Vasohibin-1 as a Novel Prognostic Factor for Head and Neck Squamous Cell Carcinoma.

AIM: We evaluated the prognostic value of vasohibin-1 (VASH1) expression in head and neck squamous cell carcinoma. MATERIALS AND METHODS: Immunohistochemistry for VASH1 and cluster of differentiation 34 (CD34) was performed on 61 head and neck squamous cell carcinoma specimens. The association between VASH1 expression in the tumour and clinical outcomes was analyzed statistically. RESULTS: VASH1 staining in normal tissue adjacent to cancerous tissue was negative, whereas it was positive in tumour blood vessels and AE1/AE3 and Ki67-positive tumour cells. Therefore, we examined the association between VASH1 expression in the tumour and clinical outcomes. Patients with high VASH1 expression in tumour had significantly shorter disease-free survival and more frequently had lymph node recurrence than those with low VASH1 expression. CONCLUSION: These results suggest that VASH1 expression is associated with tumour progression and may be useful as a prognostic marker of head and neck squamous cell carcinoma.

ALDH1 and podoplanin expression patterns predict the risk of malignant transformation in oral leukoplakia.

Oral leukoplakia (OL) is a clinically diagnosed preneoplastic lesion of the oral cavity with an increased oral cancer risk. However, the risk of malignant transformation is still difficult to assess. The objective of the present study was to examine the expression patterns of aldehyde dehydrogenase 1 (ALDH1) and podoplanin in OL, and to determine their roles in predicting oral cancer development. In the present study, the expression patterns of ALDH1 and podoplanin were determined in samples from 79 patients with OL. The association between protein expression and clinicopathological parameters, including oral cancer-free survival, was analyzed during a mean follow-up period of 3.4 years. Expression of ALDH1 and podoplanin was observed in 61 and 67% patients, respectively. Kaplan-Meier analysis demonstrated that the expression of the proteins was correlated with the risk of progression to oral cancer. Multivariate analysis revealed that expression of ALDH1 and podoplanin was associated with 3.02- and 2.62-fold increased risk of malignant transformation, respectively. The malignant transformation risk of OL was considerably higher in cases with expression of both proteins. Point-prevalence analysis revealed that 66% of patients with co-expression of ALDH1 and podoplanin developed oral cancer. Taken together, our data indicate that ALDH1 and podoplanin expression patterns in OL are associated with oral cancer development, suggesting that ALDH1 and podoplanin may be useful biomarkers to identify OL patients with a substantially high oral cancer risk.

HuR and podoplanin expression is associated with a high risk of malignant transformation in patients with oral preneoplastic lesions.

The risk of malignant transformation in oral preneoplastic lesions (OPLs) is challenging to assess. The objective of the present study was to determine the expression of ELAV like RNA binding protein 1 (HuR) and podoplanin in OPLs, and to evaluate the use of each protein as biomarkers for the risk assessment of malignant transformations. Immunohistochemistry for HuR and podoplanin was performed on the tissues of 51 patients with OPL, including cases of low grade dysplasia (LGD) and high grade dysplasia (HGD). The association between the protein expression patterns and clinicopathological parameters, including oral cancer free survival (OCFS) time, was analyzed during the follow-up period. HuR and podoplanin expression was observed in 28 (55%) and 36 (71%) of 51 patients, respectively. Kaplan-Meier analysis showed that the expression of HuR and podoplanin was associated with the risk of progression to oral cancer (P<0.05). Multivariate analysis revealed that HuR and podoplanin expression was associated with a 2.93-fold (95% confidence interval (CI), 0.98-10.34; P=0.055) and 2.06-fold (95% CI, 0.55-8.01; P=0.283) increase in risk of malignant transformation, respectively. The risk of OPL malignant transformation was considerably increased with the coexpression of HuR and podoplanin compared with the histological grading (95% CI, 1.64-23.59; P=0.005). The results of the present study demonstrated that the expression of HuR and podoplanin associates with malignant transformation and suggests that the proteins may be used as biomarkers to identify OPL patients with an increased risk of cancer development.

Receptor activator of NF-κB ligand induces cell adhesion and integrin α2 expression via NF-κB in head and neck cancers.

