A Case of Nonocclusive Mesenteric Ischemia During Bioradiotherapy With Cetuximab.

Nonocclusive mesenteric ischemia (NOMI) causes mesenteric ischemia and intestinal necrosis despite the absence of organic obstruction, such as thrombi and emboli in mesenteric blood vessels, and it has an extremely poor prognosis. We report a case of NOMI developed during bioradiotherapy (BRT) with cetuximab for cervical lymph node metastasis of tongue cancer. The patient was a 73-year-old man who underwent right radical neck dissection for neck lymph node metastasis after tongue cancer surgery. Postoperatively, the patient received BRT with cetuximab. On the 34th day after BRT, the patient had abdominal distension and a decreased level of consciousness. Contrast-enhanced computed tomography revealed mesenteric ischemia without thrombi and extensive intestinal emphysema. The patient was diagnosed with NOMI. Furthermore, he had septic shock and was treated with vasopressors and antibacterial agents; however, the condition of the patient did not improve, and he died on the same day.

Prognostic Significance of Serum Interleukin-6 Levels in Oral Squamous Cell Carcinoma.

Introduction The prognosis of oral squamous cell carcinoma (OSCC) is often poor despite standard treatments. Additionally, no useful prognostic markers are available. Therefore, we aimed to investigate the relationship between serum Interleukin-6 (IL-6) levels and prognosis and explore its local and systemic effects in patients with OSCC. Methods Ninety-five new cases of OSCC were included, and the prognosis was compared between high and low serum IL-6 groups. The localization of IL-6 in OSCC tissues was examined. Furthermore, a comprehensive gene expression analysis was performed in OSCC tissues and compared between the two groups. Results A significant difference in overall survival and disease-free survival was observed. Furthermore, a substantial expression of IL-6 was localized in the stroma. Comprehensive gene expression analysis of tumor localization showed increased expression of genes related to oxidoreductase and lipid metabolism in the primary tissues of the group with high serum IL-6 levels. Regarding the correlation between blood tests and serum IL-6 levels, a strong positive correlation was observed between inflammatory responses and nutritional factors. Conclusion These results suggest that serum IL-6 may be a prognostic factor for metabolic abnormalities in patients with OSCC and that aggressive nutritional interventions may contribute to prognosis.

KRT13 and UPK1B for differential diagnosis between metastatic lung carcinoma from oral squamous cell carcinoma and lung squamous cell carcinoma.

Oral squamous cell carcinomas unusually show distant metastasis to the lung after primary treatment, which can be difficult to differentiate from primary squamous cell carcinoma of the lung. While the location and number of tumor nodules is helpful in diagnosing cases, differential diagnosis may be difficult even with histopathological examination. Therefore, we attempted to identify molecules that can facilitate accurate differential diagnosis. First, we performed a comprehensive gene expression analysis using microarray data for OSCC-LM and LSCC, and searched for genes showing significantly different expression levels. We then identified KRT13, UPK1B, and nuclear receptor subfamily 0, group B, member 1 (NR0B1) as genes that were significantly upregulated in LSCC and quantified the expression levels of these genes by real-time quantitative RT-PCR. The expression of KRT13 and UPK1B proteins were then examined by immunohistochemical staining. While OSCC-LM showed no KRT13 and UPK1B expression, some tumor cells of LSCC showed KRT13 and UPK1B expression in 10 of 12 cases (83.3%). All LSCC cases were positive for at least one of these markers. Thus, KRT13 and UPK1B might contribute in differentiating OSCC-LM from LSCC.

MicroRNA-1289 Functions as a Novel Tumor Suppressor in Oral Squamous Cell Carcinoma.

