Binding of glyceraldehyde-3-phosphate dehydrogenase to the cis-acting element of structure-anchored repression in ccn2 mRNA.

CCN2/connective tissue growth factor (CTGF) can be induced by hypoxia and promotes tumor angiogenesis. Our previous studies revealed that hypoxia-induced gene expression of human ccn2 mRNA is regulated post-transcriptionally in human chondrosarcoma-derived cell line, HCS-2/8, in which a minimal cis-element, entitled CAESAR, in the 3'-untranslated region (UTR) of ccn2 mRNA and a 35-kDa protein counterpart play an important role by determining the stability of ccn2 mRNA. In the present study, we identified this corresponding protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by utilizing RNA affinity chromatography combined with mass spectrometry. The results of an RNA binding assay revealed the specific binding of GAPDH to this cis-element. To further characterize the interaction between GAPDH and ccn2 mRNA, we examined the roles of redox conditions and glycolytic coenzyme in the binding of GAPDH to the ccn2 mRNA. An oxidizing agent, diamide, abolished the GAPDH-RNA interaction in a concentration-dependent manner; whereas this effect could be reversed by subsequent treatment with 2-mercaptoethanol (2-ME). In addition, nicotinamide-adenine dinucleotide (NAD), a coenzyme of GAPDH, inhibited the GAPDH-RNA binding. Taken together, these findings suggest that the glycolytic enzyme GAPDH regulates the gene expression of ccn2 mRNA in trans by acting as a sensor of oxidative stress and redox signals, leading to CCN2 overexpression under the condition of hypoxia and promotion of angiogenesis.

Identification of CCDC62-2 as a novel cancer/testis antigen and its immunogenicity.

Cancer/testis (CT) antigens are expressed in normal germ line tissues and various cancers. They are considered promising target molecules for immunotherapy for patients with various cancers. To identify CT antigens, we performed serological identification of antigens by recombinant expression cloning. The humoral immune response of cancer patients against a newly defined antigen was analyzed. A testicular cDNA library was immunoscreened with serum obtained from a gastric adenocarcinoma patient whose primary cancer had regressed once and most liver metastases had disappeared transiently. We isolated 55 positive cDNA clones comprising 23 different genes. They included 4 genes with testis-specific expression profiles in the Unigene database, including coiled-coil domain containing 62 (CCDC62). RT-PCR analysis showed that the expression of 2 splice variants of CCDC62 was restricted to the testis in normal adult tissues. In malignant tissues, CCDC62 variant 2 (CCDC62-2) was aberrantly expressed in a variety of cancers, including stomach cancer. A serological survey of 191 cancer patients with a range of different cancers by ELISA revealed antibodies to CCDC62-2 in 13 patients, including stomach cancer. None of the 41 healthy donor serum samples were reactive in the same test. The serum reaction against CCDC62-2 was confirmed by western blot. CCDC62-2 is a CT antigen that is immunogenic in cancer patients.

Clinical significance and pathogenic function of connective tissue growth factor (CTGF/CCN2) in osteolytic mandibular squamous cell carcinoma.

BACKGROUND: Mandibular bone destruction is a frequent occurrence in oral squamous cell carcinoma. However, the relationship between the bone destruction and associated factors is unclear. Here, the role and diagnostic utility of connective tissue growth factor (CCN2) in bone destruction of the mandible was investigated. PATIENTS AND METHODS: The production of CCN2 was explored by using immunohistochemistry on paraffin-embedded tissues from 20 cases of mandibular squamous cell carcinoma. The effect of CCN2 on osteoclastogenesis was examined in vitro by using total bone marrow cell populations from male mice. RESULTS: Immunohistochemical analysis showed that CCN2-positive signals were closely associated with destructive invasion of the mandible by oral squamous cell carcinomas. Consistent with these results, recombinant human CCN2 (rCCN2) stimulated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cell formation in vitro. CONCLUSION: CCN2 can be considered a diagnostic marker and target for treatment in oral osteolytic mandibular squamous cell carcinoma.

[Two cases of giant metastatic cervical lymphnode from oral cancer responding to concurrent chemoradiotherapy with S-1].

We report two cases of giant metastatic cervical lymphnode from oral squamous cell carcinoma, successfully treated with concurrent chemoradiotherapy with S-1. Case 1 was a 80-year-old male who had an ipsilateral occult neck metastasis(52x46 mm mass)at level IV after surgery of the left tongue carcinoma(T2N0M0). Radiotherapy(2.0 Gy/day; 5 days/week)was given at a total dose of 66 Gy. Two courses of oral administration of S-1(61 mg/m2/day)for 2 weeks followed by 1-week rest period as one course was repeated with the concurrent radiotherapy. After the radiotherapy, the oral administration of S-1 alone was continued for 1 year under the same regimen. Case 2 was a 51-year-old male who had left tongue carcinoma(T4bN2cM0)with ipsilateral cervical lymphnode metastasis(88x44 mm mass). The same chemoradiotherapy(1.8 Gy/day; 5 days/week; a total dose of 63 Gy; S-1 53 mg/m2/day)was carried out as in case 1. These giant metastatic lymphnodes of case 1 and 2 disappeared by 3 and 6 months after radiotherapy, respectively, and achieved a complete response. This therapy may be effective not only for primary lesion of oral cancer, but also metastatic regional lymphnodes in some cases.