Cellular interactions with the extracellular matrix play critical roles in tumor progression. We previously reported that receptor activator of NF-κB ligand (RANKL) specifically facilitates head and neck squamous cell carcinoma (HNSCC) progression in vivo. Here, we report a novel role for RANKL in the regulation of cell adhesion. Among the major type I collagen receptors, integrin α2 was significantly upregulated in RANKL-expressing cells, and its knockdown suppressed cell adhesion. The mRNA abundance of integrin α2 positively correlated with that of RANKL in human HNSCC tissues. We also revealed that RANK-NF-κB signaling mediated integrin α2 expression in an autocrine/paracrine manner. Interestingly, the amount of active integrin ß1 on the cell surface was increased in RANKL-expressing cells through the upregulation of integrin α2 and endocytosis. Moreover, the RANK-integrin α2 pathway contributed to RANKL-dependent enhanced survival in a collagen gel and inhibited apoptosis in a xenograft model, demonstrating an important role for RANKL-mediated cell adhesion in three-dimensional environments.

Histological assessment for femora of ovariectomized obesity (db/db) mice carrying mutated leptin receptor.

In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12-week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.

Cytoplasmic expression of HuR may be a valuable diagnostic tool for determining the potential for malignant transformation of oral verrucous borderline lesions.

Oral verrucous carcinoma (OVC) is a low grade variant of oral squamous cell carcinoma, and oral verrucous hyperplasia (OVH) is a benign lesion without malignant features. However, pathologists are sometimes presented with borderline lesions and are indecisive as to diagnose them as benign or malignant. Thus, these lesions are tentatively termed oral verrucous lesions (OVLs). HuR is an ARE mRNA-binding protein, normally localized in the nucleus but cytoplasmic exportation is frequently observed in cancer cells. The present study aimed to elucidate whether expression of the HuR protein facilitates the diagnosis of true malignant lesions. Clinicopathological features were evaluated, and immunohistochemical analysis for p53, Ki67 and HuR proteins was performed in 48 cases of OVH, OVC and OVL, and the outcomes were correlated using appropriate statistical analysis. The association of these three proteins in relation to malignant transformation was analyzed after a 3-year follow-up of 25 OVL cases. The basal characteristics (age, gender and location) of all cases had no significant association with the types of lesions. Gingiva (39.4%) was the common site for all lesions. Distribution of the examined proteins had a significant association with the lesions. As compared with the OVLs, the number of immunostained-positive cells was significantly higher in the OVCs and lower in the OVH cases. During follow-up, 24% of the OVLs underwent malignant transformation for which high HuR expression and a diffuse staining pattern in the epithelium were observed. Taken together, the high degree of HuR expression with diffuse staining pattern in the epithelium may be an effective diagnostic tool that determines the potential of OVLs for malignant transformation.

pp32r1 controls the decay of the RNA-binding protein HuR.

pp32 is a tumor suppressor and is one of the associated proteins of the RNA-binding protein HuR. The pp32-HuR complex is exported to the cytoplasm of cells under stress conditions, and HuR is degraded by caspases in the cytoplasm. In the present study, we examined the role of pp32r1, a member of the pp32 family that has oncogenic properties, in the decay of HuR. pp32r1 was found to be abundantly expressed in cancer cells, and overexpression of pp32r1 induced colony formation in soft-agar. pp32r1 was expressed in both the nucleus and cytoplasm, whereas pp32 was predominantly localized in the nucleus. Even with lethal stress such as staurosporine (STS), HuR in the cytoplasm was never downregulated, and caspase-3 activity was inhibited when cells expressed pp32r1. pp32r1 bound to HuR without interacting with pp32. In cancer cells, HuR survived in the cytoplasm of cells overexpressing pp32r1, although HuR was not expressed in the cytoplasm of pp32-expressing cells, similar to lethal stress conditions. Taken together, these results indicate that pp32r1 binds to HuR to avoid the caspase-mediated decay of HuR in the cytoplasm of cells. We suggest that this function contributes to the oncogenic activity of pp32r1.

Factors related to the incidence of anterior disc displacement without reduction and bony changes of the temporomandibular joint in patients with anterior open bite.

