Tetsuro Ikebe

Expression of MMPs, MT-MMP, and TIMPs in squamous cell carcinoma of the oral cavity: Correlations with tumor invasion and metastasis

Matrix metalloproteinases (MMPs) that degrade the extracellular matrices (ECMs) have been thought to play an important role in both the invasion and metastasis of tumors. However, the detailed role of MMPs and TIMPs (tissue inhibitors of MMP) on the biological behavior of tumor cells has yet to be elucidated in vivo. The aim of the present study was thus to determine whether expression of MMPs on tumor cells is associated with such clinicopathological features as the invasive and metastatic potential.

Activation of NF-κB Is Involved in the Survival of Osteoclasts Promoted by Interleukin-1

We previously reported that interleukin-1 (IL-1) promoted the survival of murine osteoclast-like cells (OCLs) formedin vitro and activated a transcription factor, NF-κB, of OCLs. The present study examined whether the activation of NF-κB is directly involved in the survival of OCLs promoted by IL-1. The expression of IL-1 type I receptor mRNA in OCLs was detected by the polymerase chain reaction amplification of reverse-transcribed mRNA. An electrophoretic mobility shift assay showed that IL-1 transiently activated NF-κB in the nuclei of the OCLs, and the maximal activation occurred at 30 min. The degradation of IκBα coincided with the activation of NF-κB in the OCLs. The immunocytochemical study revealed that p65, a subunit of NF-κB, was translocated from the cytoplasm into almost all of the nuclei of the OCLs within 30 min after IL-1 stimulation. The purified OCLs spontaneously died via apoptosis, and IL-1 promoted the survival of OCLs by preventing their apoptosis. The pretreatment of purified OCLs with proteasome inhibitors suppressed the IL-1-induced activation of NF-κB and prevented the survival of OCLs supported by IL-1. When OCLs were pretreated with antisense oligodeoxynucleotides to p65 and p50 of NF-κB, the expression of respective mRNAs by OCLs was suppressed, and the IL-1-induced survival of OCLs was concomitantly inhibited. These results indicate that IL-1 promotes the survival of osteoclasts through the activation of NF-κB.

High expression levels of nuclear factor κB, IκB kinase α and akt kinase in squamous cell carcinoma of the oral cavity

BACKGROUND It has been reported that the transcription factor nuclear factor κB (NF-κB) is involved in the growth, invasion, and antiapoptotic activity of cultured tumor cells. METHODS The authors used immunohistochemistry to examine the expression of NF-κB and the signaling molecules leading to NF-κB activation in 36 untreated biopsy specimens from patients with squamous cell carcinoma (SCC) and in 15 specimens from patients with epithelial dysplasia of the oral cavity. RESULTS Among the molecules examined, the p65 subunit of NF-κB (p65) and IκB kinase α (IKKα) were expressed highly in almost all SCC specimens examined, whereas the samples of normal squamous epithelia adjacent to tumors as well as epithelial dysplasia specimens were negative in for immunohistochemical staining. The invasiveness and metastasis of SCC seemed to correlate with the degree of staining degree in the molecules. Moreover, phosphorylated Akt kinase, which may be associated with antiapoptosis signaling of NF-κB, was detected in the same areas where IKKα existed in large amounts. CONCLUSIONS The results suggest that high expression levels of p65 and IKKα contribute to malignant behavior and antiapoptotic activity in SCC of the oral squamous epithelium. Cancer 2001;92:3037–44.

Gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer

We examined whether or not the gelatinolytic activity in tumor tissue was associated with the invasion and metastasis of oral squamous cell carcinoma (OSCC). Tissue homogenates were prepared from 57 biopsy specimens of OSCC. The gelatinolytic activities in the homogenates were measured by gelatin zymography and its densitometric analysis. The Immunoblot findings revealed the major gelatinolytic activities to be due to matrix metalloproteinase (MMP)-2 and -9. The zymography-detected gelatinolytic activities of MMP-2 and MMP-9 in the tissue specimens significantly correlated with the degree of immunohistochemical staining detected in frozen sections of the same biopsy specimens. According to a histopathological analysis of the mode of invasion, highly invasive cases showed the increased gelatinolytic activities of MMP-2 as well as MMP-9 in the tissue specimens. Although no significant differences were observed in the gelatinase activities between the metastatic cases and the non-metastatic cases, the levels of tissue inhibitor of MMP (TIMP)-1 in the tumor tissue specimens were higher in the non-metastatic cases than in the metastatic cases. The cases with the high levels of MMPs and low levels of TIMP-1 thus seemed to have a high potential to metastasize. As a result, the zymographic measurement of the gelatinolytic activity in biopsy tissue specimens may therefore be useful in predicting the behavior and prognosis of OSCC.

