MicroRNA-125b regulates proliferation and radioresistance of oral squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are involved in essential biological activities, and have been reported to exhibit differential expression profiles in various cancers. Our previous study demonstrated that intercellular adhesion molecule-2 (ICAM2) inhibition induces radiosensitisation in oral squamous cell carcinoma (OSCC) cells. Thus, we hypothesised that certain miRNAs play crucial roles in radioresistance in OSCC by regulating ICAM2 expression. METHODS: Because predicted target gene analyses revealed that microRNA-125b (miR-125b) potentially regulates ICAM2 mRNA expression, we examined the association between miR-125b and radioresistance. The expression of miR-125b was investigated by real-time quantitative reverse transcriptase-PCR. For a functional analysis, miR-125b was transfected to OSCC-derived cells. RESULTS: A downregulated expression of miR-125b was found in OSCC-derived cell lines and OSCC samples. The miR-125b-transfected cells showed a decreased proliferation rate, enhanced radiosensitivity to X-ray irradiation and diminished ICAM2 mRNA expression. Moreover, miR-125b expression correlated with OSCC tumour staging and survival. CONCLUSION: These findings suggested that the downregulated miR-125b expression was associated with proliferation and radioresistance mechanisms, probably through ICAM2 signalling. Thus, controlling the expression or activity of miR-125b might contribute to suppressing proliferation and overcoming radioresistance in OSCC.

Oncolytic activity of Sindbis virus in human oral squamous carcinoma cells.

BACKGROUND: Sindbis virus (SIN) infection causes no or only mild symptoms (fever, rash, and arthralgia) in humans. However, SIN has a strong cytopathic effect (CPE) on various cancer cells. This study focuses on the oncolytic activity of SIN AR399 on oral cancer cells compared with reovirus, a well-known oncolytic virus that targets cancer cells. METHODS: We analysed the cytotoxicity and growth of SIN in 13 oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, H-1, Sa-3, KON, KOSC-2, OK-92, HO-1-N1, SCC-4, SAT, SKN-3) and normal human oral keratinocytes (NHOKs). RESULTS: Sindbis virus infection induced CPE in 12 OSCC cell lines at a low multiplicity of infection (MOI) of 0.01, but not in the OSCC cell line, HSC-4 or NHOKs. Sindbis viral growth was not observed in NHOKs, whereas high SIN growth was observed in all OSCC cell lines, including HCS-4. The cytotoxicity and growth of SIN was the same as reovirus at an MOI of 20 in 12 OSCC cell lines. The CPE was shown, by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assays, to be apoptotic cell death. Furthermore, quantitative RT-PCR of mRNA in HSC-3 and HSC-4 cells after SIN infection showed that activation of caspases, cytochrome c, and IkappaBalpha was associated with SIN-induced apoptosis. CONCLUSION: As a replication-competent oncolytic virus, SIN may be a useful therapeutic modality for oral cancers.

Aberrant Collagen Cross-linking in Human Oral Squamous Cell Carcinoma.

Tumor progression is a complex process involving extracellular matrix (ECM) remodeling and stiffening. However, the mechanisms that govern these processes and their roles in tumor progression are still poorly understood. In this study, we performed bioinformatics, immunohistochemical, and biochemical analyses to examine if collagen cross-linking is associated with tumor stage and regional lymph node metastasis (RLNM) in oral squamous cell carcinoma (OSCC). We found that the genes encoding key enzymes for cross-linking are frequently overexpressed in oral, head, and neck cancers. Specifically, the enzymes lysyl hydroxylase 2 (LH2) or lysyl oxidase (LOX) and LOX-like 2 (LOXL2) were significantly upregulated in late-stage tumors and associated with poor patient prognosis. The protein levels of these enzymes in the primary human OSCC were also significantly increased in late-stage tumors and markedly elevated in the RLNM-positive tumors. Notably, while overall LOX/LOXL2-catalyzed collagen cross-links were enriched in late-stage and RLNM-positive tumors, LH2-mediated stable cross-links were significantly increased. To our knowledge, this is the first study to investigate the association of collagen cross-linking and expression of key enzymes regulating this process with OSCC stage. The data indicate a critical role for collagen cross-linking in OSCC tumor progression and metastasis, which may provide insights into development of novel therapeutic strategies to prevent OSCC progression.