PURPOSE: We aimed to investigate factors related to the prevalence of anterior disc displacement without reduction (ADDwoR) and bony changes of the condylar head (bony changes) in the temporomandibular joints (TMJs) of patients with anterior open bite. METHODS: Subjects are comprised of 36 preoperative patients (72 joints) with skeletal anterior open bite without facial asymmetry who had undergone orthognathic surgery at the Hokkaido University Hospital; magnetic resonance imaging of the TMJ and cephalometric analysis were performed before treatment. Logistic regression analysis was performed to clarify relationships among age, overbite, overjet, ANB angle, sella to nasion (SN) to mandibular plane angle (SN-MP angle), SN to ramus plane angle (GZN angle), gonial angle, and incidence of ADDwoR or bony changes in patients with anterior open bite. RESULTS: Fifteen patients had bilateral ADDwoR, and five patients had unilateral ADDwoR; 17 patients had bilateral bony changes, and five patients had unilateral bony changes. SN-MP angle was greater in 20 patients with ADDwoR than that in 16 patients without ADDwoR (p < 0.05). GZN angle was greater in the 20 patients showing bony changes than that in the 16 patients without bony changes (p < 0.05). CONCLUSION: In terms of dentofacial morphology, SN-MP angle appears to be associated with the incidence of ADDwoR, and GZN angle appears to be associated with bony changes in the TMJ.

Expression of parathyroid hormone-related protein confers malignant potential to mucoepidermoid carcinoma.

Parathyroid hormone-related protein (PTHrP) is known to induce bone resorption by activating RANKL as well as PTH. PTHrP plays a central role in humoral hypercalcemia, and its expression has been reported to be closely associated with bone metastasis of breast carcinoma. PTHrP expression in oral squamous carcinoma cell lines was investigated, and PTHrP was expressed in oral squamous cell carcinoma cell lines similar to that in a prostate carcinoma cell line. Mucoepidermoid carcinoma is the most common malignant salivary gland tumor composed of different types of cells including a squamous component. Its clinical behavior is highly variable and ranges from slow-growing and indolent to locally aggressive and highly metastatic. We examined the PTHrP expression in mucoepidermoid carcinoma and assessed the significance of its correlation with clinicopathological features. Immunohistochemical detection of PTHrP was carried out in 21 cases of mucoepidermoid carcinoma in the head and neck region. PTHrP was highly detectable in intermediate and epidermoid cells, and abundant expression of PTHrP in intermediate cells had a significant association with cancer malignancy, including lymph node metastasis and/or tumor recurrence. These results suggest that PTHrP expression can be used as a prognostic factor for mucoepidermoid carcinoma.

E1A expression might be controlled by miR-214 in cells with low adenovirus productivity.

In this study, we explored the differences in the human adenovirus type 5 (Ad5) production efficiencies of various cell types. The rate of virus production was higher in several cell lines, such as HeLa cells, than in Saos-2 cells. The expression level of the coxsackie and adenovirus receptor (CAR) protein, an adenovirus receptor, was very similar among these cell lines. Although no significant difference in the expression of early region 1A (E1A) mRNA was detected, the amount of E1A protein in the Saos-2 cells was markedly lower than that in HeLa cells. Proteasome inhibitor treatment did not rescue the quantity of E1A in the Saos-2 cells, suggesting that their decreased E1A protein expression is not due to protein decay. To examine the different expression of E1A protein, we employed a bioinformatics approach to identify miRNA that target the 3'-untranslated region (3'-UTR) of E1A mRNA and identified miR-214 as a highly promising candidate. In Saos-2 cells, which have abundant levels of endogenous miR-214, the expression of luciferase was dramatically repressed, when the reporter gene was fused with the 3'-UTR of E1A mRNA including an miR-214 binding site. On the other hand, the activity from the same reporter was unchanged in HeLa cells, which display low-level miR-214 expression. Finally, we confirmed that the knockdown of the miR-214 upregulated the productive efficiency of the virus. These findings indicate that cellular miR-214 is capable of inhibiting adenovirus replication by regulating the translation of E1A protein.

The role of area postrema neurons expressing H-channels in the induction mechanism of nausea and vomiting.

The area postrema is one of the circumventricular organs, lacks a blood-brain barrier, and is well known as the chemoreceptor trigger zone for emesis. Area postrema neurons are sensitive to emetic chemical substances carried in the blood plasma. Our previous study demonstrated the presence of 3 types of neurons characterized by different ion channels expressed in each cell type, but the type or types of area postrema neurons involved in the induction of nausea and/or emesis have remained unclear. To clarify the role of the most populous cells, which express the hyperpolarization-activated cation channel (H-channel), in induction of nausea and/or emesis, we investigated the effects of ZD7288 (an H-channel inhibitor) on apomorphine-induced conditioned taste aversion (CTA) to saccharin and c-Fos expression in the area postrema. We found that ZD7288 inhibited the acquisition of CTA and reduced apomorphine-induced c-Fos expression in the area postrema, indicating the involvement of the cells expressing H-channels in the induction of nausea and/or emesis. Finally, we discuss the role of cells expressing H-channels in the mechanism of nausea and/or vomiting.