Recently, numerous tumor-suppressive microRNAs (TS-miRs) have been identified in human malignancies. Here, we attempted to identify novel TS-miRs in oral squamous cell carcinoma (OSCC). First, we transfected human OSCC cells individually with 968 synthetic miRs mimicking human mature miRs individually, and the growth of these cells was evaluated using the WST-8 assay. Five miR mimics significantly reduced the cell growth rate by less than 30%, and the miR-1289 mimic had the most potent growth inhibitory effect among these miRs. Subsequently, we assessed the in vivo growth-inhibitory effects of miR-1289 using a mouse model. The administration of the miR-1289 mimic-atelocollagen complex significantly reduced the size of subcutaneously xenografted human OSCC tumors. Next, we investigated the expression of miR-1289 in OSCC tissues using reverse transcription-quantitative PCR. The expression level of miR-1289 was significantly lower in OSCC tissues than in the adjacent normal oral mucosa. Furthermore, 15 genes were identified as target genes of miR-1289 via microarray and Ingenuity Pathway Analysis (IPA) microRNA target filtering. Among these genes, the knockdown of magnesium transporter 1 (MAGT1) resulted in the most remarkable cell growth inhibition in human OSCC cells. These results suggested that miR-1289 functions as a novel TS-miR in OSCC and may be a useful therapeutic tool for patients with OSCC.

The mutational spectrum in whole exon of p53 in oral squamous cell carcinoma and its clinical implications.

Mutations in p53 are common in human oral squamous cell carcinoma (OSCC). However, in previous analyses, only detection of mutant p53 protein using immunohistochemistry or mutations in some exons have been examined. Full length mutant p53 protein in many cases shows a loss of tumor suppressor function, but in some cases possibly shows a gain of oncogenic function. In this study, we investigate relationships of outcomes with the mutational spectrum of p53 (missense and truncation mutations) in whole exon in OSCC. Specimens from biopsy or surgery (67 cases) were evaluated using next-generation sequencing for p53, and other oncogenic driver genes. The data were compared with overall survival (OS) and disease-free survival (DFS) using univariate and multivariate analyses. p53 mutations were detected in 54 patients (80.6%), 33 missense mutations and 24 truncation mutations. p53 mutations were common in the DNA-binding domain (43/52) and many were missense mutations (31/43). Mutations in other regions were mostly p53 truncation mutations. We detected some mutations in 6 oncogenic driver genes on 67 OSCC, 25 in NOTCH1, 14 in CDKN2A, 5 in PIK3CA, 3 in FBXW7, 3 in HRAS, and 1 in BRAF. However, there was no associations of the p53 mutational spectrum with mutations of oncogenic driver genes in OSCC. A comparison of cases with p53 mutations (missense or truncation) with wild-type p53 cases showed a significant difference in lymph node metastasis. DFS was significantly poorer in cases with p53 truncation mutations. Cases with p53 truncation mutations increased malignancy. In contrast, significant differences were not found between cases with p53 missense mutations and other mutations. The p53 missense mutation cases might include cases with mostly similar function to that of the wild-type, cases with loss of function, and cases with various degrees of gain of oncogenic function.

Human papillomavirus-16 infection and p16 expression in oral squamous cell carcinoma.

Human papillomavirus (HPV) is a possible carcinogenetic factor in oral squamous cell carcinoma (OSCC). Previous studies have reported the prevalence of HPV in patients with OSCC. However, the association between HPV and OSCC remains controversial. The present study aimed to clarify the association between HPV infection, p16 protein expression and the clinicopathological characteristics of OSCC. The expression level of HPV-16E6 mRNA and p16 protein, a known surrogate marker of HPV infection, was investigated in 100 OSCC cases using TaqMan reverse transcription-quantitative PCR and immunohistochemistry staining, respectively. HPV-16E6 mRNA expression level was only detected in one case (1%), and positive expression of p16 was found in 10 cases (10%), including an HPV-positive case. Subsequently, the association between p16 expression level and clinicopathological characteristic factors were analyzed; however, no significant association was found. These results suggested that HPV-16 infection was less likely to cause OSCC in Japan and p16 expression was not a suitable marker for HPV infection in OSCC.

Oral squamous cell carcinoma may originate from bone marrow-derived stem cells.