Immunohistochemical study of desmosomes in oral squamous cell carcinoma: Correlation with cytokeratin and E-cadherin staining, and with tumour behaviour

Reduction or loss of the intercellular junctions known as desmosomes may contribute to the invasive and metastatic behaviour of various carcinomas. Previous studies have shown that metastasis of oral squamous cell carcinomas of the head and neck correlates with a reduction in immunohistochemical staining for desmoplakin and desmoglein at the invasion front. The primary aim of the present study was to extend these observations to include a third component of desmosomes, the glycoprotein desmocollin. An additional aim was to determine whether the differentiation status of tumours is reflected in their staining for cytokeratins 1, 13, and 19, and, if so, whether these parameters correlate with desmosomal staining and/or metastasis. The study included 54 primary tumours of which 28 showed lymph node metastases. The results of this investigation show that tumours can be divided into three groups according to whether they have lost staining for no, one or more than one desmosomal component. A statistically significant correlation was found between the number of desmosomal components lost and metastasis. Tumours could also be divided into five groups according to their staining for different combinations of cytokeratins. Furthermore, differentiation status as indicated both histologically and by cytokeratin staining correlated with reduced desmosomal staining and metastasis. Tumours were also examined for intensity of staining for the adhesion molecule E‐cadherin. Reduction in E‐cadherin staining was correlated with mode of invasion and with reduction in desmosomal staining, but not with poor differentiation as indicated by cytokeratin staining. The results of this extensive study reinforce the view that adhesive junctions and adhesion molecules contribute to the suppression of tumour invasion and metastasis.

Nationwide Survey for Bisphosphonate-Related Osteonecrosis of the Jaws in Japan

PURPOSE A nationwide retrospective cohort study was conducted by the Japanese Society of Oral and Maxillofacial Surgeons to assess the occurrence of bisphosphonate (BP)-related osteonecrosis of the jaws (BRONJ) during 2006 to 2008 and to elucidate the outcome and factors associated with remission of BRONJ. MATERIALS AND METHODS A written questionnaire, including the clinical characteristics, management, and outcome of patients with BRONJ, was sent to 248 institutions certified as training facilities by the Japanese Society of Oral and Maxillofacial Surgeons in 2008. RESULTS A total of 568 patients with BRONJ, including suspicious cases, were registered. Of these 568 patients, 263, including the maxilla in 81, the mandible in 160, and both in 22, met the working definition of BRONJ proposed by the American Association of Oral and Maxillofacial Surgeons. The patients included 219 women (83.3%) and 44 men (16.7%). Of these patients, 152 (57.8%) had received intravenous BPs, 104 (39.5%) had received oral BPs, and 7 (2.7%) had received both. The mean duration of administration until onset of BRONJ was 23.6 months for intravenous BPs and 33.2 months for oral BPs. BRONJ was stage 1 in 42 patients (16.0%), stage 2 in 187 (71.1%), stage 3 in 32 (12.2%), and unknown in 2. Of these patients, 34.2% had remission of BRONJ, 46.0% had persistent or progressive disease, and 19.7% died of malignancy or were lost to follow-up. Statistical analysis revealed that surgical treatment, including tooth extraction, sequestrectomy, and segmental mandibulectomy, contributed to the remission of BRONJ. In contrast, conservative treatment, concurrent anticancer drugs, poor oral hygiene, and the use of intravenous BPs did not. CONCLUSIONS The relative ratio of BRONJ related to the use of oral BPs was greater in Japan than in the United States and European Union. Surgical treatment contributed to remission of BRONJ, and conservative treatment, concurrent anticancer drugs, poor oral hygiene, and intravenous BPs did not.

Involvement of proteasomes in migration and matrix metalloproteinase-9 production of oral squamous cell carcinoma

We investigated whether proteasomes were involved in the invasiveness of oral squamous cell carcinoma (SCC) cells. The migration of SCC cells through a gelatin‐coated membrane was enhanced with tumor necrosis factor α (TNFα), which was strongly inhibited by a peptide aldehyde, N‐acetyl‐Leu‐Leu‐norleucinal (ALLN), but not by its structurally related compound, N‐acetyl‐Leu‐Leu‐methioninal (ALLM). Since ALLN is a more potent inhibitor against proteasomal proteolysis than ALLM, cell migration inhibited by ALLN may thus likely depend on proteasomes. The TNFα‐induced migration through gelatin appeared to be associated with the gelatinolytic activity from the cells, since TNFα strongly enhanced the production of matrix metalloproteinase (MMP)‐9/gelatinase B in the SCC cells, as detected by gelatin zymography. The production of MMP‐9 was also inhibited by pretreatment with ALLN, but not ALLM, in a dose‐dependent manner. Moreover, ALLN could block the activation and nuclear translocation of a transcription‐activating factor, NF‐κB, which is known to regulate MMP‐9 expression in TNFα‐stimulated SCC cells. The TNFα‐induced degradation of IκBα was also suppressed by ALLN treatment, thus implying that the molecule linking proteasome to MMP‐9 production should be IκBα. We finally reconfirmed the involvement of proteasomes in the invasive behavior of oral SCC using lactacystin, a specific proteasome inhibitor, which could prevent TNFα from enhancing MMP‐9 production, NF‐κB activation, induction of MMP‐9 mRNA and cell migration. Int. J. Cancer 77:578–585, 1998.

