Recent clinical studies have indicated that intra-tumoral gene expression levels of 5-fluorouracil (5-FU) metabolism-related enzymes may predict the clinical response of several cancers to 5-FU-based chemotherapy. However, few studies examining oral squamous cell carcinomas (OSCCs) have been reported. In this study, we determined the expression levels of 5-FU metabolism-related enzymes like thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylate (TP) and orotate phosphoribosyl transferase (OPRT) using reverse transcription-polymerase chain reaction (RT-PCR) combined with laser capture microdissection (LCM). We also evaluated the correlation between the mRNA expressions of these genes and clinico-pathological factors or the treatment effects of 5-FU-based chemotherapy combined with radiotherapy in 27 patients with OSCC. No significant correlation was observed between the mRNA expression levels of any of the examined genes and the T-stage, N-stage, differentiation grade or mode of tumor invasion. Although TS and OPRT mRNA were not correlated with the histopathological effects and the development of tumor recurrence, DPD and TP mRNA were significantly correlated with the histopathological effects and tumor recurrence. A significant positive correlation was also observed between the expression of TS and DPD mRNA, but no other correlations were observed among the other genes. Our results suggest that the combined evaluation of TP and DPD mRNA expression in tumor cells using LCM and RT-PCR may be a useful predictor of the efficacy of 5-FU-based chemotherapy combined with radiotherapy in patients with OSCC.

: We report a rare case of a malignant granular cell tumor in the maxilla. A 61-year-old woman presented with painless swelling and bleeding of the hard palate. Clinical examinations revealed diffuse swelling of the right side of the hard palate with a necrotic ulcer. CT and MRI showed a tumor-like mass extending from the right max-illary sinus to the palate and nasal cavity. Histopathologically, the tumor consisted of clusters of eosinophilic granular cells exhibiting marked atypia, polymorphism, and vesicular nuclei with a high nuclear-to-cytoplasmic ratio. Immunohistochemical examination revealed that the tumor cells were positive for S100, NSE, CD68, CD57, and vimentin, but not for HMB45, KL1, CAM5.2, EMA, α-SMA, desmin, or MBP. We diagnosed the tumor as a malignant granular cell tumor and performed partial maxillectomy. Seven months after the operation, multiple metastat-ic lesions were detected in the lungs, liver, scapula, vertebrae, and iliac bone on PET-CT. The patient died 14 months after surgery, even though she received 2 courses of chemotherapy with cisplatin and etoposide.

Bisphosphonates, nonmetabolized analogues of pyrophosphate, have been widely used to treat patients with hypercalcemia caused by malignancy, bone metastasis, multiple myeloma, and osteoporosis. Recent studies have documented that bisphosphonates can cause osteonecrosis of the jaws. We report a case of bisphosphonateassociated osteonecrosis of the maxilla. A 72-years-old woman was referred to our clinic for further examination of the upper left premolar region. Intraoral examination revealed necrotic alveolar bone in the upper left premolar region, with swelling of the surrounding soft tissue and exposed necrotic bone at the right side of the upper jaw. The patinet had a past medical history of breast cancer with bone metastases to the ribs and received intravenous pamidronate at a dose of 30 mg every 2 weeks. We suspected bisphosphonate-associated osteonecrosis and performed sequestrectomy after antibiotic therapy and local irrigation. Because the number of patients who receive bisphosphonates is increasing in Japan, dentists, especially oral surgeons, should be careful about bisphosphonate-associated osteonecrosis of the jaw bones.

Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by γ-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

