Overexpression of Translocation Associated Membrane Protein 2 Leading to Cancer-Associated Matrix Metalloproteinase Activation as a Putative Metastatic Factor for Human Oral Cancer.

Translocation associated membrane protein 2 (TRAM2) has been characterized as a component of the translocon that is a gated channel at the endoplasmic reticulum (ER) membrane. TRAM2 is expressed in a wide variety of human organs. To date, no information is available regarding TRAM2 function in the genesis of human cancer. The purpose of this study was to investigate the status of the TRAM2 gene in oral squamous cell carcinoma (OSCC) cells and clinical OSCC samples. Using real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting analysis, and immunohistochemistry, we detected accelerated TRAM2 mRNA and protein expression levels both in OSCC-derived cell lines and primary tumors. Moreover, TRAM2-positive OSCC tissues were correlated closely (P<0.05) with metastasis to regional lymph nodes and vascular invasiveness. Of note, knockdown of TRAM2 inhibited metastatic phenotypes, including siTRAM2 cellular migration, invasiveness, and transendothelial migration activities with a significant (P<0.05) decrease in protein kinase RNA(PKR) - like ER kinase (PERK) and matrix metalloproteinases (MMPs) (MT1-MMP, MMP2, and MMP9). Taken together, our results suggested that TRAM2 might play a pivotal role in OSCC cellular metastasis by controlling major MMPs. This molecule might be a putative therapeutic target for OSCC.

FBLIM1 enhances oral cancer malignancy via modulation of the epidermal growth factor receptor pathway.

Filamin-binding LIM protein 1 (FBLIM1) is related to regulation of inflammatory responses, such as chronic recurrent multifocal osteomyelitis; however, the relevance of FBLIM1 in oral squamous cell carcinoma (OSCC) is unknown. The aim of the current study was to elucidate the possible role of FBLIM1 in the carcinogenesis of OSCC. We analyzed FBLIM1 expression using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunoblot analysis, and immunohistochemistry. The expression levels of FBLIM1 were up-regulated significantly (P < 0.05) in OSCC-derived cell lines and primary OSCCs specimens compared with normal counterparts. FBLIM1 expression also was correlated with the primary tumoral size (P < 0.05) and vascular invasion (P < 0.05). We then assessed tumoral progression after treatment with FBLIM1 siRNA and clopidogrel, an antiplatelet agent. Similar to the FBLIM1 knockdown effect, clopidogrel-treated cells had attenuated functions of proliferation, migration, and invasiveness. Interestingly, clopidogrel treatment led to down-regulation of epidermal growth factor receptor (EGFR) and FBLIM1. These findings identify FBLIM1 as a putative therapeutic target by using clopidogrel for inhibiting over activation of EGFR signaling to prevent OSCC malignancy.

Critical role of deoxynucleotidyl transferase terminal interacting protein 1 in oral cancer.

Deoxynucleotidyl transferase terminal interacting protein 1 (DNTTIP1) forms a complex with histone deacetylase (HDAC); however, the relevance of DNTTIP1 in cancer remains unknown. The aim of this study was to examine DNTTIP1 expression and its functional mechanisms in oral squamous cell carcinomas (OSCCs). DNTTIP1 expression was analyzed by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting analysis, and immunohistochemistry. The expression of DNTTIP1 was upregulated significantly in vitro and in vivo, and in patients with OSCC in whom DNTTIP1 was overexpressed and the expression level was correlated significantly (P < 0.05) with tumoral growth. DNTTIP1 knockdown (siDNTTIP1) cells showed depressed cellular proliferation by cell-cycle arrest at the G1 phase with high acetylation of p53 and upregulation of p21Cip1. Moreover, resveratrol, a HDAC inhibitor, controlled not only acetylated p53 status but also DNTTIP1 expression, leading to a similar phenotype of siDNTTIP1 cells. A marked (P < 0.05) reduction of tumoral growth in mouse xenograft models was observed with lower DNTTIP1 expression under the presence of this chemical reagent. Taken together, our results suggested that DNTTIP1-HDAC interaction promotes tumoral growth through deacetylation of p53 and that DNTTIP1 might be a critical therapeutic target in OSCCs.

