Mitsuhiro Nakazawa

Mutations of the APC, beta-catenin, and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma

Purpose: The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. Methods: 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequecing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. Results: In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. Conclusion: The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.

Effectiveness of boron neutron capture therapy for recurrent head and neck malignancies.

It is necessary to explore new treatments for recurrent head and neck malignancies (HNM) to avoid severe impairment of oro-facial structures and functions. Boron neutron capture therapy (BNCT) is tumor-cell targeted radiotherapy that has significant superiority over conventional radiotherapies in principle. We have treated with BNCT 42 times for 26 patients (19 squamous cell carcinomas (SCC), 4 salivary gland carcinomas and 3 sarcomas) with a recurrent and far advanced HNM since 2001. Results of (1) (10)B concentration of tumor/normal tissue ratios (T/N ratio) of FBPA-PET studies were SCC: 1.8-5.7, sarcoma: 2.5-4.0, parotid tumor: 2.5-3.7. (2) Therapeutic effects were CR: 12 cases, PR: 10 cases, PD: 3 cases NE (not evaluated): 1 case. Response rate was 85%. (3) Improvement of QOL such as a relief of severe pain, bleeding, and exudates at the local lesion, improvement of PS, disappearance of ulceration, covered with normal skin and preserved oral and maxillofacial functions and tissues. (4) Survival periods after BNCT were 1-72 months (mean: 13.6 months). Six-year survival rate was 24% by Kaplan-Meier analysis. (5) Adverse-events were transient mucositis and alopecia in most of the cases; three osteomyelitis and one brain necrosis were recognized. These results indicate that BNCT represents a new and promising treatment approach for advanced HNM.

Induction of endonuclease G-mediated apopotosis in human oral squamous cell carcinoma cells by protein kinase C inhibitor safingol

PKC inhibitor safingol suppressed the growth of human oral squamous cell carcinoma (SCC) cells significantly at concentrations that inhibit PKC isoforms. Safingol inhibited the translocation of PKC following treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA) in PKC α-EGFP-transfected cells, but not in PKC β-EGFP- transfected cells, indicating selective inhibition for PKC α in oral SCC cells. Flow cytometric analysis and DNA analysis by agarose gel electrophoresis revealed an increase in the proportion of sub-G1 cells and DNA fragmentation in safingol-treated cells. Mitochondrial membrane potential was decreased, and cytochrome c was released from mitochondria. However, the safingol-induced cell death was not accompanied by activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). The broad-spectrum caspase inhibitor BD-fmk failed to prevent safingol-induced cell death. Another apoptogenic factor endonuclease G, but not apoptosis-inducing factor (AIF), was also released from mitochondria and translocated to the nucleus. These results suggest that PKC α inhibitor safingol induces an endonuclease G- mediated apoptosis in a caspase-independent manner.

Anti-HER-2/neu immune responses are induced before the development of clinical tumors but declined following tumorigenesis in HER-2/neu transgenic mice.

HER-2/neu oncogene products have been implicated as a potential target of T cell–mediated immune responses to HER-2/neu–induced tumors. Using HER-2/neu transgenic mice (oncomice), we investigated whether, and if so how, anti–HER-2/neu immune responses are induced and modulated in these oncomice from birth to tumor initiation. Female oncomice carrying the activated HER-2/neu oncogene displayed apparent hyperplasia in mammary glands at 10 weeks of age and developed mammary carcinomas around an average age of 26 weeks. Unfractionated spleen cells from 10- to 15-week-old oncomice that were cultured without any exogenous stimuli exhibited cytotoxicity against the F31 tumor cell line established from an HER-2/neu–induced mammary carcinoma mass. The final antitumor effectors were a macrophage lineage of cells. However, this effector population was activated, depending on the stimulation of oncomouse CD4+ T cells with oncomouse-derived antigen-presenting cell (APC) alone or with wild-type mouse APC in the presence of F31 membrane fractions, suggesting the presence of HER-2/neu–primed CD4+ T cells and HER-2/neu–presenting APC in 10- to 15-week-old oncomice. These antitumor cytotoxic responses were detected at ∼5 weeks of age and peaked at age 10 to 15 weeks. However, the responses then declined at tumor-bearing stages in which the expression of target proteins could progressively increase. This resulted from the dysfunction of CD4+ T cells but not of APC or effector macrophages. These results indicate that an anti–HER-2/neu CD4+ T cell–mediated immune response was generated at the pretumorigenic stage but did not prevent tumorigenesis and declined after the development of clinical tumors.

Lymphatic vessels and related factors in adenoid cystic carcinoma of the salivary gland