Mitochondrial DNA (mtDNA) repair systems are thought to be associated with the susceptibility of cancer cells to anticancer agents. The present study investigated the relationship between the susceptibility to γ‐rays and the mtDNA repair ability of oral squamous cell carcinoma (OSC) cell lines. The levels of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and mtDNA common deletion in both nuclear and mitochondrial DNA of OSC‐2, OSC‐3 and OSC‐6 cells (radio‐sensitive cell lines) after γ‐ray‐irradiation were higher than those of OSC‐1, OSC‐4 and OSC‐5 cells (radio‐resistant cell lines). Compared with OSC‐2, OSC‐3 and OSC‐6 cells, OSC‐1, OSC‐4 and OSC‐5 cells had higher levels of activity of phosphoinositide‐3 kinase (PI‐3K)/Akt and more strongly expressed 8‐hydroxyguanine DNA glycosylase (OGG1), DNA polymerase γ (POLG) and mitochondrial transcription factor A (Tfam). Down‐regulation of these mtDNA‐repair‐associated molecules by the RNA interference technique enhanced the susceptibility of OSC‐2 and OSC‐5 cells to γ‐rays, and the expression of Tfam and POLG was down‐regulated by inhibitors of PI‐3K/Akt signaling. These results indicate that the inhibition of mtDNA repair capacity by PI‐3K/Akt signal inhibitors and OGG1 down‐regulator in cancer cells may be a useful strategy for cancer treatment when combined with ionizing irradiation and chemotherapeutic drugs. ( Cancer Sci 2008; 99: 2230–2237)

We report a case of lingual osseous choristoma, a rare benign lesion. A 32-year-old woman was referred to our clinic because of a painless polypous tumor on the posterior part of the dorsum of the tongue. There was an elastic hard mass approximately 5mm in diameter on the left side of the cecal foramen of the tongue. We performed excision of the tumor with the patient under local anesthesia. Histologically, the lesion was a well-circumscribed mass of dense lamellar bone with Haversian canals. The postoperative course was uneventful, with no recurrence for about 18 months. Since 1938, when Muta reported the first case in Japan, 37 cases of oral osseous choristoma have been reported, including 34 arising on the tongue. The clinical findings of lingual osseous choristomas were reviewed, and their pathophysiology was discussed.

Osteoma, a benign proliferative bony lesion, is an uncommon lesion in the maxillary sinus. We report a case of ring-shaped osteoma with stratified squamous epithelium in the left maxillary sinus. A 55-year-old man was referred to our clinic because of a radiopaque lesion in the sinus. Computed tomography disclosed a bulky highdensity mass with a cavity. The mass was pedunculated and extended from the inferior to the posterior walls of the left maxillary sinus. The mass was extirpated by opening the maxillary sinus from the canine fossa under general anesthesia. The histopathological diagnosis was osteoma consisting mainly of spongeous bone, partially overlaid with pseudostratified ciliated epithelium and containing granulation tissue covered by stratified squamous epithelium. The clinical features, histopathology, and pathogenesis of osteoma in the maxillary sinus are discussed.

We prospectively assessed the benefits of using either a range-of-movement technique or an anatomical landmark method to determine the rotational alignment of the tibial component during total knee replacement. We analysed the cut proximal tibia intraoperatively, determining anteroposterior axes by the range-of-movement technique and comparing them with the anatomical anteroposterior axis. We found that the range-of-movement technique tended to leave the tibial component more internally rotated than when anatomical landmarks were used. In addition, it gave widely variable results (mean 7.5 degrees ; 2 degrees to 17 degrees ), determined to some extent by which posterior reference point was used. Because of the wide variability and the possibilities for error, we consider that it is inappropriate to use the range-of-movement technique as the sole method of determining alignment of the tibial component during total knee replacement.

Although adherent junctions have been extensively studied, the role of tight junctions in cancer cell invasion is not sufficiently explored. We investigated whether claudin-1, a component of tight junctions, regulated invasion activity in oral squamous cell carcinoma (OSC) cells. The expression of claudin-1, activity of matrix metalloproteinase (MMP)-2, and cleavage of laminin-5 gamma2 chains were assessed by Western blot analysis, immunohistochemistry, and zymography in OSC cell lines (OSC-4 and NOS-2, highly invasive; OSC-7, weakly invasive) and their xenografts in severe combined immunodeficient (SCID) mice. The influence of claudin-1 small interfering RNA (siRNA) on the invasion activity of the cell lines was also investigated. Compared with OSC-7, both OSC-4 and NOS-2 more strongly expressed claudin-1 and possessed high activities of MMP-2 and MMP-9. Tumors formed in the tongues of SCID mice xenografted with OSC-4, NOS-2, and OSC-7 immunohistochemically revealed strong, moderate, and weak expression of laminin-5 gamma2 chains, respectively, and laminin-5 gamma2 chains were secreted in the conditioned medium of the cancer cells in parallel with the in vivo results. Claudin-1 siRNA largely suppressed the invasion of OSC-4 and decreased the activation of MMP-2, the expression of membrane-type MMP-1 (MT1-MMP), and the cleavage of laminin-5 gamma2. In addition, not only antibodies against MT1-MMP and epidermal growth factor receptor (EGFR) but also MMP-2 and EGFR inhibitors strongly suppressed the invasion activity of OSC-4. These results suggest that claudin-1 up-regulates cancer cell invasion activity through activation of MT1-MMP and MMP-2, which results in enhanced cleavage of laminin-5 gamma2 chains.

