Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma.

BACKGROUND: G protein-coupled receptor family C group 5 member A (GPRC5A), a member of G protein-coupled receptor family, has been shown to function as a tumor suppressor in lung tissue. The biological functions of GPRC5A have therefore been linked to lung tissue. However, the biological significance of this gene product remains obscure. In this study, we investigated the expression of GPRC5A proteins in normal oral tissue and oral squamous cell carcinoma (OSCC), and we characterized its biological activity in OSCC cell lines. METHODS: Western blot analysis and immunohistochemical staining were used to investigate the expression of GPRC5A in both OSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental OSCC cells were characterized for their biological activities in anchorage-independent growth. RESULTS: High levels of immunohistochemical GPRC5A expression were detected in normal oral tissue, especially differentiated area. In contrast, GPRC5A expression was dramatically repressed in OSCCs (P < 0.01). The immunohistochemical GPRC5A expression was moderately well differentiated, but greatly repressed in moderately differentiated OSCCs and completely repressed in poorly differentiated OSCCs. Overexpression of GPRC5A in OSCC CAL27 cells resulted in a suppressed anchorage-independent growth activity, a transforming phenotype. CONCLUSIONS: GPRC5A is expressed in normal oral epithelium. Repression of GPRC5A is associated with poorly differential grade of OSCCs. Overexpression of GPRC5A in OSCC cell line reversed the malignant phenotype. Thus, GPRC5A is important for homeostasis in oral tissue, and deletion or repression of this gene may involve in tumorigenesis of OSCCs and may serve as a prognostic marker for malignant type of OSCCs.

Accuracy of Mandibular Reconstruction with a Vascularised Iliac Flap Using 3D Templates: a Systematic Review.

OBJECTIVE: To conduct a systemic review for guidance regarding the application of templates in mandibular reconstruction with vascularised iliac flaps. METHODS: By searching PubMed, EMBASE and the Cochrane Library and collecting relevant literature, information about the types and accuracy of templates was extracted. Data relating to surgical time were also included for further analysis. RESULTS: Eight studies were included. The data analysis showed that the accuracy of operations with templates was higher than that of conventional surgery. The mean deviation was between 0.70 and 3.72 mm. The operational time was shortened to 314.4 minutes and the graft ischemic time was reduced to 15.6 to 26.8 minutes. Application of functional or specifically designed templates can improve the accuracy and shorten surgical time. CONCLUSION: Templates can increase the accuracy and efficiency of mandibular reconstruction with vascularised iliac flaps, which will benefit patients' prognosis and subsequent functional restoration. Further studies should be conducted into application of templates to improve the accuracy of reconstructions.

Epigenetic silencing of MAL, a putative tumor suppressor gene, can contribute to human epithelium cell carcinoma.

BACKGROUND: To identify new and useful candidate biomarkers in head and neck squamous cell carcinoma (HNSCC), we performed a genome-wide survey and found that Myelin and lymphocyte-associated protein (MAL) was a gene that was markedly down-regulated in HNSCC. Hence, we investigated the mechanism of MAL silencing and the effects of MAL on the proliferation, invasion, and apoptotic potential in HNSCC. RESULTS: MAL was significantly down-regulated in 91.7% of HNSCC specimens at the mRNA level as compared with adjacent normal tissues (P = 0.0004). Moreover, the relative transcript levels of the MAL gene were remarkably decreased by five-fold in nine HNSCC cell lines as compared with normal head and neck epithelium cells. MAL gene expression was restored in 44%, 67%, and 89% in HNSCC cell lines treated with TSA, 5-Aza-dC, and TSA plus 5-Aza-dC, respectively. Furthermore, bisulfate-treated DNA sequencing demonstrated that the two CpG islands (that is, M1 and M2) located in MAL promoter region were completely methylated in the HNSCC cell lines (CpG methylated ratio was more than 90%), and only one CpG island (that is, M1) was partially methylated in HNSCC tissues (CpG methylated ratio between 20% and 90%). A significant reduction in cell proliferation and a change in the cell cycle profile were also observed in MAL transfectants. Matrigel assay demonstrated that the invasiveness of HNSCC cells significantly decreased. A significant increase in the population of apoptotic cells was observed in MAL transfected cells. The exogenous expression of the MAL gene suppressed malignant phenotypes, while the cell death induced by MAL gene transfer was a result of apoptosis as demonstrated by the induction of cleavage of the poly (that is, ADP-ribose) polymerase. Additionally, tumor growth was suppressed in cells expressing MAL as compared with cells not expressing MAL. CONCLUSION: Our data suggest that the epigenetic inactivation of MAL, as a candidate tumor suppressor gene, can contribute to human epithelial cell carcinoma and may be served as a biomarker in HNSCC.