Molecules that demonstrate a clear association with the aggressiveness of oral squamous cell carcinoma (OSCC) have not yet been identified. The current study hypothesized that tumor cells in OSCC have three different origins: Epithelial stem cells, oral tissue stem cells from the salivary gland and bone marrow (BM) stem cells. It was also hypothesized that carcinomas derived from less-differentiated stem cells have a greater malignancy. In the present study, sex chromosome analysis by fluorescence in situ hybridization and/or microdissection PCR was performed in patients with OSCC that developed after hematopoietic stem cell transplantation (HSCT) from the opposite sex. OSCC from 3 male patients among the 6 total transplanted patients were considered to originate from donor-derived BM cells. A total of 2/3 patients had distant metastasis, resulting in a poor prognosis. In a female patient with oral potentially malignant disorder who underwent HSCT, there were 10.7% Y-containing cells in epithelial cells, suggesting that some epithelial cells were from the donor. Subsequently, gene expression patterns in patients with possible BM stem cell-derived OSCC were compared with those in patients with normally developed OSCC by microarray analysis. A total of 3 patients with BM stem cell-derived OSCC exhibited a specific pattern of gene expression. Following cluster analysis by the probes identified on BM stem cell-derived OSCC, 2 patients with normally developed OSCC were included in the cluster of BM stem cell-derived OSCC. If the genes that could discriminate the origin of OSCC were identified, OSCCs were classified into the three aforementioned categories. If diagnosis can be performed based on the origin of the cancer cells, a more specific therapeutic strategy may be implemented to improve prognosis. This would be a paradigm shift in diagnostic and therapeutic strategies for OSCC.

Troglitazone, a Selective Ligand for PPARγ, Induces Cell-cycle Arrest in Human Oral SCC Cells.

AIM: We attempted to clarify the role of Peroxisome proliferator-activated receptor γ (PPARγ) and its ligand, troglitazone (TRO) on oral squamous cell carcinoma (SCC). MATERIALS AND METHODS: The expression of PPARγ gene was examined in 47 human oral SCC tissues and two human oral SCC cell lines, CA9-22 and HSC-4. The effects of TRO on the growth and cell-cycle progression of human oral SCC cells were examined. RESULTS: PPARγ mRNA was detected in 20 of 47 oral SCC tissues and two human oral SCC cells. TRO significantly suppressed the growth of the cells, but did not induce apoptosis. CA9-22 cells treated with TRO showed an increased fraction in the G1 phase and decreased fractions in the S and G2-M phases. CONCLUSION: TRO did not induce apoptosis in oral SCC cells, but did inhibit the growth of the cells by arresting the cell cycle at G1 phase.

MicroRNA-361-3p is a potent therapeutic target for oral squamous cell carcinoma.

MicroRNAs (miRNAs) can act not only as tumor suppressor genes but also as oncogenes. Oncogenic miRNAs (oncomiRs) could therefore provide opportunities for the treatment of human malignancies. Here, we aimed to identify oncomiRs present in oral squamous cell carcinoma (OSCC) and addressed whether targeting these miRNAs might be useful in treatment for cancer. Functional screening for oncomiRs in a human OSCC cell line (GFP-SAS) was carried out using the miRCURY LNA microRNA Knockdown Library - Human version 12.0. We identified a locked nucleic acid (LNA)/DNA antisense oligonucleotide against miR-361-3p (LNA-miR-361-3p) which showed the largest degree of growth inhibition of GFP-SAS cells. Transfection with a synthetic mimic of mature miR-361-3p resulted in an approximately 20% increase in the growth of GFP-SAS cells. We identified odd-skipped related 2 (OSR2) as a miR-361-3p target gene. Transfection of GFP-SAS cells with LNA-miR-361-3p caused a significant increase in the expression levels of OSR2. Cotransfection of a OSR2 3'-UTR luciferase reporter plasmid and LNA-miR-361-3p into GFP-SAS cells produced higher levels of luciferase activity than in cells cotransfected with the LNA-nontarget. We assessed the effect of LNA-miR-361-3p on the in vivo growth of GFP-SAS cells. We found that LNA-miR-361-3p significantly reduced the size of s.c. xenografted GFP-SAS tumors, compared to the control group treated with LNA-NT. Finally, we observed that miR-361-3p is overexpressed in OSCC tissues. These results suggest that miR-361-3p supports the growth of human OSCC cells both in vitro and in vivo and that targeting miR-361-3p could be a useful therapeutic approach for patients with OSCC.

Effect of a novel orally bioavailable CXCR4 inhibitor, AMD070, on the metastasis of oral cancer cells.