Multiple coagulation factor deficiency protein 2 as a crucial component in metastasis of human oral cancer.

Multiple coagulation factor deficiency protein 2 (MCFD2), a binding partner of lectin mannose binding 1 (LMAN1), causes combined deficiencies of coagulation factors V and VIII. MCFD2 function in inherited hematologic disorders is well elucidated; however, little is known about its role in human tumorigenesis. The aim of the current study was to investigate the states of MCFD2 in oral squamous cell carcinoma (OSCC). The expression of MCFD2 was up-regulated significantly in all cell lines examined. Evaluation of the cellular functions associated with tumoral metastasis showed that MCFD2 knockdown (shMCFD2) cells exhibited significantly lower cellular invasiveness and migration and higher cellular adhesion compared with shControl cells. Of note, shMCFD2 cells also showed weak immunoreactivity of LMAN1 and a lower secretion level of galactoside-binding soluble 3 binding protein (LGALS3BP). In addition to in vitro validation, clinical data on 70 patients with OSCC indicated that state of MCFD2 expression level is associated with regional lymph node metastasis. Altogether, we have demonstrated that MCFD2 promotes cancer metastasis by regulating LMAN1 and LGALS3BP expression levels. Hence, MCFD2 may represent a promising candidate for a novel therapeutic target for patients with metastatic OSCCs.

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression.

Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

Evaluation of tryptophan-aspartic acid repeat-containing protein 34 as a novel tumor-suppressor molecule in human oral cancer.

Tryptophan-aspartic acid (WD) repeat-containing protein 34 (WDR34), one of the WDR protein superfamilies with five WD40 domains, inhibits a transforming growth factor-beta (TGF-ß) activated kinase 1 (TAK1)-associated NF-κB activation pathway. Nevertheless, little is known about the roles of WDR34 in cancer. The current study sought to elucidate the clinical relevance of WDRsfb34 in oral squamous cell carcinoma (OSCC). We found WDR34 down-regulation in OSCCs compared with normal control tissues using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Models of overexpression of WDR34 (oeWDR34) showed depressed cellular growth through cell-cycle arrest at the G1 phase. To investigate the inhibitory function of WDR34, we challenged oeWDR34 cells with interleukin (IL)-1, a ligand for activation of the TAK1-NF-κB pathway and assessed the expression of a target gene of the pathway. oeWDR34 strongly inhibited IL-6 expression, which is closely related to tumoral growth, compared with control cells, suggesting that WDR34 would be a critical molecule for control of tumoral progression. In addition to the in vitro experiments, WDR34 negativity was correlated with tumoral growth of OSCCs. Our findings suggested that WDR34 inhibits OSCC progression and might be a potential tumor-suppressor molecule in OSCCs.

ARNT2 Regulates Tumoral Growth in Oral Squamous Cell Carcinoma.

Aryl hydrocarbon receptor nuclear translocator (ARNT) 2 is a transcriptional factor related to adaptive responses against cellular stress from a xenobiotic substance. Recent evidence indicates ARNT is involved in carcinogenesis and cancer progression; however, little is known about the relevance of ARNT2 in the behavior of oral squamous cell carcinoma (OSCC). In the current study, we evaluated the ARNT2 mRNA and protein expression levels in OSCC in vitro and in vivo and the clinical relationship between ARNT2 expression levels in primary OSCCs and their clinicopathologic status by quantitative reverse transcriptase-polymerase chain reaction, immunoblotting, and immunohistochemistry. Using ARNT2 overexpression models, we performed functional analyses to investigate the critical roles of ARNT2 in OSCC. ARNT2 mRNA and protein were down-regulated significantly (P < 0.05 for both comparisons) in nine OSCC-derived cells and primary OSCC (n=100 patients) compared with normal counterparts. In addition to the data from exogenous experiments that ARNT2-overexpressed cells showed decreased cellular proliferation, ARNT2-positive OSCC cases were correlated significantly (P < 0.05) with tumoral size. Since von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase, a negative regulator of hypoxia-inducible factor (HIF1)-α, is a downstream molecule of ARNT2, we speculated that HIF1-α and its downstream molecules would have key functions in cellular growth. Consistent with our hypothesis, overexpressed ARNT2 cells showed down-regulation of HIF1-α, which causes hypofunctioning of glucose transporter 1, leading to decreased cellular growth. Our results proposed for the first time that the ARNT2 level is an indicator of cellular proliferation in OSCCs. Therefore, ARNT2 may be a potential therapeutic target against progression of OSCCs.

