Masayasu Iwase

Role of interleukin-8 secreted from human oral squamous cell carcinoma cell lines

Interleukin-8 (IL-8) is an important cytokine involved in tumor growth and angiogenesis in a variety of malignancies. Furthermore, matrix metalloptoteinases (MMPs) also play important roles in the invasion and metastasis of carcinomas including oral squamous cell carcinoma (OSCC). We studied whether IL-8 and MMPs participate in tumorigenesis and metastasis of OSCC. First, we investigated the gene and protein expressions of IL-8 and IL-8 receptor (IL-8R), and the effect of IL-8 on proliferation, migration and invasion of OSCC. Second, we thus also investigated the effect of IL-8 on MMP release in OSCC cells. OSCC cell lines NA and HSC-4 constitutively expressed IL-8 mRNA and secreted its protein in vitro. The production of IL-8 was significantly enhanced by the addition of tumor necrosis factor (TNF)-alpha and IL-beta, but not interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-2. Flow cytometric analysis revealed the constitutive expression of both receptors of IL-8, IL-8RA and IL-8RB, in OSCC cell lines. The expression of IL-8 receptors in HSC-4 cells was stronger than that in NA cells. The intensity of IL-8RA expression was stronger than that of IL-8RB expression in each cell line. The expression of IL-8 receptors was not altered by the addition of cytokines such as TNF-alpha and IL-1beta. The conditioned medium containing IL-8 from OSCC cell lines induced migration and invasion of OSCC cells, but did not change cell proliferation. The differences in migrational and invasive ability between NA cells and HSC-4 cells were correlated with the expression intensity of IL-8 receptors in each cell line. Neutralizing antibodies to IL-8, IL-8RA and IL-8RB partially inhibited the chemotactic activity induced by conditioned medium. The expression of MMP-2, -7 and -9 was detected in culture supernatants from these OSCC cell lines. The expressions of MMP-7 protein and mRNA were enhanced by the addition of rIL-8, but that of other MMPs was not observed in a similar manner. These results suggest that IL-8 secreted from OSCC may contribute to the invasion of OSCC through the regulation of MMP-7 expression.

Changes in bite force and occlusal contacts in patients treated for mandibular prognathism by orthognathic surgery

PURPOSE The purpose of this study was to evaluate changes in bite force and occlusal contacts before and after orthognathic surgery in patients with mandibular prognathism and to compare the findings with those in controls with normal occlusion. PATIENTS AND METHODS Bite force and occlusal contacts were analyzed in 23 (7 male and 16 female) patients with mandibular prognathism before and after sagittal split ramus osteotomy, and in 20 (10 male and 10 female) controls with normal occlusion. The bite force and occlusal contacts were simultaneously measured by a computerized occlusal analysis system, the T-Scan system, immediately before surgery, and at 6 weeks, 3 months, 6 months, and 1 year postoperatively. RESULTS Both the bite force and occlusal contacts in the patients were significantly less than those in the controls before surgery. Although both the bite force and occlusal contacts in the patients were improved by the orthognathic surgery, neither approached the level in the controls within 1 year. Bite force was correlated with the number of occlusal contacts in both patient and control groups. CONCLUSION The postoperative masticatory function does not reach control levels even 1 year after the orthognathic surgery for mandibular prognathism. Therefore, further adjustment of the occlusion should be considered before the end of treatment.

Suppressive effect of selective cyclooxygenase-2 inhibitor on cytokine release in human neutrophils.

To clarify whether a selective cyclooxygenase-2 (COX-2) inhibitor can affect various functions in human peripheral blood neutrophils. For this purpose, the effects of selective COX-2 inhibitors, NS-398 and nimesulide, on the expression of COX-2, PGE2 release and respiratory burst, degranulation and cytokine release in activated neutrophils were examined. Peripheral blood neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP; 100 nM) or opsonized zymosan (OZ; 200 microg/ml). Then, the expression of COX-2 at protein and mRNA levels was detected by Western blot analysis and RT-PCR. The concentration of prostaglandin E2 (PGE2) and cytokines in the culture supernatant of neutrophils was determined using ELISA. Superoxide generation was measured by the cytochrome c reduction method. Elastase activity was measured using a chromogenic substrate assay specific for human neutrophil elastase. FMLP and OZ enhanced PGE2 release through induction of COX-2 protein and mRNA expression. FMLP- or OZ-induced PGE2 release was abolished by the addition of NS-398 or nimesulide; nevertheless, even a high concentration of COX-2 inhibitor did not change FMLP- or OZ-induced expression of COX-2 at message and protein levels. Although FMLP- or OZ-induced superoxide generation and elastase release were not affected by the addition of COX-2 inhibitor, cytokine release such as interleukin (IL)-1beta, IL-6 and IL-8 was significantly inhibited by high concentration of COX-2 inhibitor, but tumor necrosis factor-alpha (TNF-alpha) was partially attenuated. These studies showed that selective COX-2 inhibitors, NS-398 and nimesulide, suppressed PGE2 and proinflammatory cytokine release in activated neutrophils. These results suggest that selective COX-2 inhibitors may contribute to resolution of acute inflammation through the reduction of inflammatory cytokine release in activated neutrophils.