Correlation of NF-kappaB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma.

BACKGROUND: Nuclear factor-kappa B (NF-kappaB) signaling constitutes a key event in the multistep process of carcinogenesis, progression and treatment in many cancer types. However, the significance of NF-kappaB pathway for complex and tissue-specific aspects of head and neck cancer progression, such as invasion and metastasis, is less understood. METHODS: The expression of NF-kappaB p65 in squamous cell carcinoma of the head and neck (SCCHN) clinical specimens by immunohistochemistry. The role of NF-kappaB activity in head and neck squamous cell carcinoma was determined by western blot, reporter assay and EMSA analysis in vitro and metastasis assays in vivo in different metastatic potential tumor cells. Furthermore, the apoptosis rate and expression of metastasis-related protein such as MMP9 and VEGF were examined by Annexin V/PI staining and Western blot, respectively. RESULTS: A higher level of active nuclear-localized NF-kappaB was observed in the metastatic SCCHN specimens group (p < 0.01). The NF-kappaB activities of SCCHN cell lines with different metastatic potentials were then determined and in excellent agreement with results found in SCCHN specimens, highly metastatic SCCHN cell lines expressed high level of NF-kappaB activity. The treatment of highly metastatic SCCHN cells with NF-kappaB inhibitors reduced the in vitro cell invasion capacity of the cells without affecting the apoptotic rate. Additionally, the NF-kappaB inhibitors significantly inhibited the experimental lung metastasis of Tb cells and lymph node metastasis of TL cells in nude mice. Furthermore, the expression of metastasis-related proteins, such as matrix metalloproteinase 9 and vascular endothelial growth factor, was inhibited by pyrrolidine dithiocarbonate. CONCLUSIONS: This study suggests that NF-kappaB activity significantly contributes to tumor hematologic and lymphatic metastases and may aid in the development of early detection methods or therapies targeting non-conventional molecular targets.

Association between interleukin-10 gene polymorphisms and risk of oral carcinoma: A meta-analysis.

BACKGROUND: The single nucleotide polymorphisms (SNPs) of Interleukin-10 (IL-10) gene have been linked with the risk of oral carcinoma (OC) in a relatively small sample size. Our study aims to investigate the pooled associations by conducting a meta-analysis of published studies. METHODS: PubMed, Web of Science and Google Scholar databases were searched to identify eligible studies published in English before October 2019. The odds ratio (OR) with a 95% confidence interval (CI) was used to assess association. The publication bias was detected by Begg's test. Sensitivity and cumulative analyses were performed to evaluate the stability of crude results. RESULTS: The meta-analysis involved eight studies. Significant associations were certified between IL-10 gene -1082A/G polymorphism and susceptibility of OC for A vs. G (OR=1.817, 95% CI: 1.481-2.230), AA vs. GG (OR=3.436, 95% CI: 2.281-5.175), dominant genetic model (OR=2.913, 95% CI: 1.939-4.376), and recessive genetic model (OR=1.886, 95% CI: 1.372-2.594) in overall population, East Asians and South Asians. In addition, the significant association between -592A/C polymorphism of the gene and susceptibility of OC were detected in South Asians. CONCLUSIONS: The meta-analysis results support that the IL-10 gene -1082G allele is a risk factor for OC in East Asians and South Asians, and IL-10 gene -592C allele is a protective factor for the disease.