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.

Monophasic epithelial synovial sarcoma arising in the temporomandibular joint.

Synovial sarcoma is a mesenchymal spindle-cell tumour that occurs infrequently in the head and neck. It originates from unknown stem cells differentiating into mesenchymal and/or epithelial structures. Most synovial sarcomas are biphasic in character, consisting of epithelial and spindle-cell elements. Here is reported a case of monophasic epithelial synovial sarcoma arising in the temporomandibular joint. The tumour was of a predominantly epithelial pattern, although a minute area of sarcomatous cells was found. The primary mode of treatment was wide en-bloc excision. Two years after surgery, the patient died of hepatocellular carcinoma, but there was no evidence of synovial sarcoma recurrence.

Mutational state of tumor suppressor genes (DCC, DPC4) and alteration on chromosome 18q21 in human oral cancer.

In order to investigate the roles of two candidate tumor suppressor genes, DCC (deleted in colorectal carcinoma) and DPC4 (deleted in pancreatic carcinoma 4) genes in oral squamous cell carcinoma (SCC), we examined 32 primary SCCs by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Additionally, 32 pairs of normal and tumor DNA from 32 patients with oral SCCs were also analyzed for loss of heterozygosity (LOH) using 10 microsatellite markers on chromosome 18q21 where DCC and DPC4 genes are localized. We detected point mutations of DPC4 gene in two cases by PCR-SSCP analysis and sequencing. One case showed an AGC (Ser) to ACC (Thr) missense mutation at codon 1061 and the other the substitution C for A of the intron between exons 7 and 8. No mutation of DCC gene was observed in our cases. LOH at 18q21 was observed in 14 of the 32 cases (43.8%). The highest frequency (33.3%) of LOH was found at D18S46, and this was significantly correlated with the pathological results. These findings suggest that DCC and DPC4 gene may play minor roles in the genesis of oral SCC, and that another tumor suppressor gene involved in the development of oral SCC may exist in the region of D18S46 of this chromosome.

Rare mutations of the growth suppressor genes involved in negative regulation of the cell cycle.

The growth suppressing activity of the retinoblastoma susceptibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose activity is negatively regulated by CDK inhibitors of the p16 family. We have previously reported point mutations of the p16/CDKN2 gene in 4 (57%) of 7 oral squamous cell carcinoma (SCC) cell lines. In the current study, we examined the mutational status of CDK inhibitors, including 3 genes of the p16 family (p16, p15 and p18), in 50 human oral SCCs, and also additional results concerning their loss of heterozygosity in the regions of the p16, p15 and p18 genes. Our results demonstrated that 2 of 50 (4%) primary oral SCCs had nonsense mutations of the p16 gene, and 2 of 50 (4%) showed frameshift mutations of the p18 gene. However, we detected no mutation of the p15 gene in any of the 50 oral SCCs. In addition, no evidence of hypermethylation of the p16 gene was found in our series. To better understand the extent of alterations affecting chromosomes 9p21 (location of the p15/p16 genes) and 1p32 (location of the p18 gene), loss of heterozysity (LOH) on these locations was examined. LOH was detected in 16 of 34 (47%) informative samples that had no detectable mutation of the p15/p16 genes on 9p21, but we found no LOH at 1p32. These results strongly suggest that a putative tumor suppressor gene for oral SCC may be present on chromosome 9p21-22, while the p16, p15 and p18 genes play a minor role in the oncogenesis of this cancer.

Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13.

In order to understand the detail of genetic alternation on chromosome 22, we performed polymerase chain reaction analysis of microsatellite polymorphisms corresponding to 13 loci on chromosome 22. We examined 33 primary carcinoma tissues, 5 metastatic tissues and corresponding normal tissues. We detected microsatellite instability (MI) in 14 (42.4%) of 33 cases in this study. Loss of heterozygosity (LOH) was observed in at least one locus in 24 (72. 7%) of the 33 cases. Among the loci examined, LOH was restricted to D22S274 on chromosome 22q13 in 11 (40.7%) of 27 informative cases. No significant correlation between histological differentiation and LOH was observed. These observations suggest that the incidence of LOH at chromosome 22q is high and is associated with the carcinogenesis of oral squamous cell carcinoma (SCC). The D22S274 locus may play an important role in the development of oral SCC and be the site harboring a putative tumor suppressor gene.

