. In recent decades, the 5-year mortality rate is approximately 50% around the world. As reliable biomarkers of oral cancer are still lacking, it is necessary to identify new target molecules for early diagnosis, effective therapy, and monitoring of the disease. In the present work, we focused on the expression of the enzyme nicotinamide N-methyltransferase (NNMT). We analyzed enzyme activity in 37 paired tumor and non-tumor tissues and found that activity levels are significantly higher in tumor compared with adjacent normal oral mucosa. Interestingly, oral epithelium surrounding tumor of unfavorable cases (N+) seems to display higher activity levels compared with that of favorable ones (N0). Western blot analyses were performed to evaluate protein levels in saliva samples from patients with OSCC and healthy subjects. Preliminary results indicated an up-regulation of salivary NNMT in tumor. This study shows a marked increase in enzyme activity in oral cancer and suggests that adjacent normal tissue of unfavorable cases seems to change toward cancer. Moreover, it is conceivable to hypothesize that NNMT could represent a potential biomarker for early and non-invasive diagnosis of oral cancer.     

RNA-Mediated Gene Silencing of Nicotinamide N-Methyltransferase Is Associated with Decreased Tumorigenicity in Human Oral Carcinoma Cells

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. Despite progress in the treatment of OSCC, overall survival has not improved substantially in the last three decades. Therefore, identification of reliable biomarkers becomes essential to develop effective anti-cancer therapy. In this study, we focused on the enzyme Nicotinamide N-methyltransferase (NNMT), which plays a fundamental role in the biotransformation of many xenobiotics. Although several tumors have been associated with abnormal NNMT expression, its role in cancer cell metabolism remains largely unknown. In this report, 7 human oral cancer cell lines were examined for NNMT expression by Real-Time PCR, Western blot and HPLC-based catalytic assay. Subsequently, we evaluated the in vitro effect of shRNA-mediated silencing of NNMT on cell proliferation. In vivo tumorigenicity of oral cancer cells with stable knockdown of NNMT was assayed by using xenograft models. High expression levels of NNMT were found in PE/CA PJ-15 cells, in keeping with the results of Western blot and catalytic activity assay. PE/CA PJ-15 cell line was stably transfected with shRNA plasmids against NNMT and analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and soft agar Assays. Transfected and control cells were injected into athymic mice in order to evaluate the effect of NNMT silencing on tumor growth. NNMT downregulation resulted in decreased cell proliferation and colony formation ability on soft agar. In athymic mice, NNMT silencing induced a marked reduction in tumour volume. Our results show that the downregulation of NNMT expression in human oral carcinoma cells significantly inhibits cell growth in vitro and tumorigenicity in vivo. All these experimental data seem to suggest that NNMT plays a critical role in the proliferation and tumorigenic capacity of oral cancer cells, and its inhibition could represent a potential molecular approach to the treatment of oral carcinoma.     

Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas,Which Contributes to Poor Prognosis

Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival.     

Ghrelin and obestatin expression in oral squamous cell carcinoma: an immunohistochemical and biochemical study

The underlying molecular mechanism of carcinogenesis in oral squamous cell carcinoma (OSCC) is poorly understood and appears to be controlled on many genetic, environmental, and hormonal factors. Obestatin and ghrelin, two recently discovered hormones, are co-expressed in endocrine cells. The purpose of this investigation was to examine the immunohistochemical features of OSCCs in relation to the tissue concentration of ghrelin and obestatin. The association between OSCC and Epstein Barr Virus (EBV) status was also explored. The expression of ghrelin and obestatin was examined by immunohistochemistry and immunoassay in oral biopsy specimens: 10 benign squamous epithelial cell samples, 10 microinvasive squamous cell carcinomas, and seven well-differentiated and seven poorly differentiated OSCCs. The presence of EBV was evaluated in these samples using immunohistochemistry. The concentrations of ghrelin and obestatin in tissue homogenates were measured by RIA and ELISA, respectively. Squamous cell carcinomas and benign tissue samples were positive for anti-EBV antibody, and obestatin and ghrelin were shown to be co-expressed in all stratified squamous epithelium samples. Expression of ghrelin and obestatin was decreased or absent in OSCCs in relation to the invasiveness of the carcinoma; ghrelin and obestatin levels in cancerous tissue homogenates were lower than in benign tissue homogenates. These results indicate that the concentrations and distribution of immunoreactive obestatin and ghrelin might be helpful in distinguishing OSCC from benign tumors. Maintaining normal levels of these hormones might be required for regulation of normal cell division. However, detailed studies will be required for better understanding of the complex mechanism of carcinogenesis relating to OSCCs.     