Decreased expression of Annexin A1 correlates with pathologic differentiation grade in oral squamous cell carcinoma.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including the human immortalized oral epithelia cells (HIOECs) and its derived cancerous HB96 cells. In this study, comparative proteomic analysis identified that Annexin A1 was one of the significantly down-regulated genes in the cancerous HB96 cells. To investigate Annexin A1 down-regulation and its potential usefulness as a molecular marker in OSCC, we further screened Annexin A1 expressions with a panel of OSCC lines, and clinical samples of cancerous and the paired adjacent normal tissues from primary OSCC patients. By Western blot analysis and real-time PCR, we showed that both Annexin A1 mRNA and protein expressions decreased in OSCC cell lines except in two cell lines for the mRNA levels. Immunohistochemistry and real-time PCR also showed that both Annexin A1 mRNA and protein expressions decreased in the cancerous tissues from OSCC patients compared with those in the paired adjacent non-malignant epithelia. More importantly, both Annexin A1 mRNA and protein expressions negatively correlated with the pathologic differentiation grades of cancerous tissues. The lower Annexin A1 mRNA or protein expressions correlated with the poorer pathologic differentiation grades. These results suggest that decreased expression of Annexin A1 contributes to the cancerous progression of OSCC, and Annexin A1 may be a potential biomarker for pathologic differentiation grade of OSCC.

Vandetanib inhibits growth of adenoid cystic carcinoma in an orthotopic nude mouse model.

PURPOSE: Adenoid cystic carcinoma (ACC) can often be controlled with surgery and postoperative adjuvant radiotherapy but is also characterized by late local recurrence and distant metastasis. No effective systemic therapeutic agents have been found to alter the natural history of ACC. Therefore, new therapeutic approaches are needed. In this study, we evaluated whether vandetanib (Zactima), a potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, had antitumor efficacy in vitro and in an orthotopic nude mouse model of human ACC. EXPERIMENTAL DESIGN: The in vitro effects of vandetanib were assessed in three ACC cell lines on cell growth, apoptosis, and VEGFR-2 and EGFR phosphorylation levels. The in vivo antitumor activity of vandetanib was examined in nude mice bearing parotid gland ACC tumors. The mice were treated for 4 weeks with vandetanib (50 mg/kg/d) or placebo (control). Tumors were resected at necropsy, and immunohistochemical and immunofluorescence staining were done. RESULTS: In vitro, vandetanib caused dose-dependent inhibition of VEGFR-2 and EGFR phosphorylation in ACC cells. Vandetanib also inhibited the cell proliferation and induced their dose-dependent apoptosis. In vivo, mice in the vandetanib group had tumor volumes significantly lower than those in the control group (P < 0.01). In addition, immunohistochemical staining showed a decrease in microvessel density and an increase in apoptosis of both tumor cells and endothelial cells within the tumor xenografts. CONCLUSION: These results suggest that vandetanib inhibits the growth of ACC in vitro and in vivo, making it a promising novel agent for the treatment of ACC.

Overexpression of insulin-like growth factor binding protein 3 in oral squamous cell carcinoma.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC), including a human immortalized oral epithelial cell (HIOEC) line and its derived cancerous HB96 cell line. Further cDNA microarray analysis showed a significant up-regulated gene, insulin-like growth factor binding protein 3 (IGFBP3), accompanying with in vitro cancerization from HIOEC to HB96. In order to investigate IGFBP3 up-regulation and its potential usefulness as a molecular marker in OSCC, we detected the IGFBP3 expression with a panel of OSCC lines, and clinical samples of cancerous tissues and paired adjacent non-malignant epithelia from primary OSCC patients. Western blotting and real-time PCR showed increased IGFBP3 mRNA level and protein expression in OSCC cell lines compared with HIOEC in vitro; immunohistochemistry and real-time PCR also showed increased IGFBP3 mRNA level and protein expression in cancerous tissues compared with adjacent non-malignant epithelia from OSCC patients. Positive correlations were found between the IGFBP3 protein-positive grade in cancerous tissue and the tumor size as well as lymph node metastasis, a larger tumor size and positive lymph node metastasis indicating a higher level of IGFBP3 protein-positive grade. Based on these results, IGFBP3 may be used as a positive biomarker for OSCC development and progression.