Effect of FR167653 on pancreatic ischemia-reperfusion injury in dogs.

BACKGROUND: The role of inflammatory cytokines is still unclear in ischemia-reperfusion injury of the pancreas. We investigated the effect of FR167653 (FR), a newly developed compound that is a potent suppressor of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on ischemia-reperfusion injury of the isolated pancreatic tail in dogs. METHODS: The tail of the pancreas was subjected to ischemia for 90 minutes. During occlusion of the vascular inflow, the head of the pancreas was removed. A control group (n = 14) and an FR treatment group (n = 11) were evaluated for survival rate, tissue blood flow, arterial oxygen pressure (Pao(2)), serum amylase and lipase levels, glucose and insulin, liver enzymes, creatinine, IL-1beta mRNA in the peripheral blood, and histopathology. RESULTS: Six of the 14 control animals and 2 of the 11 FR-treated animals died. The FR treatment group showed lower amylase (P=.037) and lipase (P =.030) levels, lower IL-1beta mRNA expression (P =.033), and less pancreatic tissue damage (P =.041) than did the control group, but there was no remarkable change in endocrine function (P =.422). Pao(2) during the acute phase in the FR treatment group was maintained (P=.009), but pulmonary tissue was damaged. Results of biochemical and histologic examinations of the liver and kidneys were unremarkable. CONCLUSIONS: FR ameliorates ischemia-reperfusion injury of the pancreas and reduces the production of inflammatory cytokines that may contribute to secondary damage to distant organs.

Allelic loss on the short arm of chromosome 8 in oral squamous cell carcinoma.

Allelic deletions on the short arm of chromosome 8 (8p) are frequent events in many different types of malignant tumors, including head and neck tumors and oropharyngeal cancers. These regions are thought to harbor tumor suppressor genes. However, there has been no detailed analysis of loss of heterozygosity (LOH) on the chromosome arm 8p in oral squamous cell carcinoma (SCC). In order to estimate details of 8p loci involved in oral SCC, 32 patients with oral SCCs were examined for the LOH state 8p by PCR-LOH assay using 14 microsatellite markers. Based on our results a detailed deletion map of 8p showed LOH in at least one of the loci tested in 62.5% (20 of 32) of patients; and microsatellite instability (MI) was observed in 50% (16 of 32). The frequent LOH on this chromosome arm was identified at D8S87 and D8S258, mapped on 8p12 and 8p22, respectively. Fisher's exact test revealed no significant statistical correlation between the incidence of LOH or MI and clinicopathological features. Our observations indicate that the short arm of chromosome 8 may play a role in the pathogenesis of oral SCC; and that the two loci identified in this study may be tumor suppressor gene loci on 8p.

Evidence of multi-step oncogenesis of oral squamous cell carcinoma.

We examined biopsy samples from one oral cancer and three precancerous lesions of the tongue of an 81-year old woman by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequence analyses using 18 oligonucleotide primer pairs of adenomatous polyposis coli (APC) gene and 5 primers of p53 gene. Normal tongue epithelium adjacent to lesions was used as a control. The four lesions harbored the common mutation of APC gene that was not detected in the control. At codon 1621 in exon 15 of the APC gene there was a C to G substitution resulting in serine (TCA) to stop codon (TGA). No mutation of p53 gene was detected in any samples of the control and three precancerous lesions of the tongue. On the other hand, an A to G substitution at codon 170 in exon 5 of p53 gene resulting in glutamic acid (ACG) to glycine (GCG) was detected in the DNA of her tongue cancer. These results may suggest that the four lesions have the same origin, and that multi-step oncogenesis had occurred, the APC gene alteration being one of the early events in the process of tumorigenesis and p53 gene alteration involved in the late events.

