Soichi Iwai

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.

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase‐activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF‐kB, and their inhibitors diminished both proMMP9‐overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2‐Ets1, p38/HSP27, and PAR2/NF‐kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.

The effects of trichostatin A on the oncolytic ability of herpes simplex virus for oral squamous cell carcinoma cells

Combining the use of a chemotherapeutic agent with oncolytic virotherapy is a useful way to increase the efficiency of the treatment of cancer. The effect of the histone diacetylase (HDAC) inhibitor trichostatin A (TSA) on the antitumor activity of a herpes simplex virus type-1 (HSV-1) mutant was examined in oral squamous cell carcinoma (SCC) cells. Immunoblotting analysis and immunoflourescence staining revealed that a cytoplasmic nuclear factor-κB (NF-κB) component, p65, translocated into the nucleus after infection with γ134.5 gene-deficient HSV-1 R849, indicating that R849 activated NF-κB. TSA induced acetylation of p65 and increased the amount of p65 in the nucleus of oral SCC cells. Treatment of R849-infected cells with TSA also increased the amount of nuclear p65 and binding of NF-κB to its DNA-binding site and an NF-κB inhibitor SN50 diminished the increase in nuclear p65. In the presence of TSA, the production of virus and the expression of LacZ integrated into R849 and glycoprotein D, but not ICP0, ICP6 and thymidine kinase, were increased. The viability of cells treated with a combination of R849 and TSA was lower than that of those treated with R849 only. After treatment with TSA, expression of the cell cycle kinase inhibitor p21 was upregulated and the cell cycle was arrested at G1. These results indicate that TSA enhanced the replication of the HSV-1 mutant through the activation of NF-κB and induced cell cycle arrest at G1 to inhibit cell growth. TSA can be used as an enhancing agent for oncolytic virotherapy for oral SCC with γ134.5 gene-deficient HSV-1.

Carbonic Anhydrase VI in the Mouse Nasal Gland

Western blotting analysis of mouse nasal tissue using a specific anti-mouse secreted carbonic anhydrase (CA VI) antibody has shown that CA VI is present in this tissue. A single immunoreactive band of 42 kD was observed, as has been found previously for salivary tissues. RT-PCR analysis has shown that nasal mucosa expressed CA VI mRNA. By immunohistochemistry (IHC), CA VI was observed in acinar cells, in duct contents of the anterior gland of the nasal septum, and in the lateral nasal gland. The Bowmans gland, the posterior gland of the nasal septum, and the maxillary sinus gland were negative. Immunoreactivity was also observed in the mucus covering the respiratory and olfactory mucosa and in the lumen of the nasolacrimal duct. In contrast, an anti-rat CA II antibody (that crossreacts with the mouse enzyme) stained only known CA II-positive cells and an occasional olfactory receptor neuron. These results indicate that CA VI is produced by the nasal gland and is secreted over the nasal mucosa. By reversible hydration of CO2, CA VI is presumed to play a role in mucosal functions such as CO2 sensation and acid–base balance. It may also play a role in olfactory function as a growth factor in maturation of the olfactory epithelial cells.

Regenerative behavior of biomineral/agarose composite gels as bone grafting materials in rat cranial defects

The main objective of this study was to evaluate the biological behavior of Hydroxyapatite (HAp)/agarose and calcium carbonate (CaCO3)/agarose composite gels by an alternate soaking process used for the treatment of surgically produced bone defects in rat cranium. We designed the following four groups: (i) HAp (HAp/agarose composite gel), (ii) CaCO3 (CaCO3/agarose composite gel), (iii) Agarose (bare agarose gel), and (iv) Defect (no filling materials). We subdivided (i) (ii) (iii) into two application types as a (I) Homogenized Group (homogenized materials) and a (II) Disk Group (disk shaped materials). We assessed samples by radiological and histological analyses 0, 4, and 8 weeks after implantation. The results indicated that the composite gels showed higher radiopacity in microfocus-computed tomography (muCT) images and showed higher volume in quantitative analyses using Dual Energy X-ray Absorptiometry (DEXA) and Peripheral Quantitative Computed Tomography (pQCT) than the Agarose and Defect groups. The histological examination showed characteristic images due to each application form. Consequently, HAp and CaCO3/agarose composite gels can be expected to accelerate the speed of producing more new bone associated with osteogenesis. These novel biomaterials play an important role as an alternative biocompatible and biodegradable bone grafting filler material for autogenous bone.