Characteristics of a cancerous cell line, HIOEC-B(a)P-96, induced by benzo(a)pyrene from human immortalized oral epithelial cell line.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial region. The mechanism of carcinogenesis of OSCC is still unclear. In vitro study on OSCC cell lines, especially derived from immortalized oral epithelial cells, is a very useful strategy to understand the mechanism of carcinogenesis. Based on our previous human immortalized oral epithelial cell (HIOEC) line, obtained from normal oral epithelial cells by transfection of HPV16 E6/E7 gene, a new cancerous cell line, HIOEC-B(a)P-96 (HB96), was established from the HIOEC by induction with benzo(a)pyrene. The characteristics of the HB96 cells such as cell morphology, ultrastructure, proliferation ability, invasion ability, and tumorigenesis were studied. The HB96 cells lost contact inhibition with uncontrolled cell division and obvious cell overlap, they were polygonal in shape and ununiform in size with increased ratio between nucleus and plasma. Increased proliferative ability and invasion ability were confirmed by the cell proliferation analysis and cell invasion assay, respectively. The tumorigenicity of well to moderately differentiated squamous cell carcinoma was confirmed in the nude mice experiments pathologically. Increased expression of HPV16 E6/E7 proteins and obvious correlation with decreased expression of p53 and Rb proteins was also confirmed by Western blotting. Thus, this HB96 cell line induced by benzo(a)pyrene from the HIOEC line is a useful tool to study the mechanism of carcinogenesis of OSCC in vitro for future genomic and proteomic analyses. It is also the first in vitro cancerous cell line of OSCC in China derived from immortalized oral epithelial cells.

A new mutation of PTCH gene in a Chinese family with nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disease characterized by a combination of development anomalies and a predisposition to tumour formation. Mutation of patched gene (PTCH), considered the molecular defect of NBCCS, in a Chinese NBCCS family was investigated in this study. METHODS: Genomic DNA was isolated from blood samples of all 12 members of this family. The mutated PTCH gene was screened by polymerase chain reaction amplification and direct sequencing. RESULTS: A new mutation of 3 bp (GAT deletion) was found in all seven affected members of this family. This mutation caused one aspartate deletion in the fourth transmembrane domain of the PTCH protein located within the sterol sensing domain (SSD). This deletion was not found in any unaffected members of this family nor in 200 control samples. CONCLUSIONS: Our findings suggest that one 3-bp deletion in PTCH gene was the cause of nevoid basal cell carcinoma in a Chinese family through affecting the conformation and function of PTCH protein.

Benzo (a) pyrene induced tumorigenesity of human immortalized oral epithelial cells: transcription profiling.

BACKGROUND: The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene. METHODS: The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genes of human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene. Tumorigenicity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction. RESULTS: There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction, possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens. CONCLUSIONS: This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to benzo (a) pyrene exposure.

Sonic Hedgehog Signalling Activation Contributes to ALCAM Over-Expression and Poor Clinical Outcome in Patients with Oral Squamous Cell Carcinoma.

OBJECTIVE: To investigate the Sonic Hedgehog (SHH) signalling molecules and activated leukocyte cell adhesion molecule (ALCAM) expression in the mechanisms regulating invasion and metastasis in oral squamous cell carcinoma (OSCC). METHODS: The expressions of SHH signalling molecules Gli family zinc finger 1/2 (Gli1/Gli2), as well as ALCAM expression, was analysed in 101 OSCC patients by immunohistochemistry. The potential relationship between Gli1/Gli2 and ALCAM in regard to invasion and metastasis were studied by western blot, invasion and wound-healing assays. RESULTS: Gli1, Gli2 and ALCAM were expressed in 54.5%, 49.5% and 47.5% of the 101 OSCC specimens, respectively. High expression of ALCAM was associated with shorter survival in the patient population (P = 0.018), which was independent of other clinical parameters. Notably, when both ALCAM expression and positive nodal status were considered, an enhanced prediction of clinical outcomes was achieved (P = 0.001). In OSCC cell lines, down-regulation of ALCAM resulted in reduced cell invasion and metastasis. Importantly, SHH activation increased the half-life of ALCAM leading to ALCAM accumulation and increased cell invasion and migration. CONCLUSION: ALCAM over-expression in OSCC is an independent prognostic factor for OSCC patients. Its over-expression may be the result of the activation of the SHH signalling pathway and contributes to OSCC progression.

Increased Cyfra 21-1 concentration in saliva from primary oral squamous cell carcinoma patients.