We examined the effect of concomitant chemo-radio-immunotherapy on 80 patients with tongue carcinoma. Disappearance of the tumor without recurrence was observed in 21 patients (38.9%) in intravenous infusion chemotherapy group (A) and in 20 patients (76.9%) in intra-arterial infusion chemotherapy group (B) (P<0.005). A total of 41 patients (51.3%) were free from the tumor after the combined therapy. Along with the good therapeutic effect, oral function was preserved with minimal impairment of speech and mastication. Tumor stage, the mode of tumor cell invasion and tumor cell differentiation were not correlated with the therapeutic effect. In addition, the expression of p53, p21Cip1/WAF1 and proliferating cell nuclear antigen did not differ between the patients with lethal and non-lethal effects. The 5-year-survival rate was 56.8% in Group A, 76.9% in Group B and 59.6% overall. Thus, combined chemo-radio-immunotherapy, especially intra-arterial infusion, may bring a universal therapeutic effect in tongue carcinoma regardless of the tumor stage and the expression of cell phase-regulating proteins.

OBJECTIVES:To establish a novel strategy of fungal infection control.METHODS:We examined the influences of antimicrobial peptides including a synthesized short lactoferrin peptide (FKCRRWQWRM, Peptide 2; Pep2) on the synthesis of Candida cell wall polysaccharides, ergosterol synthesis, membrane permeability and the efflux of ATP.RESULTS:Colony formation of Candida albicans was synergistically suppressed by a combination of low concentrations of each drug and peptide. All peptides and amphotericin B, but not itraconazole, revealed weak inhibitory activities against ergosterol synthesis and the peptides weakly suppressed the synthesis of Candida cell wall components, glucan, mannan and chitin. Cell membrane permeability was not only increased by these peptides but also clearly increased by both amphotericin B and itraconazole. ATP efflux was however up-regulated by low concentrations of the peptides, especially by Pep2 and Hst5, although both antifungal drugs did not exert any influence on ATP efflux. The expression of the Candida drug resistance genes 1 and 2 (CDR1 and CDR2) was increased by both drugs, but this increase was suppressed by each peptide. In addition, larger amounts of amphotericin B and itraconazole remained in Candida cells in the presence of Pep2 or Hst5 due to the lower excretion. The effects of both peptides on ATP efflux and increase of intercellular amphotericin B and itraconazole were blocked by anion channel inhibitors 4,4'-diisothiocyanatestilbene-2, 2'-disulphonic acid and 5-nitro-2-(3-phenylpropylamino) benzoic acid.CONCLUSIONS:The examined peptides, especially Pep2 and Hst5, enhance the candidacidal activity of antifungal drugs by promoting anion channel-associated ATP efflux from Candida cells and decreasing efflux of the drugs, which could be useful clinical applications.

Aspiration of oropharyngeal bacteria and fungi is occasionally suspected in patients with pneumonia. A patient with oral carcinoma underwent chemoradioimmunotherapy and, about 4 weeks from the start of the therapy, the patient suffered from severe oral mucositis induced by chemoradiotherapy, and candidal pneumonia was subsequently induced. The candidal pneumonia was insufficiently improved by potent antifungal drugs, taking a lethal course. Randomly amplified polymorphic DNA analysis and DNA sequence examination of strains isolated from the oral cavity 1 week before the onset of pneumonia and autopsied lung revealed the identity of both strains as Candida albicans, and the DNA analysis supported aspiration of oral Candida. These results indicate that the pathogen of the pneumonia, C. albicans, was aspirated from the oral cavity and that oral Candida is easily aspirated and becomes the pathogen of pneumonia.