An immunohistochemical evaluation of BMP-2, -4, osteopontin, osteocalcin and PCNA between ossifying fibromas of the jaws and peripheral cemento-ossifying fibromas on the gingiva.

The present study examined histological difference between ossifying fibromas (OF, n=5) and peripheral cemento-ossifying fibromas (PCOF, n=7). Bone morphogenetic proteins (BMP)-2 and -4, osteopontin (OPN), osteocalcin (OCN) and proliferating cell nuclear antigen (PCNA) were used for the immunohistochemical examinations. Oxytalan fibers present at the periodontal tissue were stained to determine the tumor cell origin. Many OFs showed high immunohistochemical reactions for BMP-2, -4 and OPN compared to those of PCOFs. PCNA index (IP) of OFs was significantly higher than that of PCOFs. All the PCOFs showed a high expression of oxytalan fibers. Only two OFs exhibited a small number of oxytalan fibers. These results suggest that PCOF has only little ability to form hard tissue and seems to be a reactive lesion. The expression of oxytalan fibers reveals that OF does not only originate from periodontal tissue.

[A case of acute respiratory distress syndrome (ARDS) induced by docetaxel administration for lung metastases from oral cancer].

We reported a case of acute respiratory distress syndrome (ARDS) induced by docetaxel in treating lung metastases from oral cancer. The patient was an 84-year-old man who had undergone partial mandibulectomy and radical neck dissection for lower gingival carcinoma. The patient developed ARDS after docetaxel administration (40 mg/body) for multiple lung metastases.

Prognostic significance of heat shock protein 27 (HSP27) in patients with oral squamous cell carcinoma.

Heat shock proteins (HSPs) have been defined as proteins induced by heat shock and other environmental and pathophysiologic stress. Heat shock protein 27 (HSP27) is one of the small heat shock proteins. HSP27 is implicated in protein-protein interactions such as folding, translocation, and prevention of inappropriate protein aggregation. Many of their functions suggest that they play important roles in cancers. Archival tissues from 40 patients with oral squamous cell carcinoma who received primary surgical resection were examined for HSP27 by immunohistochemistry and correlated with clinical stage, lymph node metastasis, histological grade and survival period. HSP27 expression was positive staining (+) in 20 (50%), weak or negative staining (-) in 20 (50%) of total 40 cases. There was no correlation between HSP27 expression and clinical stage, lymph node metastasis and histological grade. However, when compared with clinicopathological features, the expression of HSP27 correlated inversely with survival period. This study suggests that the expression of HSP27 is frequently promoted in patients with oral squamous cell carcinoma and should be considered an independent prognostic factor of oral squamous cell carcinoma patients.

Expression of vascular endothelial growth factor-C predicts regional lymph node metastasis in early oral squamous cell carcinoma.

The purpose of this study was to investigate the association of the expression of vascular endothelial growth factor-C (VEGF-C) with regional lymph node metastasis in oral squamous cell carcinoma (OSCC). The expression of VEGF-C in biopsy specimens obtained from 62 patients with OSCC was examined by immunohistochemistry. In the early stages of T1 and T2 (38 cases), VEGF-C expression strongly correlated with lymph node metastasis (P < 0.001), and the percentage of VEGF-C-positive staining was 88.2% for patients with lymph node metastasis (15 of 17 cases) and 28.6% for those without lymph node metastasis (6 of 21 cases). However, in the advanced stages of T3 and T4, no significant correlation between VEGF-C expression and lymph node metastasis was observed. These results indicate that VEGF-C expression in biopsy specimens can be used as a reliable predictor of regional lymph node metastasis, particularly in early OSCC, and may become an important factor in the selection of appropriate treatment.

Tumor protein D54 is a negative regulator of extracellular matrix-dependent migration and attachment in oral squamous cell carcinoma-derived cell lines.

PURPOSE: Tumor protein D54 (TPD54) belongs to the TPD52 family of proteins and is expressed in several types of cancer, including oral squamous cell carcinoma (OSCC). Here, we investigated relationships between various OSCC-related characteristics and TPD54 expression in vitro. METHODS: The expression of TPD54 in several OSCC-derived cell lines and normal, non-malignant, cells was assessed. Based on the results obtained, OSCC-derived SAS cells were subsequently subjected to exogenous over-expression of alternative splice variants (ASVs) of TPD54 and to TPD54 knock-down, mediated by siRNA. Next, the role of TPD54 in cellular growth, apoptosis, invasion, migration and extracellular-matrix (ECM)-dependent migration and attachment was investigated, as also the concomitant expression of integrins and integrin-related proteins by the OSCC-derived cells. RESULTS: Western blot analysis and RT-PCR revealed that several TPD54 ASVs were expressed in the OSCC-derived cell lines tested. Neither exogenous ASV over-expression nor TPD54 knock-down modulated the proliferation or invasion of SAS cells in a monolayer culture assay. However, exogenous ASV over-expression did decrease anchorage-independent growth and TPD54 knock-down did increase anchorage-independent growth, irrespective of caspase activities. The same effects were observed on ECM-dependent cellular migration and cell attachment to the ECM. The expression levels of the major α and ß integrin subunits, and of E-cadherin, were found to be similar to those observed in the non-transfected control cells, whereas talin1 expression was found to be increased after TPD54 knock-down. Also Akt was found to be activated after TPD54 knock-down, even in the absence of serum stimulation. Very similar effects were observed in the OSCC-derived cell lines HSC 2 and HSC 3. CONCLUSIONS: Our results show that TPD54 affects OSCC cell attachment to the ECM, OSCC cell migration, and Akt/PKB activation by modulating integrin activation via a talin1-mediated inside-out signal of the ECM. Based on these results, we suggest that TPD54 may serve as a novel biomarker for OSCC and as a putative target for OSCC therapy.