We have previously demonstrated that the stromal cell­derived factor (SDF­1)/CXCR4 system is involved in the metastasis of head and neck cancer. Additionally, it has been revealed that the blockade of CXCR4 by subcutaneous daily injection with AMD3100, a CXCR4 antagonist, may be effective in preventing metastasis in CXCR4­related head and neck cancer. Recent investigations have suggested that AMD070, a novel orally bioavailable inhibitor of CXCR4, may be minimally invasive compared with AMD3100. In the present study, we examined the effect of AMD070 on metastasis induced by the SDF­1/CXCR4 axis in B88­SDF­1 oral cancer cells, which express high levels of SDF­1 and CXCR4. Although treatment with AMD070 did not affect the anchorage­dependent growth of B88­SDF­1 cells, it significantly suppressed the anchorage­independent growth. Moreover, the SDF­1/CXCR4­dependent migration and invasion of B88­SDF­1 cells was significantly inhibited following treatment with AMD070. Subsequently, we performed an experimental therapy using AMD070 to prevent the distant metastasis of B88­SDF­1 cells in vivo. Daily oral administration of AMD070 significantly inhibited the lung metastasis of B88­SDF­1 cells in nude mice. These results indicated that AMD070 could be useful as a novel orally bioavailable inhibitor of oral cancer metastasis.

Involvement of miR-518c-5p to growth and metastasis in oral cancer.

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral cancer. Recently, small non coding RNAs, microRNAs (miRNAs) have been shown to be involved in the metastatic process of several types of cancers. However, the miRNAs that contribute to metastases induced by the SDF-1/CXCR4 system in oral cancer are largely unknown. In this study, we examined the metastasis-related miRNAs induced by the SDF-1/CXCR4 system using B88-SDF-1 oral cancer cells, which exhibit functional CXCR4 and distant metastatic potential in vivo. Through miRNA microarray analysis, we identified the upregulation of miR-518c-5p in B88-SDF-1 cells, and confirmed the induction by real-time PCR analysis. Although an LNA-based miR-518c-5p inhibitor did not affect cell growth of B88-SDF-1 cells, it did significantly inhibit the migration of the cells. Next, we transfected a miR-518c expression vector into parental B88 cells and CAL27 oral cancer cells and isolated stable transfectants, B88-518c and CAL27-518c cells, respectively. The anchorage-dependent and -independent growth of miR-518c transfectants was significantly enhanced compared with the growth of mock cells. Moreover, we detected the enhanced migration of these cells. The LNA-based miR-518c-5p inhibitor significantly impaired the enhanced cell growth and migration of miR-518c transfectants, indicating that these phenomena were mainly dependent on the expression of miR-518c-5p. Next, we examined the function of miR-518c-5p in vivo. miR-518c transfectants or mock transfectants were inoculated into the masseter muscle or the blood vessels of nude mice. Tumor volume, lymph nodes metastasis, and lung metastasis were significantly increased in the mice inoculated with the miR-518c transfectants. These results indicated that miR-518c-5p regulates the growth and metastasis of oral cancer as a downstream target of the SDF-1/CXCR4 system.

The role of metabotropic glutamate receptor 5 on the stromal cell-derived factor-1/CXCR4 system in oral cancer.

We have demonstrated that blocking CXCR4 may be a potent anti-metastatic therapy for CXCR4-related oral cancer. However, as CXCR4 antagonists are currently in clinical use to induce the mobilization of hematopoietic stem cells, continuous administration as an inhibitor for the metastasis may lead to persistent leukocytosis. In this study, we investigated the novel therapeutic downstream target(s) of the SDF-1/CXCR4 system, using B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and exhibit distant metastatic potential in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. We identified a gene that is highly upregulated in B88-SDF-1 cells, metabotropic glutamate receptor 5 (mGluR5), which was downregulated following treatment with 1,1' -[1,4-Phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. The upregulation of mGluR5 mRNA in the SDF-1/CXCR4 system was predominately regulated by the Ras-extracellular signal-regulated kinase (ERK)1/2 pathway. Additionally, the growth of B88-SDF-1 cells was not affected by the mGluR5 agonist (S)-3,5-DHPG (DHPG) or the mGluR5 antagonists 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP). However, we observed that DHPG promoted B88-SDF-1 cell migration, whereas both MPEP and MTEP inhibited B88-SDF-1 cell migration. To assess drug toxicity, the antagonists were intraperitoneally injected into immunocompetent mice for 4 weeks. Mice injected with MPEP (5 mg/kg) and MTEP (5 mg/kg) did not exhibit any side effects, such as hematotoxicity, allergic reactions or weight loss. The administration of antagonists significantly inhibited the metastasis of B88-SDF-1 cells to the lungs of nude mice. These results suggest that blocking mGluR5 with antagonists such as MPEP and MTEP could prevent metastasis in CXCR4-related oral cancer without causing side effects.