Suppression of metastasis by mirtazapine via restoration of the Lin-7C/ß-catenin pathway in human cancer cells.

No definitive therapy exists to treat human metastatic tumors. We reported previously that down-regulation of Lin-7C is essential for metastasis of human squamous cell carcinomas (hSCCs). In this study, we investigated the chemical restoration of Lin-7C expression and demonstrated its effectiveness for suppressing the metastatic potential in human cancer cells. Ingenuity Pathway Analysis (IPA) identified candidate chemical agents, i.e., apomorphine, caffeine, risperidone, quetiapine, and mirtazapine. Among them, mirtazapine, an antagonist of HTR2C, an upstream molecule of Lin-7C, caused substantial up-regulation of the Lin-7C/ß-catenin pathway in a metastatic hSCC cell line and human melanoma-derived cell line in vitro, and up-regulation did not contribute to cellular proliferation. Moreover, the antimetastatic effect of mirtazapine in these metastatic cell lines in vivo also was evident in multiple organs of immunodeficient mice with no marked side effects. The current data offer novel information for further study of antimetastatic activity in association with enhanced Lin-7C/ß-catenin pathway activation with mirtazapine.

Lipocalin-2 is associated with radioresistance in oral cancer and lung cancer cells.

The aim of the present study was to identify a target molecule that could predict the efficacy of radiotherapy in oral squamous cell carcinoma (OSCC). We used DNA microarray analysis to identify differences in gene expression after X-ray irradiation. We compared the gene expression profiles between X-ray (8 Gy)-irradiated Ca9-22 cells (an OSCC-derived cell line) and unirradiated Ca9-22 cells. A total of 167 genes with a 2-fold higher level of expression induced by X-ray irradiation were identified. Lipocalin-2 (LCN2) had the greatest increase in expression after X-ray irradiation, and it was categorized in a network that has cancer-related functions with the Ingenuity Pathway Analysis tool. Upregulated expression of LCN2 mRNA was validated by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. When the LCN2 gene was knocked down in OSCC cells (Ca9-22 and HSC-2) and lung cancer cells (A549) by using small interfering RNA, the radiosensitivity of these cells was enhanced. Our findings suggest that the overexpression of LCN2 is likely associated with radioresistance in oral cancer and lung cancer cells, and that LCN2 expression levels could be used to predict radioresistance. Thus, regulating the expression or function of LCN2 could enhance the radiation response, resulting in a favorable outcome of radiotherapy.

Expression status of Zic family member 2 as a prognostic marker for oral squamous cell carcinoma.

PURPOSE: To determine the involvement of ZIC2 in oral squamous cell carcinoma (OSCC). METHODS: ZIC2 mRNA and protein expression in primary OSCCs (n = 74), oral premalignant lesions (OPLs, n = 20) and five OSCC-derived cell lines (HSC-2, HSC-3, OK-92, H1, and Sa3) were analyzed by quantitative reverse transcriptase-polymerase chain reaction, Western blot and immunohistochemistry (IHC). In addition, we evaluated the correlation between ZIC2 IHC scores in OSCCs and the clinicopathologic status. RESULTS: Significant up-regulation of ZIC2 was detected in OSCC-derived cell lines (P < 0.05), primary OSCCs (P < 0.05) and OPLs (P < 0.05) compared with normal counterparts. Among the clinical variables analyzed, ZIC2 expression was associated with the histopathologic types of OSCC. Furthermore, the survival rates differed significantly between ZIC2-positive cases and ZIC2-negative cases. CONCLUSIONS: These results suggested that ZIC2 expression is correlated with the differentiation type of OSCC and diagnosis and might be a potential prognostic indicator and therapeutic target for OSCCs.