Multicenter phase II trial of preoperative chemoradiotherapy with S-1 for locally advanced oral squamous cell carcinoma.

PURPOSE: We evaluated whether preoperative chemotherapy with S-1 and concurrent radiotherapy is feasible and efficacious in the treatment of advanced oral squamous cell carcinoma. METHODS: Participants comprised 39 patients with oral carcinoma (stage III, n = 15; stage IVA, n = 24). All patients received a total radiation dose of 40 Gy, in once-daily 2-Gy fractions, and received S-1 at 65 mg/m(2)/day for 5 consecutive days, over 4 consecutive weeks with concurrent radiotherapy. RESULTS: Hematological toxicity was mild and reversible. The most common non-hematological toxicity was grade 3 mucositis, but this was transient and tolerable. Radical surgery was performed for 37 patients, with the remaining 2 patients declining the surgery. Postoperatively, local failure developed in 1 patient, and neck failure in 2 patients. Distant metastases were identified in 4 patients. At a median follow-up of 38.0 months (range 23-88 months), locoregional control, disease-specific survival, and overall survival rates at 3 years were 91.5, 83.8, and 83.8 %, respectively. CONCLUSION: Concurrent administration of S-1 and radiotherapy combined with surgery offers a well-tolerated method of successfully treating advanced oral squamous cell carcinoma. The locoregional control rate remains high even at 3 years of follow-up, and no serious adverse effects have been encountered.

The potential anticancer activity of extracts derived from the roots of Scutellaria baicalensis on human oral squamous cell carcinoma cells.

Various herb products derived from plants have potent biological effects including anticancer activity. In the present study, the antitumor activity of a herbal product derived from the Scutellaria baicalensis (S. baicalensis) was examined, using in vitro assays in a human oral squamous cell carcinoma (OSCC) cell line. Results showed that S. baicalensis root extract at the concentration of 100 µg/ml inhibited monolayer- and anchorage-independent growth in human OSCC cell lines, while not affecting the adhering abilities of cells. This suggested that it did not alter the expression of any of the adhesion receptors that mediate cell-extracellular matrix (ECM) interactions. The S. baicalensis root extract demonstrated potent cytostatic and apoptotic effects due to the downregulation of the cyclin-dependent kinase 4 expression and its partner cyclin D1, resulting in G1 arrest and poly (ADP-ribose) polymerase (PARP) cleavage. Additionally, the S. baicalensis root extract was found to have blocked vascular endothelial growth factor (VEGF)-induced migration and tube formation in human endothelial cells. Taken together, these results demonstrate that as a herbal product, the S. baicalensis root extract is a potential inhibitor of tumori- and angiogenesis and may be valuable in the development of pharmaceutical medications for the treatment of oral squamous cell carcinoma.

Bone destruction by invading oral squamous carcinoma cells mediated by the transforming growth factor-beta signalling pathway.

BACKGROUND: Gingival squamous cell carcinoma (SCC) cells frequently invade mandibular bone, and this destruction is associated with a worse prognosis. However, the relationship between bone destruction and associated factors is unclear. In this study, the role and diagnostic utility of transforming growth factor-beta (TGF-beta) type I receptor (TbetaRI) in bone destruction of the mandible was investigated. PATIENTS AND METHODS: The expression of TbetaRI was explored by using an immunohistochemical method on paraffin-embedded tissues from 21 cases of mandibular SCC. An inhibitor of the kinase activity of the TbetaRI (TbetaRI-I) was used to assess the role of TbetaRI in bone destruction by a human oral SCC cell line (HSC-2) that highly expresses TbetaRI. RESULTS: TbetaRI-positive signals were closely associated with destructive invasion of the mandible by oral SCC cells. Consistent with these results, TbetaRI-I greatly reduced HSC-2 cell-induced bone destruction and osteoclast formation in vivo and in vitro. TbetaRI-I treatment reduced the expression of TNF-alpha, RANKL and connective tissue growth factor (CTGF/CCN2), all of which were up-regulated by TGF-beta in HSC-2 cells. CONCLUSION: These data demonstrated an important role for TGF-beta signalling in bone invasion by oral SCC cells, and suggest that the bone destruction is mediated by RANKL, TNF-alpha and CCN2.