Bortezomib-enhanced radiosensitization through the suppression of radiation-induced nuclear factor-κB activity in human oral cancer cells.

Oral cancer cells have a significantly augmented nuclear factor-κB (NF-κB) activity and the inhibition of this activity suppresses tumor growth. Bortezomib is a proteasome inhibitor and a drug used for molecular-targeted therapy (targets NF-κB). In this study, we investigated whether bortezomib would be effective as an inhibitor of proliferation and a radiosensitizer for the treatment of oral cancer. We demonstrate that bortezomib inhibits NF-κB activity and cell proliferation. The combined treatment with bortezomib and radiation (RT) suppressed NF-κB activity and cell growth in vitro and in vivo compared with RT treatment alone. To investigate the mechanisms by which bortezomib suppresses tumor growth, the expression of signaling molecules downstream of NF-κB were examined by ELISA. The combined treatment significantly inhibited the radiation-induced production of angiogenic factors and decreased the number of blood vessels in the tumor tissues. Although the expression of anti-apoptotic proteins was upregulated by RT, bortezomib downregulated the RT-induced expression of these proteins. Moreover, the expression of cleaved poly(ADP-ribose) polymerase in vitro and in vivo was enhanced by bortezomib, indicating that bortezomib inhibits tumor growth by inducing apoptosis. This study clearly demonstrates that bortezomib significantly inhibits tumor growth and that the combined treatment with bortezomib and RT results in a significant inhibition of tumor growth. The mechanisms underlying the inhibition of tumor growth by bortezomib include the suppression of angiogenesis and the induction of apoptosis. A novel molecular targeting therapy including bortezomib may be effective in the treatment of oral cancer by suppressing NF-κB activity.

Expression and function of CXCR4 in human salivary gland cancers.

Salivary gland cancers (SGCs) frequently metastasize to cervical lymph nodes and distant organs. Currently, the mechanisms responsible for the metastatic behavior of SGC cells are not fully understood. We previously demonstrated that the stromal cell-derived factor-1 (SDF-1; also known as CXCL12)/CXCR4 system is involved in the establishment of metastasis in oral squamous cell carcinoma. In the present study, we investigated the role of CXCR4 in the metastatic behavior of SGCs. We examined the expression of CXCR4 mRNA and protein in human SGC cell lines by quantitative RT-PCR and western blotting, respectively. The expression of CXCR4 mRNA and protein were frequently upregulated in 5 out of 6 SGC cell lines. Functional CXCR4 expression was demonstrated by the ability of these SGC cell lines to migrate toward an SDF-1 gradient. SDF-1 rapidly activated extracellular signal-regulated kinase (ERK)1/2 in SGC cell lines. Immunohistochemical analysis revealed that CXCR4 protein expression was detected in either the nucleus or cytoplasm of cancer cells in 16 out of 20 tissues of adenoid cystic carcinoma (ACC) and in 4 out of 6 tissues of mucoepidermoid carcinoma, which are representative of SGC. Furthermore, ACC cell lines exhibited dramatic metastasis to the lung following intravenous inoculation, whereas AMD3100, a CXCR4 antagonist, significantly inhibited lung metastasis of the cells, ameliorated body weight loss and improved the survival rate of tumor-bearing nude mice. These results indicate that CXCR4 expression contributes to the metastatic potential of SGCs.

Antitumor efficacy of sequential treatment with docetaxel and 5-fluorouracil against human oral cancer cells.