Overexpression and clinicopathological significance of homeobox gene Quox-1 in oral squamous cell carcinoma

The expression and clinicopathological significance of Quox-1 gene was studied in oral squamous cell carcinoma (OSCC). Immunocytochemistry and western blot analysis were used to examine the different expressions of Quox-1 protein in 114 OSCC specimens, 34 oral epithelial dysplasia specimens, and 16 normal oral mucosa specimens. RT-PCR and virtual Northern Blot were also used to examine the expression of Quox-1 mRNA. It was found that Quox-1 was not expressed in normal epithelium. However, as dysplastic lesions progressed Quox-1 expression increased (p < 0.01), and Quox-1 expression was not significantly different between severe dysplasia and highly differentiated OSCCs (p > 0.05). As the degree of differentiation decreased, Quox-1 positivity increased in OSCC (p < 0.01), and the rate of Quox-1 (81.58%) positivity in OSCC was higher than that in normal oral mucosa (p < 0.01). Our findings imply that the positive expression of Quox-1 is correlated with the histological classification of OSCCs. Thus, the expression of Quox-1 in OSCC may serve as a significant predicting factor of proliferative status and malignant degree, and it may also be a biological detection marker of oral mucosas initial cancer and of OSCC.     

MiR-34a inhibits the proliferation,migration, and invasion of oral squamous cell carcinoma by directly targeting SATB2

In various kinds of carcinomas, the special AT-rich sequence-binding protein 2 (SATB2) with its atypical expression promotes the metastasis and progression of the tumor, though in the oral squamous cell carcinoma (OSCC) its inherent mechanism and the status of SATB2 remain unclear. The role played by the SATB2 expression in the OSCC cell lines and tissue samples in the target of miR-34a downstream is the intended endeavor of this study. In te OSCCs the miR-34a expression was determined by quantitative real-time polymerase chain reaction (q-PCR), while the SATB2 expression in the cell lines and tissue samples in OSCC was analyzed with the q-PCR and the western blot. Studies in both in vitro and in vivo of the effects of miR-34a on the initiation of OSCC were conducted. As a direct target of the miR-34a the SATB2 was verified with the luciferase reporter assay. In cases where the miR-34a levels were low, the SATB2 in OSCCs seemed to be overexpressed. Besides, both in the in vitro and in vivo a suppression of migration, invasion, and cell growth was caused by miR-34a by down regulating the SATB2 expression. The SATB2 being a direct target of miR-34a was confirmed by the cotransfection of miR-34a mimics specifically the decrease in the expression of luciferase of SATB2–3′UTR-wt reporter. As a whole, our study confirmed the inhibition of miR-34a in the invasion, proliferation, and migration of the OSCCs, playing a potential tumor suppressor role with SATB2 as its downstream target.     

Search for the tumor-associated proteins of oral squamous cell carcinoma collected in Taiwan using proteomics strategy

Squamous cell carcinoma (SCC) accounts for more than 90% of malignant tumors of the oral cavity. In Taiwan, oral squamous cell carcinoma (OSCC) is among the most frequent malignancies, largely due to betal quid chewing. Despite the recent improvement in treatment results, the long-term outcome of OSCC generally remains poor, especially for those with advanced diseases. It is therefore desirable to identify potential biomarkers that may aid in risk stratification and perhaps the development of therapeutic targets. In this study, we exploited two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry to compare the proteome maps of 10 OSCC specimens with their adjacent nontumorous epithelia to identify differentially expressed proteins. Comparative proteomics indicated that 17 proteins were differentially expressed in OSCC with 11 up-regulated and 6 down-regulated proteins. These deregulated proteins participated in cytoskeletal functions, cell signaling, antiapoptosis, angiogenesis, lipid metabolism, drug metabolism, and protein translation/turnover. They were all associated with tumor development in various cancers. Among the dys-regulated proteins, the immunoexpression of three proteins including nicotinamide N-methyltransferase, apolipoprotein AI, and 14-3-3 zeta were evaluated in 38 OSCCs of testing cohort to confirm the proteomics data. Subsequently, the expression of 14-3-3 zeta, as the most relevant to OSCC progression determined by testing cohort, was further assessed in 80 OSCCs of independent validation cohort to identify the clinical relevance of its expression. By this comprehensive study, we identified 14-3-3 zeta as the only prognosticator of local recurrence-free survival (LRFS) and also an independently predicted factor of disease-specific survival (DSS).     

