Relationship between hyposalivation and oxidative stress in aging mice.

The increase in oxidative stress that accompanies aging has been implicated in the abnormal advance of aging and in the onset of various systemic diseases. However, the details of what effects the increase in oxidative stress that accompanies aging has on saliva secretion are not known. In this study, naturally aging mice were used to examine the stimulated whole saliva flow rate, saliva and serum oxidative stress, antioxidant level, submandibular gland H-E staining, and immunofluorescence staining to investigate the effect of aging on the volume of saliva secretion and the relationship with oxidative stress, as well as the effect of aging on the structure of salivary gland tissue. The stimulated whole saliva flow rate decreased significantly with age. Also, oxidative stress increased significantly with age. Antioxidant levels, however, decreased significantly with age. Structural changes of the submandibular gland accompanying aging included atrophy of parenchyma cells and fatty degeneration and fibrosis of stroma, and the submandibular gland weight ratio decreased. These results suggest that oxidative stress increases with age, not just systemically but also locally in the submandibular gland, and that oxidative stress causes changes in the structure of the salivary gland and is involved in hyposalivation.

In vitro/in vivo evaluation of the efficacy of gatifloxacine-loaded PLGA and hydroxyapatite composite for treating osteomyelitis.

Molten 10 wt% gatifloxacine (GLFX-loaded poly(lactide-co-glycolide) (PLGA) was introduced into three-dimensionally interconnected pores and onto the surfaces of hydroxyapatite (HA) granules. The composite granules exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 3 h to 10 days. The composite granules were implanted in bone defects created by debridement of osteomyelitis lesions in rabbit mandibles. After 4-week implantation, inflammation in the composite granule-implanted group was significantly smaller than that in the debridement group (p<0.05). Moreover, newly formed bone was observed in the pores and on the surface of HA granules of the composite. These findings show that GFLX/HA composite controls bacterial infection and supports bone regeneration for osteomyelitis treatment.

Astaxanthin affects oxidative stress and hyposalivation in aging mice.

Oral dryness, a serious problem for the aging Japanese society, is induced by aging-related hyposalivation and causes dysphagia, dysgeusia, inadaptation of dentures, and growth of oral Candida albicans. Oxidative stress clearly plays a role in decreasing saliva secretion and treatment with antioxidants such astaxanthin supplements may be beneficial. Therefore, we evaluated the effects of astaxanthin on the oral saliva secretory function of aging mice. The saliva flow increased in astaxanthin-treated mice 72 weeks after administration while that of the control decreased by half. The plasma d-ROMs values of the control but not astaxanthin-treated group measured before and 72 weeks after treatment increased. The diacron-reactive oxygen metabolites (d-ROMs) value of astaxanthin-treated mice 72 weeks after treatment was significantly lower than that of the control group was. The plasma biological antioxidative potential (BAP) values of the control but not astaxanthin-treated mice before and 72 weeks after treatment decreased. Moreover, the BAP value of the astaxanthin-treated group 72 weeks after treatment was significantly higher than that of the control was. Furthermore, the submandibular glands of astaxanthin-treated mice had fewer inflammatory cells than the control did. Specifically, immunofluorescence revealed a significantly large aquaporin-5 positive cells in astaxanthin-treated mice. Our results suggest that astaxanthin treatment may prevent age-related decreased saliva secretion.

Effects of gatifloxaine content in gatifloxacine-loaded PLGA and ß-tricalcium phosphate composites on efficacy in treating osteomyelitis.

Composites of gatifloxacin (GFLX)-loaded poly (lactic-co-glycolic) acid (PLGA) and ß-tricalcium phosphate (ßTCP) containing 0, 1, and 10 wt % GFLX (0, 1, and 10 wt % GFLX composites), and GFLX-loaded PLGA containing 1, 5, and 10 wt % GFLX (1, 5, and 10wt % GFLX-PLGA) as controls were fabricated and characterized in vitro and in vivo. On in vitro evaluation, the 10 wt % GFLX composite released GFLX over at least 28 days in Hanks' balanced solution and exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 1 h to 10 days. The 0, 1, and 10 wt % GFLX composites and 10 wt % GFLX-PLGA were implanted in bone defects created by debridement of osteomyelitis lesions induced by S. milleri and B. fragilis in the mandible of rabbits (n = 5). Four weeks after implantation of the 10 wt % GFLX composite, inflammation in the debrided area disappeared in all the rabbits, while inflammation remained in all the rabbits after implantation of the 0 wt % GFLX composite and 10 wt % GFLX-PLGA, and in three rabbits after implantation of the 1 wt % GFLX composite. Bone formation appears to be less intense for the 10 wt % GFLX composite than for the 1 wt % GFLX composite probably owing to the rapid degradation of the 10 wt % GFLX composite. These findings show that the GFLX composite is effective for the local treatment of osteomyelitis.