A detailed deletion map of chromosome 20 in human oral squamous cell carcinoma.

Loss of heterozygosity (LOH) on the long arm of chromosome 20 (20q) has been detected in several human cancers. However, little is known about LOH on chromosome 20 in oral squamous cell carcinoma (OSCC). To determine which loci of chromosome 20 were involved in OSCC tumorigenesis, 41 cases of OSCC were examined for LOH state on chromosome 20 at 17 microsatellite loci by PCR-LOH assay. LOH occurred in 41.5% of tumors in at least one locus. Among the 17 loci, D20S48 on 20p11.2 and RPN2 on 20q12-13.1 exhibited higher frequencies of LOH, 27.6% and 31.4%, respectively. The LOH incidence was significantly higher in tumors in which the primary site was on gingiva compared with other oral sites (p=0.012). Our results indicate that allelic deletions on 20q12-13.1 and 20p11.2 may play roles in OSCC carcinogenesis, and suggest that allelic deletions on 20q might have some relation with the primary site of OSCC.

Localization of a novel tumor suppressor gene loci on chromosome 9p21-22 in oral cancer.

Allelic imbalance or loss of heterozygosity (LOH) studies have been used to identify regions on chromosomes that may contain putative tumor suppressor genes. Deletions of chromosome 9 regions have been observed at high frequency in many other types of sporadic tumor, whereas in oral cancer no decisive information about the allelic loss on chromosome 9 has been reported. To provide detailed understanding of the genetic alterations in oral cancer, 24 highly polymorphic markers mapped on chromosome 9 were used to examine 34 cases of oral squamous cell carcinoma (SCC). LOH was detected in 18 (53%) of 34 informative samples at one or more loci examined. On the basis of our results, three commonly deleted regions were identified and a detailed deletion map was constructed. One of the novel regions was on 9p22, where a tumor suppressor gene, interferon a cluster (IFNA) gene, was identified before. Another region was D9S157 locus at 9p22, telomeric to IFNA locus and p15/16 genes, and the third was located on 9p21 of the D9S104 locus, centromelic to methylthioadenosine phosphorylase (MTAP) gene and p15/16 genes. Thus, our data suggest that, except for p15/16 and MTAP gene, there were at least two candidate tumor suppressor genes located at chromosome 9p, and that the alteration of these genes is associated with the tumorigenesis of oral SCC.

Targeting fibroblast growth factor receptor 3 enhances radiosensitivity in human squamous cancer cells.

Conventional therapies including radiation therapy cannot cure squamous cell carcinoma (SCC), and new treatments are clearly required. Our recent studies have shown that SCC cell lines exhibiting radioresistance show significant upregulation of the fibroblast growth factor receptor 3 (FGFR3) gene. We hypothesized that inhibiting FGFR3 would suppress tumor cell radioresistance and provide a new treatment approach for human SCCs. In the present study, we found that RNA interference-mediated FGFR3 depletion in HSC-2 cells, a radioresistant cell line, induced radiosensitivity and inhibited tumor growth. Use of an FGFR3 inhibitor (PD173074) obtained similar results with suppression of the autophosphorylation extracellular signal-regulated kinase pathway in HSC-2 cells and lung cancer cell lines. Moreover, the antitumor growth effect of the combination of PD173074 and radiation in vivo was also greater than that with either drug alone or radiation alone. Our results provided novel information on which to base further mechanistic study of radiosensitization by inhibiting FGFR3 in human SCC cells and for developing strategies to improve outcomes with concurrent radiotherapy.

Clinical observations of postoperative delirium after surgery for oral carcinoma.

The aim of the present study was to clarify the clinical characteristics of postoperative delirium and to determine appropriate postoperative management for its prevention. The authors analysed 132 cases of primary surgery for oral carcinoma and observed 24 (18%) cases of postoperative delirium. Univariate analysis revealed that significant risk factors for postoperative delirium were older age, male gender, extensive surgery and morphine pain control. Logistic regression analysis showed that older age and male gender were significant risk factors for postoperative delirium, while patient-controlled analgesia with fentanyl was effective for prevention of postoperative delirium. There was a trend for postoperative delirium to be associated with extensive surgery. In those who had delirium, blood tests revealed that alkaline phosphatase, total protein, sodium, chlorine, red blood cell count, haemoglobin and haematocrit were significantly diminished after surgery. These results indicate that general condition is closely related to the onset of postoperative delirium, and suggest that appropriate postoperative management can reduce the incidence of this complication.