Docetaxel (DOC) and 5-fluorouracil (5-FU) are important anticancer agents widely used in the treatment of a variety of cancers including oral squamous cell carcinoma (OSCC). The purpose of this study was to determine the antitumor efficacy of the sequential administration of DOC and 5-FU against OSCC cells (B88 and CAL27 cells) in vitro and in vivo. In in vitro growth inhibition assays, sequential treatment with DOC followed by 5-FU was more effective in inhibiting cancer cell growth than 5-FU followed by DOC, single treatment with DOC or 5-FU, or combined treatment with DOC and 5-FU. Furthermore, DOC followed by 5-FU significantly inhibited tumor growth in vivo compared to 5-FU followed by DOC. To understand the mechanisms underlying the enhanced growth inhibitory effect of the administration sequence, DOC followed by 5-FU, we examined the expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), which were known to regulate the antitumor effect of 5-FU, by real-time RT-PCR and western blot analysis. Downregulation of TS and DPD expression and upregulation of OPRT expression were induced by DOC treatment, suggesting that DOC enhanced the efficacy of 5-FU by altering the expression of its metabolic enzymes. These results indicate that sequential treatment with DOC followed by 5-FU could be a promising therapeutic strategy for oral cancer.

Blockade of CXCR4 in oral squamous cell carcinoma inhibits lymph node metastases.

We have previously demonstrated that a stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (OSCC). In this study, we investigated whether the blockade of CXCR4 inhibits lymph node metastasis in B88 OSCC cells. These cells harbour a functional CXCR4 and have the potential to metastasise to the lymph node in vivo. Following introduction of a vector that expresses short hairpin small interfering RNA (shRNA) against CXCR4, we isolated three clones (shCXCR4-16, -17 and -21) that showed decreased expression of CXCR4 mRNA. These clones also had reduced CXCR4 protein levels and showed impairments in calcium flux and cell migration in response to SDF-1. These cells were orthotopically inoculated into the masseter muscle of nude mice. Lymph node metastases, loss in body weight and tumour volumes were significantly inhibited in mice inoculated with shCXCR4-17 cells compared to mice inoculated with control cells. SDF-1-induced migration of B88 cells was significantly inhibited in vitro by the treatment with 1,1'-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), a CXCR4 antagonist. Subcutaneous administration of AMD3100 significantly inhibited the lymph node metastases of B88 cells when they were orthotopically inoculated into the masseter muscle of nude mice. Moreover, the enhanced production of interleukin (IL)-6 and IL-8 in response to SDF-1 was inhibited by shRNA against CXCR4 or by treatment with AMD3100. These results suggest that blockade of CXCR4 may be a potent anti-metastatic therapy against lymph node metastases in cases of CXCR4-related OSCC.

Vesnarinone downregulates CXCR4 expression via upregulation of Krüppel-like factor 2 in oral cancer cells.

BACKGROUND: We have demonstrated that the stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system is involved in the establishment of lymph node metastasis in oral squamous cell carcinoma (SCC). Chemotherapy is a powerful tool for the treatment of oral cancer, including oral SCC; however, the effects of chemotherapeutic agents on the expression of CXCR4 are unknown. In this study, we examined the expression of CXCR4 associated with the chemotherapeutic agents in oral cancer cells. RESULTS: The expression of CXCR4 was examined using 3 different chemotherapeutic agents; 5-fluorouracil, cisplatin, and vesnarinone (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2-(1H)-quinolinone) in B88, a line of oral cancer cells that exhibits high levels of CXCR4 and lymph node metastatic potential. Of the 3 chemotherapeutic agents that we examined, only vesnarinone downregulated the expression of CXCR4 at the mRNA as well as the protein level. Vesnarinone significantly inhibited lymph node metastasis in tumor-bearing nude mice. Moreover, vesnarinone markedly inhibited 2.7-kb human CXCR4 promoter activity, and we identified the transcription factor, Krüppel-like factor 2 (KLF2), as a novel vesnarinone-responsive molecule, which was bound to the CXCR4 promoter at positions -300 to -167 relative to the transcription start site. The forced-expression of KLF2 led to the downregulation of CXCR4 mRNA and impaired CXCR4 promoter activity. The use of siRNA against KLF2 led to an upregulation of CXCR4 mRNA. CONCLUSION: These Results indicate that vesnarinone downregulates CXCR4 via the upregulation of KLF2 in oral cancer.