Lysophosphatidylcholine Acyltransferase1 Overexpression Promotes Oral Squamous Cell Carcinoma Progression via Enhanced Biosynthesis of Platelet-Activating Factor

Background

The relevance of lysophosphatidylcholine acyltransferase1 (LPCAT1), a cytosolic enzyme in the remodeling pathway of phosphatidylcholine metabolism, in oral squamous cell carcinoma (OSCC) is unknown. We investigated LPCAT1 expression and its functional mechanism in OSCCs.

Methods

We analyzed LPCAT1 mRNA and protein expression levels in OSCC-derived cell lines. Immunohistochemistry was performed to identify correlations between LPCAT1 expression levels and primary OSCCs clinicopathological status. We established LPCAT1 knockdown models of the OSCC-derived cell lines (SAS, Ca9-22) for functional analysis and examined the association between LPCAT1 expression and the platelet-activating factor (PAF) concentration and PAF-receptor (PAFR) expression.

Results

LPCAT1 mRNA and protein were up-regulated significantly (p<0.05) in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemistry showed significantly (p<0.05) elevated LPCAT1 expression in primary OSCCs compared with normal counterparts and a strong correlation between LPCAT1-positive OSCCs and tumoral size and regional lymph node metastasis. In LPCAT1 knockdown cells, cellular proliferation and invasiveness decreased significantly (p<0.05); cellular migration was inhibited compared with control cells. Down-regulation of LPCAT1 resulted in a decreased intercellular PAF concentration and PAFR expression.

Conclusion

LPCAT1 was overexpressed in OSCCs and correlated with cellular invasiveness and migration. LPCAT1 may contribute to tumoral growth and metastasis in oral cancer.     

微小RNAmiR-21和miR-31在13腔鳞癌组织中的表达及意义

目的：检测口腔鳞癌中微小RNA miR-21和miR-31的表达,探讨其与肿瘤发展的关系。方法：应用实时荧光定量PCR的方法检测72例口腔鳞癌,38例正常口腔粘膜miR-21和miR-31的表达,统计学分析其表达与肿瘤临床分期和病理分型的关系。结果：①口腔鳞癌组织与对照组比较,微小RNA miR-21和miR-31的表达都有显著增高（P〈0.05）,其中miR-21的增高更为显著（P〈0.001）。②统计学分析表明,miR-21的表达在晚期鳞癌组织较早中期鳞癌组织增高更为显著（P〈0.01）,在低分化鳞癌组织较中、高分化鳞癌组织增高更为显著（p〈0.001）;MiR-31的表达水平在不同肿瘤临床分期和病理分型鳞癌组织中无明显差异（P〉0.05）。结论：miR-21和miR-31在口腔鳞癌发生发展过程中表达有明显增高,其中miR-21表达水平可作为潜在的口腔鳞癌临床分期分级和预后的指标。     

Nuclear survivin promoted by acetylation is associated with the aggressive phenotype of oral squamous cell carcinoma

Defects in apoptotic pathway contribute to development and progression of oral cancer. Survivin, a member of the inhibitors of apoptosis protein (IAP) family, is increased in many types of cancers. However, it is unclear whether increased survivin is associated with oral squamous cell carcinomas (OSCC), and what mechanisms may involve in. In this study, we examined survivin expression in OSCC compared with normal oral tissues via immunohistochemical staining. The results showed that, not only total survivin is increased in OSCCs, but also the subcellular location of survivin is changed in OSCCs compared with normal oral tissues. In most of normal oral tissues, survivin staining was either negative, or cytoplasmic positive/nuclear negative; whereas in most of OSCC tissues, survivin staining was nuclear positive. Statistic analysis indicates that nuclear survivin, rather than total or cytoplasmic one, correlates with tumor TNM stage and differentiation grade. Consistently, in vitro analysis showed that survivin is in cytoplasm in normal human oral kinotinocyte (HOK) cells; whereas it is in nucleus in OSCC HN6 cells. Importantly, treatment of HOK cells with HDAC inhibitor Trichostatin A (TSA) induces survivin acetylation and promotes its nuclear localization. Moreover, nuclear survivin in OSCC cells was acetylated at K129 in its C-terminal, suggesting that the acetylation is important for nuclear location of survivin. Our study demonstrates that it is nuclear survivin, rather than total or cytoplasmic one, associates with TNM stage and tumor grade of OSCC. Thus, we propose nuclear survivin as a prognostic marker for the progression of OSCC.     