Objective: To elucidate the immunohistochemical characteristics of oral verruciform xanthomas and the aetiology of foam cells.

ABSTRACT To establish a novel strategy for the control of fungal infection, we examined the antifungal and neutrophil-activating activities of antimicrobial peptides. The duration of survival of 50% of mice injected with a lethal dose of Candida albicans (5 × 108 cells) or Aspergillus fumigatus (1 × 108 cells) was prolonged 3 to 5 days by the injection of 10 μg of peptide 2 (a lactoferrin peptide) and 10 μg of α-defensin 1 for five consecutive days and was prolonged 5 to 13 days by the injection of 0.1 μg of granulocyte-monocyte colony-stimulating factor (GM-CSF) and 0.5 μg of amphotericin B. When mice received a combined injection of peptide 2 (10 μg/day) with amphotericin B (0.5 μg/day) for 5 days after the lethal fungal inoculation, their survival was greatly prolonged and some mice continued to live for more than 5 weeks, although the effective doses of peptide 2 for 50 and 100% suppression of Candida or Aspergillus colony formation were about one-third and one-half those of amphotericin B, respectively. In vitro, peptide 2 as well as GM-CSF increased the Candida and Aspergillus killing activities of neutrophils, but peptides such as α-defensin 1, β-defensin 2, and histatin 5 did not upregulate the killing activity. GM-CSF together with peptide 2 but not other peptides enhanced the production of superoxide (O2−) by neutrophils. The upregulation by peptide 2 was confirmed by the activation of the O2−-generating pathway, i.e., activation of large-molecule guanine binding protein, phosphatidyl-inositol 3-kinase, protein kinase C, and p47phox as well as p67phox. In conclusion, different from natural antimicrobial peptides, peptide 2 has a potent neutrophil-activating effect which could be advantageous for its clinical use in combination with antifungal drugs.

We present a rare case of an ossifying adenocarcinoma originating in the parotid gland. Of a 52-year-old female with complaints of preauricular swelling, jaw trismus and mental nerve paralysis. Panoramic radiography and computed tomography revealed a tumor in the right preauricular region extending to the mandibular head and radiopaque miliary shadows in the lungs. Histopathologically, the tumor revealed characteristics of adenocarcinoma with prominent ossification. The immunostaining for bone morphogenetic protein-2 and osteopontin suggested that the ossification was induced by the tumor cells. Despite chemoimmunotherapy with TS-1® and OK-432® and later with an epidermal growth factor-receptor antagonist, gefitinib, the original tumor and metastatic pulmonary foci continued to growth and the patient eventually died about 15 months after the start of therapy.

We examined the influence of ROS on the phosphorylation and complex formation of Bcl‐2 family proteins in Mn‐superoxide dis‐mutase (SOD) antisense‐transfected squamous cell carcinoma cells, OSC‐4 cells. The increase of intracellular ROS level induced by cis‐diamminedichloroplatinum (CDDP) and γ‐ray treatment was greater in antisense‐transfected cells than in control vector‐trans‐fected cells, and apoptosis was more extensively induced in the former. Antisense‐transfected cells expressed high levels of Bax and Bak, but low levels of Bcl‐2 and Bcl‐XL when treated with CDDP, peplomycin, 5‐fluorouracil or γ‐rays. After treatment with these agents, the phosphorylation of protein kinase A, Bcl‐2 (Thr56) and Bad (Ser155) was increased, especially in antioxidant (N‐acetylcysteine and pyrrolidine dithiocarbamate)‐pretreated control cells, but the phosphorylation levels were very low in the antisense‐transfected cells. Bcl‐2 ubiquitination was increased, but ubiquitination of Bad and Bax was decreased in the antisense‐transfected cells, although their ubiquitination was increased by the antioxidants. These results reveal that ROS induce apoptosis by regulating the phosphorylation and ubiquitination of Bcl‐2 family proteins, resulting in increased proapoptotic protein levels and decreased antiapoptotic protein expression.