Alteration of extracellular superoxide dismutase expression is associated with an aggressive phenotype of oral squamous-cell carcinoma.

Oxidative stress results in damage to cellular structures and has been linked to numerous diseases, including cancer. Extracellular superoxide dismutase (EC-SOD) is a principal enzymatic antioxidant in extracellular space. The purpose of this study was to determine whether the expression of EC-SOD protein is altered in the carcinogenetic process of oral squamous-cell carcinoma (OSCC). Immunohistochemical analysis was carried out in matched normal and tumour specimens collected from 58 OSCCs and 20 oral premalignant lesions (OPLs). Correlations between the EC-SOD expression levels and clinicopathological features of OSCC patients were evaluated by Fisher's exact test. Although EC-SOD protein was consistently expressed on the plasma membrane of cells in normal tissues, plasma membranous EC-SOD expression was lost in almost all the OSCC specimens examined (98%). Instead, positive EC-SOD expression was detected in the cytoplasmic compartments of cancerous cells in both OPLs (65%) and OSCCs (52%), together with a high incidence of lymph node metastasis (p=0.0397). These results suggest that the dysregulation of EC-SOD protein expression is a frequently occuring and early event in oral carcinogenesis, and that cytoplasmic EC-SOD may contribute to the increased aggressiveness of OSCC.

Characterization of intracellular superoxide dismutase alterations in premalignant and malignant lesions of the oral cavity: correlation with lymph node metastasis.

PURPOSE: The purpose of this study was to characterize changes in the expression of copper-zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD (Mn-SOD) in oral squamous-cell carcinoma (OSCC). METHODS: Real-time quantitative reverse transcriptase-polymerase chain reaction analysis of Cu/Zn-SOD and Mn-SOD mRNA expression was carried out in 50 pairs of OSCC tissue specimens and corresponding normal tissues. Mn-SOD protein expression was evaluated further in 65 OSCC tissue samples and 33 oral premalignant lesions (OPLs) using immunohistochemistry. RESULTS: Significant (P < 0.001) upregulation of Mn-SOD mRNA expression was observed in OSCC tissues compared with the normal tissue counterparts, whereas no significant difference was detected in Cu/Zn-SOD expression. Significant increases in Mn-SOD protein expression were seen in both OPLs (P < 0.001) and OSCC tissue (P < 0.001) together with a high incidence of lymph node metastasis (P = 0.04). CONCLUSIONS: Our findings suggested that Mn-SOD overexpression is a frequent and early event during oral carcinogenesis and could contribute to aggressive OSCC.

Downregulation of carcinoembryonic antigen-related cell adhesion molecule 1 in oral squamous cell carcinoma: correlation with tumor progression and poor prognosis.

OBJECTIVE: To identify genes associated with therapeutic targets of oral squamous cell carcinoma (OSCC), we compared gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes. METHODS: We analyzed the gene expression profiles of OSCCs using Affymetrix GeneChip analysis. The identified genes were analyzed by an Ingenuity Pathway Analysis tool to identify networks of interacting genes. A candidate gene was further evaluated for the expression status of the mRNA and protein in OSCC-derived cell lines and primary OSCCs. RESULTS: The microarray data identified 188 genes downregulated in OSCC-derived cell lines, and the genetic pathways associated with expression changes were generated. Among the genes mapped to the network with the highest significance, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was analyzed further. CEACAM1 mRNA and protein were frequently downregulated in OSCC-derived cell lines compared with human normal oral keratinocytes. Immunohistochemical analysis showed that primary OSCCs were significantly decreased in CEACAM1. Moreover, CEACAM1 expression was correlated with the TNM staging. We also found that CEACAM1-negative expression was significant both for disease-free (p = 0.036) and overall survival (p = 0.032). CONCLUSION: Repression of CEACAM1 could contribute to cancer progression and may indicate a poor prognosis for patients with OSCC.