CMTM5 exhibits tumor suppressor activity through promoter methylation in oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is one of the most common types of malignancies in the head and neck region. CKLF-like MARVEL transmembrane domain-containing member 5 (CMTM5) has been recently implicated as a tumor suppressor gene in several cancer types. Herein, we examined the expression and function of CMTM5 in oral squamous cell carcinoma. CMTM5 was down-regulated in oral squamous cell lines and tumor samples from patients with promoter methylation. Treatment with the demethylating agent 5-aza-2′-deoxycytidine restored CMTM5 expression. In the OSCC cell lines CAL27 and GNM, the ectopic expression of CMTM5-v1 strongly inhibited cell proliferation and migration and induced apoptosis. In addition, CMTM5-v1 inhibited tumor formation in vivo. Therefore, CMTM5 might act as a putative tumor suppressor gene through promoter methylation in oral squamous cell carcinoma.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

Chemoresistance to Concanamycin A1 in Human Oral Squamous Cell Carcinoma Is Attenuated by an HDAC Inhibitor Partly via Suppression of Bcl-2 Expression

V-ATPase is involved in the acidification of the microenvironment around/in solid tumors, such as oral squamous cell carcinoma (OSCC). V-ATPase is thought to induce tumor invasion and multi-drug resistance in several malignant tumors, and it also contributes to maintaining the intracellular pH under an acidic microenvironment by inducing proton extrusion into the extracellular medium. However, there is little information regarding the effects of V-ATPase inhibitors on OSCCs. In this study, the effects of a V-ATPase inhibitor, concanamycin A1 (CMA), on the proliferation and apoptosis of OSCC were investigated in vitro. We used four OSCC cell lines, MISK81-5, SAS, HSC-4 and SQUU-B. Acridine orange staining revealed that the red fluorescence was reduced in all of the low concentration CMA-treated OSCC cells, indicating that the acidification of vesicular organelles in the OSCCs was prevented by the treatment with low-concentration of CMA. CMA treatment induced apoptosis in MISK81-5, SAS and HSC-4 cells, but not in SQUU-B cells. The p-p38 expression was not altered in CMA-treated SQUU-B cells, but their levels were increased in the other cells. The Bax/Bcl-2 ratio in CMA-treated SQUU-B cells was dramatically decreased in comparison with that in the other cell lines treated with CMA. However, when the SQUU-B cells were treated with CMA and a histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), the SQUU-B cells became more susceptible to the CMA-induced apoptosis. SAHA treatment led to a significantly decrease in the Bcl-2 expression in CMA-treated SQUU-B cells, resulting in a dramatically increased Bax/Bcl-2 ratio in comparison with that observed in the SQUU-B cells treated with CMA alone. These findings suggest that CMA could have an anti-tumor effect on OSCCs. In addition, combination of CMA with other agents, such as SAHA, could help improve the pro-apoptotic effects of CMA even in CMA-resistant OSCC cells.     

Survivin phosphorylation and M-phase promoting factor in oral carcinogenesis

Survivin is a recently described inhibitor of apoptosis and mitotic regulator which is selectively over-expressed in human tumors. Its expression rate is predictive of disease progression, early recurrences and resistance to therapy. Up-regulation of survivin in oral pre-malignant lesions (OPL) and in oral squamous cell carcinoma (OSCC) has already been demonstrated in previous studies. A critical step for activation of survivin has been identified in the phosphorylation on Thr34 by the main mitotic kinase p34cdc2-cyclin B1. The aim of this work was to investigate the relationship between survivin, its phosphorylated active form (p-survivin) and M-phase promoting factor (MPF), p34cdc2-cyclin B1 in oral carcinogenesis. 32 OSCCs and 17 OPLs from surgical specimens were studied for cyclin B1, p-survivin, survivin, and p34cdc2 expression by immunohistochemistry. All cases of OSCC expressed survivin and its expression rate was correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 80% of cases, while p-34cdc2 was over-expressed in all OSCCs. All OPLs associated with OSCC expressed survivin and its levels were correlated to p-survivin levels (P<0.05). Cyclin B1 was positive in 70% of cases, while p-34cdc2 was positive in all OPLs. In conclusion, this study demonstrated that MPF, survivin and p-survivin are expressed during early and late phase of oral carcinogenesis. MPF proteins, which are co-expressed on mitotic apparatus, could represent a potential target for therapies based on manipulation of survivin phosphorylation, which would induce apoptosis in cancer cells.     