BACKGROUND: Saliva is the body fluid in the oral cavity and contacts directly with oral mucosa. As a detective media, it is acceptable and non-traumatic. Cyfra 21-1, being the soluble fragment of cytokeratin 19(CK19), correlates well with oral squamous cell carcinoma (OSCC). OBJECTIVE: To investigate the saliva Cyfra 21-1 concentrations in OSCC patients and healthy persons, and the correlation between the Cyfra 21-1 concentration in saliva and the CK19 expression in tissue from OSCC patients. DESIGN: Saliva Cyfra 21-1 concentration was detected by ELISA in 30 OSCC patients and 30 healthy persons; CK19 protein expression and CK19 mRNA level were, respectively, detected by immunohistochemistry and fluorescent real-time RT-PCR in cancerous and paracancerous tissues from 33 OSCC patients. RESULTS: Saliva Cyfra 21-1 concentration in OSCC patients (85.95+/-78.00 microg/L) was significantly higher than that in healthy persons (42.27+/-40.84 microg/L) (P=0.009); it was also significantly higher in the patients suffering later tumour recurrence (130.95+/-66.38 microg/L) than that in the patients without tumour recurrence (74.84+/-63.45 microg/L) (P=0.023). CK19 protein expression increased significantly in OSCC tissues (P<0.001) with positive rate of 90.9%, CK19 mRNA level in cancerous tissues was 2.21 folds higher than that in paracancerous tissues (P=0.020); significant correlation was found between tissue CK19 protein expression and tissue CK19 mRNA level (P=0.003), and great correlation was found between tissue CK19 protein expression and saliva Cyfra 21-1 concentration (P=0.051). CONCLUSIONS: The increased CK19 expression in OSCC tissues plays an important role in the increase of saliva Cyfra 21-1 concentration. Potential clinical value of saliva Cyfra 21-1 detection is suggested for OSCC. Further studies are encouraged to reveal the real diagnostic and prognostic value of detecting saliva Cyfra 21-1 concentration for OSCC.

A20 inhibits human salivary adenoid cystic carcinoma cells invasion via blocking nuclear factor-kappaB activation.

BACKGROUND: A20, also known as tumor necrosis factor alpha induced protein 3 (TNFaip3), is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B (NF-kappaB) activity and prevents tumor necrosis factor (TNF)-mediated programmed cell death. NF-kappaB is a transcription factor that regulates expression of genes involved in cell proliferation, cell survival and anti-apoptosis. Several studies have implicated that the NF-kappaB signal pathway is associated with angiogenesis and clinico-pathological process of adenoid cystic carcinoma (ACC) of the salivary glands. METHODS: The ability of overexpression of A20 to influence the biological behavior and invasion of ACC cells was examined. The cells were stably transfected with full-length A20 cDNA. Stable gene transfer was verified by realtime-polymerase chain reaction (PCR) and Western blot analysis. The change of cell biological behavior was examined by methyl thiazolyl tetrazolium (MTT) and NF-kappaB luciferase reporter assay and the invasion of the cells was examined by a Matrigel invasion chamber. RESULTS: pEGPFN3-A20 gene was stably transferred into ACC-2 cells and overexpressed. When cells were treated with TNFalpha, the NF-kappaB activity of ACC-2-A20 cells could be down-regulated about 46.32% in contrast to ACC-2-GFP cells (P < 0.05). A20 potently inhibited growth of A20 transfectant ACC-2-A20 compared with control vector transfected groups and the ACC-2 empty control group (P < 0.05). The ACC-2-A20 cells showed significantly reduced ability to invade through Matrigei-coated filters compared to ACC-2-GFP and ACC-2 cells. The inhibition rate was up to 71.05% (P < 0.05). CONCLUSIONS: A20 gene transfer is associated with decreased tumor invasion, in part via the down-regulation of NF-kappaB expression, providing evidence for a potential application of A20 in designing a treatment modality for salivary gland cancers such as ACC.

A three-lncRNA signature derived from the Atlas of ncRNA in cancer (TANRIC) database predicts the survival of patients with head and neck squamous cell carcinoma.