Controlled release of simvastatin from biodegradable hydrogels promotes odontoblastic differentiation.

The objective of this study was to investigate the odontoblastic differentiation of dental pulp stem cells (DPSC) by biodegradable hydrogels incorporating simvastatin micelles, both in vitro and in vivo. Simvastatin (ST) was incorporated into the micelles of gelatin grafted with L-lactic acid oligomers (LAo) to allow water-solubilization. The simvastatin-LAo-grafted gelatin (LAo-g-gelatin) micelles were mixed with gelatin, followed by chemical crosslinking to form gelatin hydrogels (ST Mi/GH). The ST Mi were released from the gelatin hydrogel granules (GH) through enzymatic degradation. The ST Mi enhanced alkaline phosphatase activity, calcium deposition, and bone morphogenic protein-2 secretion of DPSC. When implanted subcutaneously into mice, the ST Mi/GH treated group exhibited increased dentin sialoprotein and calcium deposition, compared with those treated with GH plus free ST. It is possible to achieve odontoblastic differentiation of DPSC through the controlled release of ST from GH.

Effects of bovine lactoferrin to oral Candida albicans and Candida glabrata isolates recovered from the saliva in elderly people.

The effects of bovine lactoferrin (bLF) on the growth of Candida species and on inflammatory cytokine production in gingival keratinocytes, NDUSD-1 co-cultured with Candida strains were investigated. The results showed that bLF at 10 and 100 lg/mL significantly inhibits the growth of two C. albicans strains and two C. glabrata strains isolated from the saliva of elderly people requiring nursing care, respectively. The levels of inflammatory cytokines, interleukin (IL)-6, and IL-8 in NDUSD-1 cocultured with each of these four Candida strains were measured. C. albicans tend to have a more potent capacity than C. glabrata to induce the production of the inflammatory cytokines in NDUSD-1. The levels of IL-6 and IL-8 in NDUSD-1 co-cultured with each of Candida species were measured after addition of bLF. bLF at concentrations from 1 to 100 lg/mL significantly inhibited the production of these cytokines in NDUSD-1 co-cultured with Candida species. These findings suggest that bLF may be useful in reducing the risk of aspiration pneumonia among elderly people requiring care for whom oral care is difficult.

Enhanced bone regeneration by gelatin-ß-tricalcium phosphate composites enabling controlled release of bFGF.

The objective of this study was to investigate the feasibility of biodegradable gelatin-ß-tricalcium phosphate (ß-TCP) composites as a cell scaffold and controlled-release carrier of basic fibroblast growth factor (bFGF) suitable for inducing bone regeneration at a segmental bone defect. The composite of gelatin sponge and ß-TCP granules had an interconnected pore structure with an average size of 340 µm. The composite provided the controlled release of bFGF over 2 weeks. Segmental, critical-sized, bone defects of 20 mm length were created in the ulnas of New Zealand white rabbits and the gelatin-ß-TCP composites, with or without incorporated bFGF, were implanted into the defects. Bone regeneration and ß-TCP resorption profiles were evaluated by microcomputed tomography scanner analysis and haematoxylin and eosin staining. The composites incorporating bFGF promoted significantly higher bone regeneration at the defect site as compared to the bFGF-free composites. The controlled release of biologically active bFGF from the composites may possibly be achieved through the biodegradation of the composites, resulting in the promotion of bone regeneration. We conclude that the biodegradable gelatin-ß-TCP composite is a promising scaffold for bone regeneration that enables the controlled release of bFGF.

Effects of bovine lactoferrin to oral Candida albicans and Candida glabrata isolates recovered from the saliva in elderly people.