Induction of apoptosis of detached oral squamous cell carcinoma cells by safingol. Possible role of Bim, focal adhesion kinase and endonuclease G

The protein kinase C (PKC) inhibitor safingol increased rounding and detachment of human oral squamous cell carcinoma (SCC) cells in monolayer cultures. When dissociated cells were incubated in the presence of safingol, cell adhesion was prevented and cell viability was lost gradually, while most cells survived in the absence of safingol even if their attachment was blocked by coating the culture plates with polyhydroxyethyl methacrylate. Flow cytometric analysis and agarose gel electrophoresis of cellular DNA revealed an increase in the proportion of sub-G1 cells and DNA fragmentation, indicating that safingol induced apoptosis of dissociated cells. During the induction of apoptosis in cell suspensions by safingol, there was an increase of the pro-apoptotic BH-3 only protein Bim and decrease of pro-survival Bcl-2 family proteins Bcl-xL and mitochondrial pro-apoptogenic factor endonuclease G translocated to the nucleus. The level of phosphorylated focal adhesion kinase (FAK) required for cell survival also rapidly decreased, followed by a decrease in the protein level. The introduction of siRNA against PKCα into SAS cells resulted in an increase of Bim, a decrease of Bcl-xL, the translocation of endonuclease G, and a decrease in the phosphorylation of FAK. These results suggest that Bim, Bcl-xL, FAK and endonuclease G are involved in safingol-induced apoptosis of detached oral SCC cells. Safingol can be used to induce apoptosis with cell detachment, anoikis, of oral SCC cells.

Effect of neutron capture therapy on the cell cycle of human squamous cell carcinoma cells

Purpose: The effects of boronophenylalanine (BPA)-mediated boron neutron capture therapy (BNCT) on the growth potential and cell cycle of human oral squamous cell carcinoma (SCC) cells were examined. Materials and methods: SAS cells expressing a functional wild-type p53 were exposed to neutron beams in the presence of BPA and growth potential was measured by colony formation assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cell cycle and cell cycle-related proteins were examined by flow cytometry and immunoblot analysis. Results: BNCT affected the colony-forming ability and viability of SAS cells. In the flow-cytometric analysis of BNCT-treated cells, the cell cycle was arrested at the G1 and G2 checkpoints, and sub-G1 cells appeared. Apoptotic cells were detected by nuclear DNA staining. Immunoblot analysis revealed the phosphorylation of p53, up-regulation of p21, and down-regulation of retinoblastoma (Rb) gene protein at 6 h after BNCT. Twelve hours after BNCT, the up-regulation of Wee1, phosphorylation of cdc2, and up-regulation of cyclin B1 were observed. Cleavage of poly (ADP-ribose) polymerase (PARP) occurred from 6 h after BNCT. Conclusion: These results indicate that the early inhibitory effect of BNCT on the growth of human oral SCC cells can be ascribed to arrest at the G1 and G2 checkpoints and apoptosis associated with G1 arrest.

A novel fusogenic herpes simplex virus for oncolytic virotherapy of squamous cell carcinoma

BackgroundR849 is a neurovirulent γ134.5 gene-deficient form of herpes simplex virus type 1 (HSV-1) and has LacZ genes at the deleted sites of the γ134.5 gene. HF is a spontaneously occurring, fusogenic HSV-1 strain. The purpose of this work was to generate a virus that has the syncytial character of HF, while preserving the γ134.5 gene inactivation profile of R849 virus.ResultsVero cells were infected with R849 and HF simultaneously and two viruses, RH1 and RH2, expressing the LacZ gene and inducing extensive cell fusion were selected. A polymerase chain reaction (PCR)-based analysis suggested that one copy of the γ134.5 gene is lost in RH1, whereas both copies are lost in RH2, and that the γ134.5 gene is replaced by a R849-derived DNA fragment with the LacZ gene. These viruses produced larger plaques and more progeny than the parental viruses. Infection with RH2 decreased the viability of oral squamous cell carcinoma (SCC) cells most strongly. When RH2 was injected into xenografts of oral SCC in nude mice, multinucleated cells were produced and the growth of the tumors was suppressed significantly.ConclusionThese results indicate that novel oncolytic HSV-1 vectors can be produced with the genetic background of the oncolytic HSV-1 HF, and that RH2 is deficient in γ134.5 genes and shows extensive cytopathic effects in oral SCC cells. RH2 may be useful in oncolytic virotherapy for oral SCC.

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.