Does swallowing function recover in the long term in patients with surgically treated tongue carcinomas?

PURPOSE: The present study aimed to measure postsurgical swallowing function in patients 5 years after the surgical treatment of tongue carcinoma. PATIENTS AND METHODS: Using a retrospective cohort study design, the investigators enrolled postsurgical patients treated for tongue carcinomas in Hokkaido University Hospital. The primary outcome variable was oropharyngeal swallow efficiency (OPSE) determined by videofluoroscopic evaluation, and OPSE at follow-up was compared with that at discharge. Other variables included current nutritional status (body mass index, serum albumin), dietary intake, self-rating of current swallowing function, and occurrence of pneumonia. Statistical analysis used the paired t test and the Spearman rank correlation. RESULTS: Swallowing function was assessed in 20 patients (11 men and 9 women) who underwent the surgical treatment of tongue carcinomas; the median age was 70 years (range, 56 to 90 yrs). The mean OPSE values for liquid and paste at follow-up were 26.6 ± 21.2 and 21.9 ± 22.5, respectively. The mean values for the body mass index and serum albumin at presentation were 22.2 ± 3.4 kg/m(2) and 4.5 ± 0.3 g/dL, respectively. All patients had a full oral intake of foods, with a mean self-rated value of 6.4 ± 2.5, a value acceptable to the patients. Pneumonia requiring hospitalization did not occur in these patients. CONCLUSIONS: The long-term follow-up of patients after the surgical treatment of tongue carcinomas showed acceptable levels of oral function and nutritional status despite objective measurements of poor swallowing efficiency assessed using videofluoroscopy.

Dental pulp can be a good candidate for nerve grafting in a xeno-graft model.

Dental pulp is discarded after extirpation of dental pulp and after tooth extraction. However, it contains nerve tissue abundantly and could be used more effectively. This study was designed to examine whether a dental pulp could be a candidate of donor for nerve grafting in xenografting model. The dental pulp was obtained from a human vital extracted tooth for orthodontic treatment, and treated with freezing and thawing method for reducing antigenicity. The treated sample was inserted into chitosan mesh tube for easy suturing, and then the complex was implanted into transected sciatic nerve in Sprague-Dawley (SD) rats (dental pulp group). As controls, chitosan tubes with and without sciatic nerve harvested from another SD rats were implanted (isograft group and tube group, respectively). As early as 4 weeks after grafting, regenerating axons accompanied by host Schwann cells were found to grout out through basal laminae by electron microscopy. The intact structure of basal laminae at this period suggested that they were derived from the original structure of donor graft. Twelve weeks after grafting, sporadic axonal regeneration was confirmed by light microscopy in the dental pulp group. Thirty-two weeks after implantation, aggregation of axons was observed in this group and matched that in isograft group. The average diameter of axons in dental pulp group was comparable to that in isograft group, whereas number of minifascicles and axon proportion were smaller. It was suggested that some delay occurred in dental pulp group because of the phagocytosis and absorption of tissue debris components remained after the freezing and thawing treatment. These findings clearly demonstrate that even dental pulp can act as conduits for regenerating axons.

Involvement of suppressor of cytokine signalling-1-mediated degradation of MyD88-adaptor-like protein in the suppression of Toll-like receptor 2-mediated signalling by the murine C-type lectin SIGNR1-mediated signalling.

Dendritic cells recognize pathogens through pattern recognition receptors such as Toll-like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross-talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C-type lectin SIGNR1 ligand lipoarabinomannan and the Toll-like receptor 2 ligand FSL-1. The co-stimulation significantly suppressed FSL-1-mediated activation of NF-κB as well as production of TNF-α, IL-6 and IL-12p40 in a dose-dependent manner. The suppression was significantly but not completely recovered by knock-down of SIGNR1. SIGNR1 was associated with Toll-like receptor 2 in XS106 cells. The co-stimulation upregulated the expression of suppressor of cytokine signalling-1 in XS106 cells, the knock-down of which almost completely recovered the suppression of the FSL-1-mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88-adaptor-like protein in XS106 cells was degraded by co-stimulation with FSL-1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock-down of suppressor of cytokine signalling-1. This study suggests that Toll-like receptor 2-mediated signalling is negatively regulated by SIGNR1-mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling-1-mediated degradation of the MyD88-adaptor-like protein.