The effects of bovine lactoferrin (bLF) on the growth of Candida species and on inflammatory cytokine production in gingival keratinocytes, NDUSD-1 co-cultured with Candida strains were investigated. The results showed that bLF at 10 and 100 µg/mL significantly inhibits the growth of two C. albicans strains and two C. glabrata strains isolated from the saliva of elderly people requiring nursing care, respectively. The levels of inflammatory cytokines, interleukin (IL)-6, and IL-8 in NDUSD-1 co-cultured with each of these four Candida strains were measured. C. albicans tend to have a more potent capacity than C. glabrata to induce the production of the inflammatory cytokines in NDUSD-1. The levels of IL-6 and IL-8 in NDUSD-1 co-cultured with each of Candida species were measured after addition of bLF. bLF at concentrations from 1 to 100 µg/mL significantly inhibited the production of these cytokines in NDUSD-1 co-cultured with Candida species. These findings suggest that bLF may be useful in reducing the risk of aspiration pneumonia among elderly people requiring care for whom oral care is difficult.

Preparation of thermoplastic poly(L-lactic acid) membranes for guided bone regeneration.

PURPOSE: The objective of this study was to evaluate the feasibility of application of thermoplastic poly-L lactic acid (PLLA) membranes for guided bone regeneration in rabbit parietal bone. MATERIALS AND METHODS: PLLA membranes with a molecular weight of 100,000 (PLLA-100,000) and a molecular weight of 380,000 (PLLA-380,000) were dissolved in chloroform to prepare concentrations of 8% by weight and 4% by weight, respectively. The compression strength, temperature, and time to prepare each formulation were measured. Moreover, the pH was noted and cytotoxicity of the membrane was determined by monotetrazolium assay. In vivo experiments were performed to measure the volume of newly formed bone tissue in hematoxylin and eosin-stained tissue sections 4 and 12 weeks after implantation. RESULTS: The membrane prepared from PLLA-380,000 showed excellent thermoplasticity at 75°C to 80°C and the compressive strength was equal to that of titanium mesh, in contrast to that of PLLA-100,000 and poly(lactic acid-co-glycolic acid). There was a significant change in the pH of an aqueous solution in which the PLLA-380,000 membrane was placed, but there was no cytotoxic activity. The membrane made of PLLA-380,000 induced new bone formation in a dome shape without any membrane deformation. CONCLUSION: Thermoplastic PLLA membrane shows promise for guided bone regeneration in vertical bone augmentation.

Isolation and characterization of epithelial and myogenic cells by "fishing" for the morphologically distinct cell types in rat primary periodontal ligament cultures.

The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.

In vitro evaluation of H2O2 hydrothermal treatment of aged titanium surface to enhance biofunctional activity.

Surface modification of titanium has been extensively investigated in implant science and technology in an effort to improve its osteoconductivity. The rate of protein adsorption on titanium surfaces is known to vary depending on the chemistry, structure, morphology, and titanium-specific biological aging of the surface. It is thus desirable to modify smooth titanium surfaces of miniimplants used as orthodontic anchors immediately prior to use. In this study, we have developed a simple surface modification of titanium alloy that improves its biofunctional activity. The surface of a Ti-6Al-4V disk was modified by applying 3% H(2)O(2) hydrothermal treatment using an autoclave. A nanostructured porous network TiO(2) was observed on the treated surface. Treated surfaces exhibited higher hydrophilicity, protein adsorption, and cell proliferation than untreated surfaces. 3% H(2)O(2) hydrothermal treatment is thought to provide biofunctional activity for aged titanium surface.

Electrodeposition of pronectin for titanium to augment gingival epithelium adhesion.

This paper is one trial of surface modification of titanium with pronectin F+ (PN) of an artificial protein to enhance gingival adhesion. Titanium plates were electrodeposited in the PN solution to prepare PN-electrodeposited titanium plates. When PN detachment from the PN-electrodeposited titanium plates was investigated, no detachment was observed, in contrast to the case of titanium plates simply coated with PN. A cell culture experiment demonstrated that electrodeposited PN had an inherent ability to enhance the initial attachment of gingival epithelial cells. The PN-electrodeposited titanium plates were implanted between the gingival epithelium and the underlying bone tissue of rabbits to evaluate epithelial growth on the plates and their gingival adhesion. Non-treated and PN-coated titanium plates were used as controls. PN electrodeposition enhanced epithelial growth and adhesion of titanium plates to a significantly great extent compared with PN-coated plates. These findings demonstrate that PN electrodeposition is a promising method to enhance epithelium adhesion onto a titanium surface.