Effect of 5-fluorouracil on G1 phase cell cycle regulation in oral cancer cell lines.

5-fluorouracil (5-FU) has been widely used for chemotherapy of head and neck cancer, and is known to affect the cell cycle and induce apoptotic death of cancer cells. However, the molecular actions of 5-FU on the cell cycle regulatory mechanism have not been fully explained. Herein we analyzed the effects of 5-FU on the expression of G1/S-related cell cycle regulators in oral cancer cell lines. In vitro 5-FU treatment of oral cancer cells resulted in an increase in G1/S phase cells. p21 expression was augmented by 5-FU without any notable changes in p53 expression. A remarkable up-regulation of cyclin E and a concomitant down-regulation of cyclin D were observed after 24 h 5-FU treatment. Our results suggest that 5-FU-induced changes in cell cycle regulation of oral cancer cells might associate with an alteration of G1 cyclins expression. p21 was remarkably up-regulated, but it was speculated that its activity might be cancelled by an increased binding to CDK4.

Positional changes in the mandibular condyle and amount of mouth opening after sagittal split ramus osteotomy with rigid or nonrigid osteosynthesis

PURPOSE The purpose of this study was to investigate postoperative positional changes in the mandibular condyle and mouth opening in patients undergoing sagittal split ramus osteotomy with either rigid or nonrigid osteosynthesis. PATIENTS AND METHODS The forty-six patients with mandibular prognathism underwent sagittal split ramus osteotomy for mandibular set back followed by fixation with one of four methods: circumferential wire (n = 11), lag screw technique (n = 10), positional screw technique (n = 10), or miniplates (n = 15). The changes in the condylar position were assessed by measuring the angle of the condylar long axis (the condylar angle) on submentovertex radiographs. Mouth opening was evaluated by measuring the interincisal distance immediately after the release of maxillomandibular fixation and by monitoring the duration of trismus. RESULTS Regardless of the procedure used the condylar angle increased in most patients after surgery (80 of 92 condyles). Although the amount of increase tended to be higher with rigid osteosynthesis than with nonrigid osteosynthesis, no significant differences were observed among the groups. Mouth opening was not significantly influenced by the type of osteosynthesis, and no patient complained of limitation 1 year after surgery. CONCLUSIONS Although inward rotation of the condyle frequently occurs after osteosynthesis regardless of the procedure used, the changes in condylar position are within the range of adaptability of the patient.

Changes in susceptibility to Fas-mediated apoptosis during differentiation of HL-60 cells.

The Fas‐mediated pathway has been implicated as an important cellular pathway mediating apoptosis in diverse cell types. We conducted studies to examine the susceptibility to Fas‐mediated apoptosis of HL‐60 cells treated with differentiation‐inducing factors such as dimethyl sulfoxide (DMSO), retinoic acid (RA), and 1α, 25 dihydroxyvitamin D3 (VD3). Although the expression of Fas antigen (Ag) and its mRNA showed a marked increase in HL‐60 cells with cell differentiation, that of Bcl‐2 protein and its mRNA revealed the reverse. The expression of caspase proteins such as caspases‐3 and ‐8 was also enhanced during cell differentiation. DNA fragmentation, annexin V binding, and caspase activities increased in differentiated HL‐60 cells with the addition of anti‐Fas Ag antibody. These findings were more clearly demonstrated in DMSO‐ or RA‐induced neutrophil‐like cells than in VD3‐induced monocyte‐like cells. Therefore, susceptibility to Fas‐mediated apoptosis showed an increase with differentiation of HL‐60 cells, especially in the neutrophil lineage. These results suggest that the difference of susceptibility to Fas‐mediated apoptosis among cell populations depends on the expression of Fas Ag, Bcl‐2, and caspases. Cell maturation and susceptibility to Fas‐mediated apoptosis may be linked in hematopoietic cells. J. Leukoc. Biol. 67: 374–380; 2000.