Down-regulation of plasma membranous Annexin A1 protein expression in premalignant and malignant lesions of the oral cavity: correlation with epithelial differentiation.

PURPOSE: To determine the potential involvement of ANXA1 in oral squamous-cell carcinoma (OSCC), we evaluated the ANXA1 protein expression in oral premalignant lesions (OPLs) and OSCCs and correlated the results with clinicopathologic variables. METHODS: Matched normal and tumour specimens of 44 primary OSCCs and 28 OPLs were analyzed for ANXA1 subcellular localization and protein expression level by immunohistochemistry (IHC). Correlations between ANXA1-IHC staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. RESULTS: Markedly down-regulation of ANXA1 protein expression was identified on the plasma membrane of epithelial cells in OSCCs (P < 0.001) and OPLs (P = 0.001) compared with normal counterparts. Moreover, loss of plasma membranous ANXA1 expression was significantly correlated with the poorly differentiated status of OSCC cells (P = 0.012). CONCLUSIONS: Our findings suggest that loss of ANXA1 is frequent and early event during oral carcinogenesis and that ANXA1 could contribute to maintaining epithelial differentiation in OSCC.

Overexpression and altered subcellular localization of autophagy-related 16-like 1 in human oral squamous-cell carcinoma: correlation with lymphovascular invasion and lymph-node metastasis.

Autophagy is a dynamic process of subcellular degradation, which has recently sparked great interest because it is involved in various developmental processes and various diseases including cancer. Autophagy-related 16-like 1 is a component of a large protein complex essential for autophagosome formation. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous cell carcinoma cells and detected significantly high expression levels of autophagy-related 16-like 1 in oral squamous cell carcinoma-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of autophagy-related 16-like 1 in oral squamous cell carcinoma, we evaluated the state of autophagy-related 16-like 1 protein expression in human oral premalignant lesions and primary oral squamous cell carcinomas, and correlated the results with clinicopathologic variables. Autophagy-related 16-like 1 immunoreaction was predominant in a variety of subcellular components of oral squamous cell carcinoma tissues, including the cytoplasm and plasma membrane of malignant cells (45% and 39%, respectively) and peritumoral and intratumoral stroma (52%), whereas all of the components in normal tissues had no or faint autophagy-related 16-like 1 expression. In addition, high stromal expression of autophagy-related 16-like 1 was associated significantly with lymphovascular invasion of tumor cells (P = .037) and positive lymph node status (P = .015). Furthermore, cytoplasmic and plasma membranous autophagy-related 16-like 1 were also expressed in abundance in the oral premalignant lesion cells (74% and 32%, respectively). Our finding suggests that dysregulation of autophagy-related 16-like 1 protein expression is a frequent and early event during oral carcinogenesis and could affect the malignant behavior of oral squamous cell carcinoma cells.

Susceptible genes and molecular pathways related to heavy ion irradiation in oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Heavy ion beams are high linear energy transfer (LET) radiation characterized by a higher relative biologic effectiveness than low LET radiation. The aim of the current study was to determine the difference of gene expression between heavy ion beams and X-rays in oral squamous cell carcinoma (OSCC)-derived cells. MATERIALS AND METHODS: The OSCC cells were irradiated with accelerated carbon or neon ion irradiation or X-rays using three different doses. We sought to identify genes the expression of which is affected by carbon and neon ion irradiation using Affymetrix GeneChip analysis. The identified genes were analyzed using the Ingenuity Pathway Analysis Tool to investigate the functional network and gene ontology. Changes in mRNA expression in the genes were assessed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The microarray analysis identified 84 genes that were modulated by carbon and neon ion irradiation at all doses in OSCC cells. Among the genes, three genes (TGFBR2, SMURF2, and BMP7) and two genes (CCND1 and E2F3), respectively, were found to be involved in the transforming growth factor beta-signaling pathway and cell cycle:G1/S checkpoint regulation pathway. The qRT-PCR data from the five genes after heavy ion irradiation were consistent with the microarray data (P < 0.01). CONCLUSION: Our findings should serve as a basis for global characterization of radiation-regulated genes and pathways in heavy ion-irradiated OSCC.