Pathological analysis of the Candida albicans-infected tongue tissues of a murine oral candidiasis model in the early infection stage.

OBJECTIVE: The early pathological process of Candida infection and immunological responses in tongues of the mice with experimental oral candidiasis was analysed. METHODS: CD-1 mice, pretreated by prednisolone were orally inoculated with Candida albicans. Symptoms were monitored by measuring the area of white tongue coating and number of viable Candida cells in oral cavity. The histopathological analysis was carried by PAS-stain and immunofluorescent staining. IL-4, IL-12p70, IFN-γ, TNF-α in recovered from the homogenates of the tongues were measured by ELISA. RESULTS: The fungus invaded the tongue surface of the mice and white patches developed within 24h after inoculation. Histopathological examination indicated the presence of local acute inflammation in superficial tissues of tongues covered by mycelium of C. albicans. Pathological exacerbation was observed from 24 to 48 h after the inoculation and from then the symptoms of oral candidiasis appeared to move into the recovery phase. Inflammatory cells mainly consisting of neutrophils was accumulated and located under the lesions covered by Candida-hyphae. An increase in IL-12p70 and IFN-γ in tongue homogenates was observed at 48 h after inoculation. CONCLUSIONS: The worst condition in the pathological process in experimental oral candidiasis was found 48 h after C. albicans inoculation. When the surface of the Candida-inoculated tongues was covered with Candida-hyphae, a dense accumulation of neutrophils was observed under the lesions and homogenates of the tongues contained increased levels of IL-12p70 and IFN-γ. These suggested that local pathological condition of Candida-infected tongues may be affected by neutrophils accumulation and increased levels of some cytokines.

Gatifloxacine-loaded PLGA and ß-tricalcium phosphate composite for treating osteomyelitis.

Gatifloxacine (GFLX)-containing poly(lactide-co-glycolide) (PLGA) was introduced to the pores and surfaces of porous ß-tricalcium phosphate (ßTCP) granules by melt compounding whereby no toxic solvent was used. The granular composite of GFLX-loaded PLGA and ßTCP released GFLX for 42 days in Hanks' balanced solution and exhibited sufficient in vitro bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 21 days. For in vivo evaluation, the granular composite was implanted in the dead space created by the debridement of osteomyelitis lesion induced by S. milleri and B. fragilis in rabbit mandible. After a 4-week implantation, the inflammation area within the debrided area was markedly reduced accompanied with osteoconduction and vascularization in half of the rabbits, and even disappeared in one of the six rabbits without any systemic administration of antibiotics. Outside the debrided area, inflammation and sequestrum were observed but the largest of such affected areas amounted to only 0.125 times of the originally infected and debrided area. These findings showed that the granular composite was effective for the local treatment of osteomyelitis as well as an osteoconductive scaffold which supported and encouraged vascularization.

E6AP ubiquitin ligase mediates ubiquitin-dependent degradation of peroxiredoxin 1.

E6-associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high-risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin-dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP-binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25-kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull-down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi-mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin-dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1-dependent intracellular oxidative signal transduction pathways.

Effect of an injectable 3D scaffold for osteoblast differentiation depends on bead size.

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.

Identification and characterization of the human inhibitor of caspase-activated DNase gene promoter.

DNA fragmentation factor is a heterodimer complex of the nuclease CAD and its specific inhibitor ICAD, which can be activated during apoptosis to induce DNA fragmentation. Although ICAD expression levels have been quantified in a variety of human cancer cells, the mechanism of ICAD gene regulation remains unknown. In this study, we identified a 106-bp TATA-less region upstream of the transcription start site as a basal promoter of the human ICAD gene. An E-Box motif, which binds transcription factors of the basic helix-loop-helix/leucine zipper family, is responsible for transcriptional activity, as demonstrated using mutated promoter-reporters. A chromatin immunoprecipitation assay further demonstrated that Myc binds to an endogenous ICAD promoter. The functional importance of Myc in the regulation of ICAD transcription was also demonstrated by knock-down of c-Myc and N-Myc gene expression, as well as their ectopic expression. Structural analysis of the human ICAD promoter and identification of factors which regulate its activity might further our understanding of the biological role of ICAD with respect to regulation of apoptosis and cancer development.