Pro-inflammatory Genes as Biomarkers and Therapeutic Targets in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is a major health problem worldwide, and patients have a particularly poor 5-year survival rate. Thus, identification of the molecular targets in OSCC and subsequent innovative therapies are greatly needed. Prolonged exposure to alcohol, tobacco, and pathogenic agents are known risk factors and have suggested that chronic inflammation may represent a potential common denominator in the development of OSCC. Microarray analysis of gene expression in OSCC cell lines with high basal NF-κB activity and OSCC patient samples identified dysregulation of many genes involved in inflammation, wound healing, angiogenesis, and growth regulation. In particular IL-8, CCL5, STAT1, and VEGF gene expression was up-regulated in OSCC. Moreover, IL-8 protein levels were significantly higher in OSCC cell lines as compared with normal human oral keratinocytes. Targeting IL-8 expression by siRNA significantly reduced the survival of OSCC cells, indicating that it plays an important role in OSCC development and/or progression. Inhibiting the inflammatory pathway by aspirin and the proteasome/NF-κB pathway by bortezomib resulted in marked reduction in cell viability in OSCC lines. Taken together our studies indicate a strong link between inflammation and OSCC development and reveal IL-8 as a potential mediator. Treatment based on prevention of general inflammation and/or the NF-κB pathway shows promise in OSCCs.     

Allicin Could Potentially Alleviate Oral Cancer Pain by Inhibiting “Pain Mediators” TNF-alpha,IL-8, and Endothelin

To evaluate the effects of allicin on mediators of pain secreted by oral cancer cells in vitro, single-cell suspensions were prepared by enzymatic method from oral squamous cell carcinoma (OSCC). Cancer stem cells were isolated by the CD133+ selection method with magnetic cell sorting. Stemness markers were checked in both cancer cells and cancer stem cells by RT-PCR. Comparative analysis of pain mediators TNF-alpha, IL-8, and endothelin at both RNA and protein levels for normal epithelial cells, cancer cells, and cancer stem cells was carried out with and without allicin treatment. CD133 and CD44 expression levels were checked in cancer cells and cancer stem cells flow cytometrically. Allicin inhibited both gene and protein expression of TNF-alpha, IL-8, and endothelin in both cancer cells and cancer stem cells. Allicin is more likely to be a promising treatment in alleviating the levels of pain and inflammation in OSCCs.     

Study of feline oral squamous cell carcinoma: potential target for cyclooxygenase inhibitor treatment

Oral squamous cell carcinoma (OSCC) is associated with high morbidity and mortality. A potential target for OSCC treatment is cyclooxygenase-2 (cox-2). Pet cats with naturally occurring OSCC may offer the opportunity to study anticancer activity of cox inhibitors. Cox-2 expression in feline OSCC was determined by immunohistochemistry. High intensity cox-2 immunoreactivity was detected in 6 of 34 (18%) feline OSCC samples. Weak immunoreactivity was noted in 22 OSCCs and in epithelial cells from oral mucosa of clinically normal cats. Pharmacokinetics of a cox inhibitor (piroxicam, 0.3 mg/kg) were studied in carcinoma-bearing cats to confirm a dose for follow-up trials. The average peak serum piroxicam concentration (948 ng/ml, which inhibited cox-2 activity) and serum half-life (15.9 h) were similar to that in normal cats. These results provide information (cox-2 expression as an inclusion criteria, 0.3 mg/kg daily piroxicam) for the design of follow-up trials of cox inhibitor treatment in pet cats with OSCC.     

Potential involvement of MYC- and p53-related pathways in tumorigenesis in human oral squamous cell carcinoma revealed by proteomic analysis

Oral squamous cellular carcinoma is a malignant tumor with poor prognosis. Discovery of early markers to discriminate between malignant and normal cells is of high importance in clinical diagnosis. Subcellular fractions from 10 oral squamous cell carcinoma and corresponding control samples, enriched in mitochondrial and cytosolic proteins, as well as blood from the tumor were analyzed by proteomics, two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Three-hundred and fifty different gene products were identified. Twenty proteins showed deranged levels in oral squamous cell carcinoma in comparison with the control samples and are potentially involved in tumor growth and metastasis. Of these, 16 proteins were upregulated. By applying pathway analysis, we found 8 of the upregulated gene products to be linked to three main locus genes, p53, MYC, and MYCN, and could be candidate biomarkers for OSCC. The findings of this pilot study show that OSCC gene ontology combined with proteomic analysis is a powerful tool in systems biology for the elucidation of the complexity of expression profiles in cellular processes. Application of such pathway analysis has the potential to generate new insights into complex molecular mechanisms underlying disease related processes and could therefore significantly contribute to the efficient performance of the entire discovery process.