Enhanced susceptibility of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5-fluorouracil

Our study was conducted to investigate whether anticancer drugs, cisplatin (CDDP) and/or 5‐fluorouracil (5‐FU), can modulate Fas‐mediated apoptosis in oral squamous cell carcinoma (OSCC) cell lines. When OSCC cell lines, NA and HSC‐4, were treated with CDDP and/or 5‐FU, Fas and its mRNA expression on the plasma membrane were enhanced. An increase in caspase‐3 and ‐8 activities was then observed by the addition of agonistic anti‐Fas antibody, CH‐11. Apoptosis of OSCC cells treated with anticancer drugs were significantly enhanced by CH‐11, whereas untreated cells were nearly resistant to apoptosis. Moreover, the combination of CDDP and 5‐FU resulted in an increasing susceptibility to apoptosis. Caspase‐3 and ‐8 inhibitors, but not caspase‐9 inhibitor, reduced Fas‐mediated apoptosis enhanced by the anticancer drugs. Furthermore, OSCC cells treated with anticancer drugs exhibited decreased cellular FADD‐like interleukin 1‐converting enzyme‐inhibitory protein (c‐FLIP) levels, whereas neither the Fas‐associated death domain‐containing protein (FADD) nor procaspase‐8 changed the expression. Moreover, antisense oligonucleotide to c‐FLIP confirmed that down‐regulation of c‐FLIP induced sensitization to Fas‐mediated apoptosis. These results suggest that CDDP and 5‐FU may enhance the susceptibility to Fas‐mediated apoptosis through down‐regulation of c‐FLIP. From these findings, a new potential strategy may be developed to improve the efficacy of anticancer drugs.

Lytic effects of Actinobacillus actinomycetemcomitans leukotoxin on human neutrophil cytoplasts

Actinobacillus actinomycetemcomitans leukotoxin lysed human neutrophil cytoplasts. The reaction was associated with a rapid influx of extracellular calcium, a collapse in membrane potential, release of lactate dehydrogenase, and overt disintegration of the plasma membrane. These functional and structural alterations in the plasmalemma of neutrophil cytoplasts reinforce the hypothesis that A. actinomycetemcomitans leukotoxin acts as a pore‐forming, membranolytic agent and indicate that neutrophil cytoplasts are useful tools in studying the biology of membrane‐active toxins.

Proteasome inhibitor sensitizes oral squamous cell carcinoma cells to TRAIL-mediated apoptosis

Oral squamous cell carcinoma (OSCC) cells are relatively resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis during culture. We investigated the role of a proteaosome inhibitor in the survival and apoptosis of these cells. We found that the proteasome inhibitor MG132 markedly accelerated TRAIL-mediated apoptosis in OSCC cell lines HSC-2 and HSC-3. Addition of TRAIL to MG132-treated cells resulted in Bid cleavage. Furthermore, the inhibitors of caspase-3, caspase-8 and caspase-9 reduced the accelerative effect of MG132 on TRAIL-mediated apoptosis. These results suggest that the pro-apoptotic effect of a proteasome inhibitor on TRAIL-mediated apoptosis may contribute to both extrinsic and intrinsic pathways. MG132 enhanced the expression of the TRAIL receptors DR4 and DR5, and neutralization of DR5 receptors showed a marked reduction of TRAIL-mediated apoptosis, whereas that of DR4 was a partial reduction. MG132 also markedly reduced cellular FLICE-inhibitory protein (c-FLIP), cellular inhibitor of apoptosis protein-1 (cIAP-1), X-linked IAP (XIAP) and survivin. Therefore, MG132 provides partial regulation of TRAIL-mediated apoptosis in OSCC cells via modulation of DR5, c-FLIP, cIAP-1, XIAP and survivin. The proteasome inhibitor MG132 may therefore represent a novel strategy for overcoming resistance to TRAIL-mediated apoptosis in OSCC cells.

Traumatic Displacement of Maxillary Permanent Canine into the Vestibule of the Mouth

Dentoalveolar injuries are common and are caused by many factors. Dental trauma requires special consideration when a missing tooth or tooth fracture accompanies soft tissue laceration. A tooth or its fragment occasionally penetrates into soft tissue and may cause severe complications. This report presents a case of delayed diagnosis and management of a displaced tooth in the vestibule of the mouth following dentoalveolar injury. This report suggests that radiography can lead to an early diagnosis and surgical removal of an embedded tooth in the soft tissue.