Preparation of injectable 3D-formed beta-tricalcium phosphate bead/alginate composite for bone tissue engineering.

A novel, injectable bone tissue engineering material was developed that consisted of beta-tricalcium phosphate (beta-TCP) beads as the solid phase and alginate as the gel phase. To prepare the instantaneously formed composite scaffold, an aqueous calcium chloride solution was dried on the surface of beta-TCP beads and crosslinked with an alginic acid sodium solution, thereby forming stable beta-TCP beads and alginate gel which were injectable via a syringe. This biodegradable composite was a three-dimensional (3D) material that could be used as an injectable scaffold for bone tissue engineering. In particular, the composite with 2.0 wt% alginate concentration exhibited a compressive strength of 69 kPa in dry conditions, which was significantly higher than that exhibited by 1.0 wt%. Furthermore, mesenchymal stem cells (MSC) were 3D-cultured within the composite and then investigated for osteogenic markers. MSC-loaded composite was subjected to scanning electron microscope (SEM) examination and implanted subcutaneously for in vivo experiment. Results showed that the scaffold provided support for osteogenic differentiation. In light of the encouraging results obtained, this novel injectable composite material may be useful for bone tissue engineering.

Antibiotic-loaded poly-epsilon-caprolactone and porous beta-tricalcium phosphate composite for treating osteomyelitis.

A composite of poly-epsilon-caprolactone (PCL) loaded with gatifloxacine (GFLX), an antibiotic, and a beta-tricalcium phosphate (betaTCP) porous ceramic body was prepared by a solvent-free process in which no toxic solvent was used. GFLX mostly retained its bactericidal property after the processing. The composite of GFLX-loaded PCL and betaTCP ceramic released GFLX for 4 weeks in Hanks' balanced solution, and had sustained bactericidal activity against Streptococcus milleri and Bacteroides fragilis for at least 1 week. The composite of the GFLX-loaded PCL and betaTCP ceramic was implanted in an osteomyelitis lesion induced by S. milleri and B. fragilis in the rabbit mandible. The osteomyelitis lesion expanded in the mesial-distal direction when no composite was implanted or when the lesion was treated with debridement only. The composite of GFLX-loaded PCL and betaTCP showed efficacy in controlling infection at the bone defect formed by debridement, and supported bone tissue reconstruction at the bone defect. Twelve and 50 weeks after the implantation, the inflammation even disappeared. New bone formation was observed on the surface of the composite after 4 weeks. After 50 weeks, ingrowth of bone tissues with vascular channels was observed along the PCL and betaTCP interface, which indicated degradation of PCL and/or betaTCP ceramic at the ceramic/polymer interface followed by replacement by bone tissues. The GFLX concentrations in the serum and soft tissues were very low. Therefore, the composite of GFLX-loaded PCL and betaTCP ceramic would help arrest osteomyelitis when it is used in addition to intravenous antibiotic administration, and help new bone formation and osteoconduction.

Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.

PURPOSE: The loss of epithelial phenotypes in the process of carcinoma progression correlates with clinical outcome, and genetic/epigenetic changes accumulate aggressive clones toward uncurable disease. IkappaB kinase-alpha (IKKalpha) has a decisive role in the development of the skin and establishes keratinocyte phenotypes. We assessed clinical implications of IKKalpha expression in oral carcinomas and epigenetic aberrations for the loss of expression. EXPERIMENTAL DESIGN: We examined IKKalpha expression in oral carcinomas by immunostaining (n = 64) and genetic instability by microsatellite PCR (n = 46). Promoter methylation status was analyzed by bisulfite-modified sequence (n = 11). RESULTS: IKKalpha was expressed in the nucleus of basal cells of normal oral epithelium, but not or marginally detected in 32.8% of carcinomas. The immunoreactivity was significantly decreased in less differentiated carcinomas (P < 0.05) and correlated to long-term survival of patients (P < 0.01) with an independent prognostic value (P < 0.05). Although allelic/biallelic loss of the gene was limited to four cases, we detected microsatellite instability in 63.0% cases in which the immunoreactivities were decreased and the promoter was hypermethylated. CONCLUSION: These results showed that oral carcinomas exhibiting genetic instability and promoter hypermethylation down-regulate expression of IKK and suggest that the epigenetic loss of the expression closely associates with disease progression toward unfavorable prognosis.