Background: Antimicrobial peptides in saliva appear to play a crucial role in the regulation of oral Candida growth, and study on antimicrobial excretion in saliva and oral candidiasis appears useful for the analysis of pathophysiology of oral candidiasis.Methods: To clarify the role of saliva in the regulation of oral Candida growth, the levels of antimicrobial proteins and peptides and their excretion rates were examined in saliva obtained from 50 patients with oral candidiasis and 35 healthy individuals.Results: The inhibitory activities of patients' saliva against Candida adhesion with HeLa cells and against Candida growth (radiolabeled glucose incorporation) were lower than those of saliva from the healthy controls. The salivary levels of lactoferrin (Lf; 11 +/- 9 mug/ml), secretory immunoglobulin A (sIgA; 160 +/- 37 mug/ml), beta-defensin 1 (375 +/- 37 ng/ml), and beta-defensin 2 (412 +/- 51 ng/ml) in the patients were largely lower than those in the control group (33 +/- 14 mug/ml, 204 +/- 51 mug/ml, 452 +/- 89 ng/ml, and 530 +/- 142 ng/ml, respectively), although the transferrin (Tf) and secretory component (SC) levels were almost same in both groups, and alpha-defensin 1 was slightly increased in the patient group (660 +/- 115 ng/ml vs. 467 +/- 168 ng/ml). In addition, the excretion rates of the proteins and peptides were largely decreased in the patients (Tf: 14 +/- 2 mug/10 min vs. 34 +/- 7 mug/10 min; Lf: 18 +/- 11 mug/10 min vs. 139 +/- 43 mug/10 min; sIgA: 300 +/- 132 mug/10 min vs. 900 +/- 207 mug/10 min; SC: 112 +/- 46 mug/10 min vs. 292 +/- 64 mug/10 min; alpha-defensin 1: 1223 +/- 431 ng/10 min vs. 2044 +/- 612 ng/10 min; beta-defensin 1: 687 +/- 243 ng/10 min vs. 1985 +/- 295 ng/10 min; and beta-defensin 2: 784 +/- 299 ng/10 min vs. 2288 +/- 278 ng/10 min).Conclusion: These results conclusively suggest that oral candidiasis is associated with salivary gland hypofunction and that decreases of salivary antibacterial proteins induce Candida overgrowth.

Cigarette smoking causes many chronic diseases but is a preventable risk factor in developing countries. However, it may be possible to relieve the smoke-induced damage by increasing the protective defense system. As vitamin C intake reduces smoking risk, it is recommended that smokers should take more vitamin C. However, the molecular mechanism of vitamin C intake on smokers has not been thoroughly investigated. We have found there to be suppression of smoke-induced cytochrome P-450 1A1 (CYP1A1) mRNA expression by high-dose ascorbic acid administration. Therefore, we surveyed other genes, the expressions of which were altered by the administration of high-dose ascorbic acid. As cigarette smoking increases oxidative stress, we investigated the effect on antioxidative enzyme expression. The osteogenic disorder Shionogi (ODS), rat, which lacks ascorbic acid synthesis enzyme, was administered either minimal amounts (4 mg/day, S4) or high-dose amounts (40 mg/day, S40) of ascorbic acid, and were exposed to cigarette smoke daily for 25 days. The effect on antioxidative enzymes mRNA expression in the liver was measured by competitive reverse transcription-polymerase chain reaction method (competitive RT-PCR). CuZn-superoxide dismutase (SOD), MnSOD, catalase and protein disulfide isomerase (PDI) were significantly decreased by high-dose ascorbic acid administration, and plasma glutathione peroxidase was also decreased, but not significantly. Cigarette smoke exposure slightly increased gene expression of PDI and catalase, but not significantly. The differently expressed 27 genes in the liver were found by differential display methods. From 27 genes, altered expression of plasma proteinase inhibitor, alpha-1-inhibitor III and CYP1A2 were confirmed by competitive RT-PCR. These results show that ascorbic acid intake influences gene expression of antioxidative enzymes, an ascorbic acid recycle enzyme, and xenobiotic metabolizing enzymes.