PTEN expression and methylation status in oral squamous cell carcinoma.

PTEN is a tumor suppressor gene located on chromosome10q23.3. In addition to genetic mutations and deletions, the down-regulation of PTEN has been found in various malignant tumors. However, little is known about the profile of PTEN gene in oral carcinomas. In this study, the expression profiles and genetic alterations of PTEN were examined in 113 oral squamous cell carcinoma (OSCC) cases and 9 OSCC-derived cell lines. An immunohistochemical analysis showed statistically significant differences in the immunohistochemical (IHC) scores for PTEN protein in normal tissues and in cancerous regions (P=0.0104), suggesting that PTEN protein expression is down-regulated in OSCC. No significant correlations existed between the down-regulation of PTEN protein expression and the clinicopathological features of the tumor. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed a lower PTEN mRNA expression in the 9 OSCC-derived cell lines examined, as compared to the normal oral epithelium cells. However, treatment with a demethylating reagent restored PTEN mRNA expression in 4 cell lines. No genetic mutations were detected in these cell lines by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The results suggest that epigenetic changes may be related to the down-regulation of PTEN expression. We therefore conclude that PTEN is a crucial molecule in the tumorigenesis of OSCC.

Hyaluronan-mediated motility: a target in oral squamous cell carcinoma.

To characterize cancer-related gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with human normal oral keratinocytes (HNOKs). Microarray analysis identified 166 genes that were up-regulated in OSCC-derived cell lines. Gene ontology analysis showed that cancer-related function had the highest significance. Among the genes mapped to the cancer-related network with the highest significance, the receptor for hyaluronan-mediated motility (RHAMM) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs. Overexpression of RHAMM protein was observed in all cell lines compared to HNOKs. Immunohistochemical analysis showed highly expressed RHAMM in primary OSCCs, whereas most corresponding normal tissues had no or significant down-regulation of protein immunoreactivity. Real-time quantitative reverse transcriptase-polymerase chain reaction data agreed with the protein expression. Moreover, the RHAMM expression status was correlated with the TNM stage (P<0.001). The results suggested that RHAMM expression may be correlated with tumor aggressiveness and offer clues to the development of new treatments for human OSCCs.

Establishment and characterization of a cisplatin-resistant cell line, KB-R, derived from oral carcinoma cell line, KB.

To investigate the mechanism of the resistance to cisplatin (CDDP), we established the CDDP-resistant cell line, KB-R, from CDDP-sensitive oral carcinoma cell line, KB. The 3-(3, 4-dimethyl-thiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay indicated that KB-R is 5.5-fold more resistant to CDDP than KB. Microarray analysis indicated that the expression levels of 1,718 genes were elevated at least five-fold or more in KB-R, compared with KB. The expression status of ATP binding cassette (ABC) transporter genes, which belong to multi-drug resistance genes, was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction and real-time PCR. MRP1 and MRP2 were up-regulated, whereas MDR1 was down-regulated. Pathway and ontology analysis using the Ingenuity Pathway Analysis tool indicated three highly significant genetic networks including 105 of the 1,718 overexpressed genes and one network including 35 'cell-to-cell signaling and interaction' related genes. Our results suggested that these cell lines, KB and KB-R, may be useful for searching the candidate genes responsible for CDDP-resistance and for further study to understand the mechanism of CDDP-resistance.