Network-based analysis of calcium-binding protein genes identifies Grp94 as a target in human oral carcinogenesis.

To characterise Ca(2+) -binding protein gene expression changes in oral squamous cell carcinomas (OSCCs), we compared the gene expression profiles in OSCC-derived cell lines with normal oral tissues. One hundred Ca(2+) -binding protein genes differentially expressed in OSCCs were identified, and genetic pathways associated with expression changes were generated. Among genes mapped to the network with the highest significance, glucose-regulated protein 94 kDa (Grp94) was evaluated further for mRNA and protein expression in the OSCC cell lines, primary OSCCs, and oral premalignant lesions (OPLs). A significant (P<0.001) overexpression of Grp94 protein was observed in all cell lines compared to normal oral epithelium. Immunohistochemical analysis showed highly expressed Grp94 in primary OSCCs and OPLs, whereas most of the corresponding normal tissues had no protein immunoreaction. Real-time quantitative reverse transcriptase-PCR data agreed with the protein expression status. Moreover, overexpression of Grp94 in primary tumours was significantly (P<0.001) correlated with poor disease-free survival. The results suggested that Grp94 may have potential clinical application as a novel diagnosis and prognostic biomarker for human OSCCs.

Extruded lumbar osseous endplate causing long-term radiculopathy in an adult: an endoscopic excision.

In this report, we described an adult case that had a long-term radiculopathy due to an extruded osseous endplate of the lumbar spine at the L5-S1 intervertebral disc level. The osseous material inside the extruded material was not absorbed, and it had continued compressing the nerve root for one year. Endoscopically, the bony fragment was successfully removed. After the surgery, the patient's symptom disappeared, and neurological deficits became normalized. In conclusion, we propose that surgical intervention should be taken into account for the treatment of HNP, when the extruded material contains bony fragment such as osseous endplate.

Overexpression of stathmin in oral squamous-cell carcinoma: correlation with tumour progression and poor prognosis.

Stathmin is an intracellular phosphoprotein that is overexpressed in a number of human malignancies. Our previous study using proteomic profiling showed that significant upregulation of stathmin occurs in oral squamous-cell carcinoma (OSCC)-derived cell lines. In the current study, to determine the potential involvement of stathmin in OSCC, we evaluated the state of stathmin protein and mRNA expression in OSCC-derived cell lines and human primary OSCCs. A significant increase in stathmin expression was observed in all OSCC-derived cell lines examined compared to human normal oral keratinocytes. In immunohistochemistry, 65% of the OSCCs were positive for stathmin, and no immunoreaction was observed in corresponding normal tissues. Real-time quantitative reverse transcriptase-polymerase chain reaction data were consistent with the protein expression status. Moreover, stathmin expression status was correlated with the TNM stage grading. Furthermore, we found a statistical correlation between the protein expression status and disease-free survival (P=0.029). These results suggest that expression of stathmin could contribute to cancer progression/prognosis, and that stathmin may have potential as a biomarker and a therapeutic target for OSCC.

Monitoring of circulating tumour-associated DNA as a prognostic tool for oral squamous cell carcinoma.

Frequent allelic imbalances (AIs) including loss of heterozygosity and microsatellite instability on a specific chromosomal region have been identified in a variety of human malignancies. The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma (SCC) by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral SCC was examined at nine microsatellite loci. In all, 38 (59%) DNA samples from tumorous tissues and 52% from serum showed AIs in at least one locus. Patterns of AIs in the serum DNA were matched to those detected in tumour DNA. Of them, AIs were frequently detected preoperatively (44%, 28 of 64), and postoperatively (20%, 13 of 64). Moreover, among 12 cases with AIs during the postoperative period, six had no evidence of an AI 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with AI-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in the serum DNA could be a useful predictive tool to monitor disease prognosis.