Cyclooxygenase-2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells.

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.

RANKL expression specifically observed in vivo promotes epithelial mesenchymal transition and tumor progression.

Recent findings have focused attention on the molecular consequences of the microenvironment in tumor progression, but events occurring in cancer cells themselves in response to their ambient conditions remain obscure. Here, we identify receptor activator of nuclear factor κB ligand (RANKL) as a microenvironment-specific factor essential for tumorigenesis in vivo, using head and neck squamous cell carcinoma (HNSCC) as a model. In human HNSCC tissues, RANKL is abundantly expressed, and its expression level correlates with the histological grade of differentiation. RANKL levels are significantly higher in poorly differentiated SCCs than in well or moderately differentiated SCCs. In contrast, all HNSCC cell lines tested displayed extremely low RANKL expression; however, RANKL is efficiently up-regulated when these cell lines are inoculated in the head and neck region of mice. RANKL expression is restored in a microenvironment-specific manner, and cannot be observed when the cells are inoculated in the hindlimbs. Forced expression of RANKL compensates for tumor growth in the hindlimb milieu, promotes epithelial mesenchymal transition, and induces tumor angiogenesis, in a manner independent of vascular endothelial growth factor (VEGF). These results implicate RANKL expression causatively in tumor growth and progression in HNSCC in vivo. RANKL may provide a novel functional marker for biological malignancy and a therapeutic target based on the specific nature of the microenvironment.

In vitro biomechanical evaluation of the effect of an additional L-shaped plate on straight or box plate fixation in sagittal split ramus osteotomy using a bioabsorbable plate system.

PURPOSE: Relapse caused by clockwise (opening) rotation of the distal segment (dentate segment) in the sagittal plane is one of the postoperative complications sometimes seen after sagittal split ramus osteotomy. The force involved in this movement is primarily exerted by the masticatory and suprahyoid muscles. For postoperative stability, we have used two plates on each side: a straight plate placed over the vertical osteotomy line at the buccal surface of the first molar and an L-shaped plate placed at the distal or lateral part of the last molar over the osteotomy line, just crossing over the upper-lateral edge of the buccal shelf, to prevent the tail of the distal segment from moving upward. Although postoperative stability was clinically fine, experimental evaluations have not previously been performed. To clarify the effects of the L-shaped plate, we performed an experimental study using a bioabsorbable plate system. METHODS: A custom-fabricated jig was made to simulate rotational movement of the segments. Two segments made from polyoxymethylene resin were fixed with a four-hole straight or four-hole box poly-L-lactate bioabsorbable plate. An L-shaped plated was then added for rigidity, and mechanical testing was performed. RESULTS: The yield load exerted by the four-hole straight plate alone was 152.4 ± 11.0 N. This increased significantly to 273.8 ± 43.7 N with addition of an L-shaped plate (P < 0.05). CONCLUSION: Addition of an L-shaped plate significant improves the rigidity of four-hole straight plate fixation in a bioabsorbable plate system.

Postoperative pressure-induced alopecia after segmental osteotomy at the upper and lower frontal edentulous areas for distraction osteogenesis.

INTRODUCTION: Postoperative alopecia is a relatively rare event, and therefore both patients and surgeons are puzzled once it develops even though it is said to improve spontaneously with time in most cases. We report a parieto-occipital pressure-induced alopecia firstly developed in a patient who had undergone repeated surgery for 10 years after a traffic accident. CASE REPORT: A 29-year-old male underwent segmental osteotomy at the upper and lower frontal edentulous areas for distraction osteogenesis. Throughout the operation, he was in the supine position with the hair covered with a paper cap and the head on a plastic vinyl chloride-covered soft foam horseshoe-shaped urethane sponge placed on the horseshoe-shaped headrest. About 2 weeks after the surgery, two patches of parieto-occipital alopecia were observed at the barber's. It cured gradually during the follow-up visits. CONCLUSION: The pathophysiology of the condition is thought to be mostly pressure-induced ischemia of hair follicles, and then we have to try not to make the situation. Scalp massages and the head repositioning during the surgery should be one of the means of prevention.