Adenoid cystic carcinoma of the salivary gland preferentially metastasizes to distant organs. It rarely metastasizes to lymph nodes. Recently, lymphangiogenesis has been associated with lymph node metastasis. Therefore, lymphangiogenesis in adenoid cystic carcinoma was evaluated from the number of lymphatic vessels and the expression of lymphangiogenic factors. Immunohistochemistry and molecular analysis were performed on clinical materials (29 cases for immunohistochemistry and 9 cases for molecular analysis). Normal submandibular gland was used as a negative control of lymphangiogenesis (10 cases for immunohistochemistry and 5 cases for molecular analysis). In adenoid cystic carcinoma, podoplanin-positive lymphatic vessels were small and often constricted, and localized to the tumor periphery. They did not have Ki67-positive endothelial cells. The lymphatic vessel density of the tumor did not exceed that of the salivary gland. By reverse transcriptase–polymerase chain reaction, adenoid cystic carcinoma and the salivary gland expressed vascular endothelial growth factor receptor-3 (VEGFR-3) similarly but VEGF-C and VEGF-D differently. Adenoid cystic carcinoma expressed VEGF-C, whereas the salivary gland expressed both VEGF-C and VEGF-D. VEGF-C was weak in adenoid cystic carcinoma and strong in the salivary gland. Real-time reverse transcriptase–polymerase chain reaction of VEGF-C showed that the ratio of the tumor to the salivary gland was 1 to 30 (P<0.01). Immunohistochemistry barely detected VEGF-C in adenoid cystic carcinoma. VEGF-C was expressed faintly by the tumor cells. VEGF-C and VEGF-D were detected in the serous acinar and duct cells and in the duct contents in the salivary gland. VEGFR-3 appeared to be expressed by lymphatic vessels in both adenoid cystic carcinoma and the salivary gland. These results indicate that lymphangiogenesis does not occur in adenoid cystic carcinoma. This condition would lead to the uncommon lymphatic metastasis.

Sonoporation as an enhancing method for boron neutron capture therapy for squamous cell carcinomas

BackgroundBoron neutron capture therapy (BNCT) is a selective radiotherapy that is dependent on the accumulation of 10B compound in tumors. Low-intensity ultrasound produces a transient pore on cell membranes, sonoporation, which enables extracellular materials to enter cells. The effect of sonoporation on BNCT was examined in oral squamous cell carcinoma (SCC) xenografts in nude mice.Materials and methodsTumor-bearing mice were administrated boronophenylalanine (BPA) or boronocaptate sodium (BSH) intraperitoneally. Two hours later, tumors were subjected to sonoporation using microbubbles followed by neutron irradiation.ResultsThe 10B concentration was higher in tumors treated with sonoporation than in untreated tumors, although the difference was not significant in BPA. When tumors in mice that received BPA intraperitoneally were treated with sonoporation followed by exposure to thermal neutrons, tumor volume was markedly reduced and the survival rate was prolonged. Such enhancements by sonoporation were not observed in mice treated with BSH-mediated BNCT.ConclusionsThese results indicate that sonoporation enhances the efficiency of BPA-mediated BNCT for oral SCC. Sonoporation may modulate the microlocalization of BPA and BSH in tumors and increase their intracellular levels.

Central acinic cell carcinoma of the mandible. Report of a case.

Central acinic cell carcinoma is extremely rare; only six cases have been reported in the literature. An unusual case of central acinic cell carcinoma of the mandible in an 84-year-old Japanese woman is presented. This is thought to be the seventh case of central acinic cell carcinoma described in the English literature.

Nodular fasciitis in the buccal region with rapid growth after incisional biopsy mimicking sarcoma.

Nodular fasciitis (NF) is a reactive and proliferative fibroblastic lesion that occurs predominantly in the upper limbs but rarely develops in the oral cavity. This lesion can be misdiagnosed as malignant owing to its frequent display of rapid growth, rich cellularity, and high mitotic activity. Unlike a sarcoma, NF can resolve spontaneously or after an incisional biopsy. We describe a challenging case involving a lesion in the buccal region that rapidly enlarged after incisional biopsy. This variation of clinical behavior illustrates the difficulty in predicting whether NF will continue to grow or regress. Clinicians dealing with cases of an enlarging fibrous lesion of short duration should remain aware of this disease entity and its potential diagnostic dilemma.

Transcription factors related to chondrogenesis in pleomorphic adenoma of the salivary gland: a mechanism of mesenchymal tissue formation.

In salivary gland pleomorphic adenoma, expression of extracellular matrix (ECM) substances indicates that tumor epithelial cells are becoming chondrogenic and will produce cartilage-like mesenchymal tissues. Sox9, the master transcription factor of chondrogenesis, is expressed in mouse salivary gland cells. To clarify the mechanism behind chondrogenesis in tumor epithelial cells, we examined the expression of transcription factors related to chondrogenesis in tumors and salivary glands. Reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR, and immunostaining were performed on pleomorphic adenoma tissues, salivary gland tissues, and human submandibular gland (HSG) cells. The mRNAs of essential transcription factors for chondrogenesis—Sox9, Sox6, and Sox5—were detected in both tumor and salivary gland tissues. The mRNAs of aggrecan and type II collagen—cartilage-specific ECM substances—were detected only in tumors. Sox9 and Sox6 proteins were colocalized in many epithelial cells in tumors and salivary glands. Tumor epithelial cells also possessed aggrecan protein and occasionally type II collagen protein. Moreover, mRNAs for transcription repressors of chondrogenesis δEF1 and AP-2α were detected in both tumors and salivary glands, whereas Twist1 mRNA was detected only in salivary glands and was at significantly low-to-undetectable levels in tumors. Twist1 protein was localized in the Sox9-expressing salivary gland cells. HSG cells expressed Sox9, Sox6, and Twist1, but not aggrecan or type II collagen, and thus were similar to salivary gland cells. Twist1 depletion by Twist1 siRNA led to the upregulation of aggrecan and type II collagen mRNA expression in HSG cells. In contrast, forced expression of Twist1, using Twist1 cDNA, resulted in the downregulation of both these genes. Taken together, these results indicate that salivary gland cells have a potential for chondrogenesis, and Twist1 depletion concomitant with neoplastic transformation, which would permit tumor epithelial cells to produce cartilage-like mesenchymal tissues in salivary gland pleomorphic adenoma.