Immunohistochemical staining of desmosomal components in oral squamous cell carcinomas and its association with tumour behaviour.

Desmosomes are intercellular junctions that have been shown to be down-regulated in certain types of carcinomas and that may play a role in suppression of invasion and metastasis. We have shown previously that immunohistochemical staining for the major desmosomal glycoprotein, desmoglein (Dsg), is reduced in some cases of squamous cell carcinoma (SCC) of the head and neck, and that reduced staining correlates with lymph node involvement. Desmosomes are multicomponent organelles. We therefore sought to determine whether another major desmosomal molecule, desmoplakin (Dp), showed similar reduced expression to that shown by desmoglein. We have stained 65 specimens of primary SCC of the oral cavity (37 non-metastatic and 28 metatastic) with monoclonal antibodies to both desmoglein and desmoplakin. We show that reduction of Dp staining correlates with loss of differentiation of the primary tumour, degree of invasion and presence of lymph node metastases. Similar correlations were found with Dsg staining. There was also correlation between reduction in Dp staining and reduction in Dsg staining. It is concluded that down-regulation of desmosomal expression occurs in some cases of SCC of the oral cavity and is associated with invasion and metastasis. Desmosomes may have an invasion and metastasis suppressor function.

Assessment of the Relationship Between Impacted Mandibular Third Molars and Inferior Alveolar Nerve With Dental 3-Dimensional Computed Tomography

PURPOSE The purpose of this study was to assess the capacity of dental 3-dimensional computed tomography (3D-CT; limited cone-beam CT) to predict the exposure and injury of the inferior alveolar nerve (IAN) after mandibular third molar extractions. MATERIALS AND METHODS This study was a retrospective case series of patients who presented for extraction of mandibular third molars. Subjects eligible for study enrollment were those who underwent preoperative dental 3D-CT because the mandibular third molars were determined to be extremely close to the IAN on panoramic radiogram. The predictive variable was the anatomic relation of the IAN and third molar apices and was a binary variable, contact or noncontact. The primary outcome variable was IAN exposure, and the secondary outcome variable was IAN injury. RESULTS From January 2006 to August 2007, 1,853 mandibular third molars in 1,539 patients were extracted. Among them, dental 3D-CT was performed on 53 third molars in 47 patients. The mandibular third molars were judged to make contact with the mandibular canal on dental 3D-CT images in 35 cases (66%). Intraoperative IAN exposure was observed in 17 (49%) contact cases and 2 (11%) noncontact cases on dental 3D-CT images. Of 53 cases extracted after dental 3D-CT examinations, IAN injury occurred in 8 cases (15%). IAN exposure led to IAN injury in 36.8% of cases, whereas IAN injury occurred in only 2.9% of cases without IAN exposure. Although the incidence of IAN injury in the molar-canal contact cases was 23%, all 8 cases with IAN injury (100%) were included in these contact cases. CONCLUSION When viewing the anatomic relation between the IAN and mandibular third molar root apices using dental 3D-CT, contact of the 2 anatomic structures results in an increased risk for IAN exposure or injury.

Dual Regulatory Effects of Interferon-α, -β, and -γ on Interleukin-8 Gene Expression by Human Gingival Fibroblasts in Culture upon Stimulation with Lipopolysaccharide from Prevotella intermedia, Interleukin-1α, or Tumor Necrosis Factor-α

In a previous study, we demonstrated that the amount of interleukin (IL)-8 mRNA expressed by human gingival fibroblasts stimulated with lipopolysaccharide (LPS) from Prevotella intermedia ATCC 25611 is increased by pre-treatment with beta or gamma interferon (IFN-β or --y). In the present study, we identified the regulatory effects of these IFNs on IL-8 mRNA expression and IL-8 production by human gingival fibroblasts. Priming with IFN-alpha (a), -β, or --y upregulated the IL-8 mRNA expression in response to P. intermedia LPS, whereas co-stimulation with these IFNs reduced the amount of mRNA expressed by the cells. The regulation of IL-8 mRNA expression induced by recombinant human tumor necrosis factor-a (rHuTNF-a) or rHuIL-1α was similar to that induced by LPS. The IL-8 mRNA expression in response to P. intermedia LPS was enhanced by IFN-γ independently of de novo protein synthesis, and was regulated, at least in part, at the transcriptional level. The IL-8 mRNA accumulation in response to P. intermedia LPS was inhibited by tosylphenyl-alanyl chloromethyl-ketone, an inhibitor of NF-KB activation, although the NF-KB activation itself was not altered by IFN-γ. These findings suggest that IFNs might be capable of both enhancing and inhibiting inflammatory responses in periodontal tissues through the dual regulation of IL-8 production by gingival fibroblasts in response to bacterial components and cytokines.