OBJECTIVE: Long non-coding RNAs (lncRNAs) have important biological functions and can be used as prognostic biomarkers in cancer. To identify a lncRNA prognostic signature for head and neck squamous cell carcinoma (HNSCC). METHOD: We analysed RNA-seq data derived from the TANRIC database to identify a lncRNA prognostic signature model using the orthogonal partial least squares discrimination analysis (OPLS-DA) and 1.5-fold expression change criterion methods. The prognosis prediction model based on the lncRNA signatures and clinical parameters were evaluated using the 5-fold cross validation method. RESULTS: A total of 84 out of 3199 lncRNAs were significantly associated with the survival of patients with HNSCC (log-rank test P<0.01). Using the OPLS-DA and 1.5-fold change selection criterion, 5 lncRNAs (KTN1-AS1, LINC00460, GUSBP11, LINC00923 and RP5-894A10.6) were further selected. The prediction power of each combination of the 5 lncRNAs was evaluated through the receiver operating characteristic (ROC) curve and a three-lncRNA panel (KTN1-AS1, LINC00460 and RP5-894A10.6) achieved the highest prognostic prediction power (AUC 0.68, 95% CI 0.60-0.76, P<0.0001) in the cohort. The patients were categorized into high- and low-risk groups based on their three-lncRNA profiles. Patients with high-risk scores had worse overall survival than those with low risk scores in the cohort (log-rank test P=0.0003). Multivariable Cox regression analyses showed that the lncRNA signature and tumour grade were independent prognostic factors for patients with HNSCC. CONCLUSIONS: Our findings showed that the three-lncRNA signature might be a novel biomarker for the accurate prognosis prediction of patients with HNSCC.

CD73 is associated with poor prognosis in HNSCC.

CD73 is a cell surface immunosuppressive enzyme involved in tumor progression and metastasis. While patients whose cancer cells express elevated CD73 are typically associated with an unfavorable outcome, the clinical impact of CD73 expression in patients with Head and neck squamous cell carcinoma (HNSCC) remains unclear. In the present study, we investigated the prognostic significance of CD73 in HNSCC using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with reduced overall survival in patients with HNSCC. We also investigated the functional role of CD73 in vitro and demonstrated that CD73 promotes HNSCC migration and invasion through adenosine A3R stimulation and the activation of EGF/EGFR signaling. Moreover, in vivo xenograft studies demonstrated that CD73 promotes tumorigenesis. In conclusion, our study highlights a role for CD73 as a poor prognostic marker of patient survival and also as a candidate therapeutic target in HNSCCs.

Synergistic down-regulation of telomerase by all-trans retinoic acid and antisense oligonucleotide in oral squamous cell carcinoma cell line (Tca8113).

Human telomerase, activated in about 90% of cancers, is mainly composed of hTR, hTERT and TP1. The exposed RNA template of hTR is an ideal target for antisense oligonucleotides (As-ODN); while recent findings indicate all-trans retinoid acid (ATRA) could effectively inhibit the expression of catalytic subunit-hTERT. The aim of this study was to investigate the effect of ATRA and As-ODN in oral squamous cell carcinoma and whether telomerase activity could be synergistically inhibited by them and thus therapeutically exploited in the future. As-ODN-hTR was transfected into human tongue squamous cell carcinoma cell line (Tca8113) with or without ATRA. Telomerase activity was examined by PCR-Elisa; viability was compared with growth curve; apoptotic rate was analyzed by Annexin V/PI double staining and hTERT expression was tested with western blot. Tca8113 cells displayed significant growth inhibition during the 9-day exposure to ATRA/As-ODN, especially to a combination of As-ODN-hTR and 5muM ATRA, correlating with the inhibition of telomerase expression. The relative telomerase activity was inhibited during treated with As-ODN-hTR alone, ATRA alone, or a combination of them. While without ATRA, the effect of As-ODN would disappear at 96h after transfection. As-ODN-hTR alone or combined with ATRA also significantly increase the apoptotic rate. Our findings provided direct evidence, in oral squamous cell carcinoma, As-ODN-hTR and ATRA could synergistically inhibit telomerase activity and telomerase protein in human tongue squamous cell carcinoma cells, which correlated with the induction of growth arrest.

[Effects of transient transfection of human BMP-4 gene on rabbit bone marrow stromal cells].

OBJECTIVE: Bone marrow stromal cells (MSCs) were transfected with human bone morphogenetic protein-4 (hBMP-4) gene in vitro to provide BMP gene modified cells for tissue-engineered bone. METHODS: MSCs were cultured and transfected with pEGFP-hBMP4, pEGFP plasmids respectively or left uninfected as control. Transcription of BMP-4 gene as well as gene transfection efficiency was tested. Morphological and growth feature of the transfected cells were valued. Alkaline phosphatase (ALP), von Kossa, and Osteocalcin (OC) were tested to determine the phenotypes of osteoblast. RESULTS: The gene transfection efficiency was 20%-30%, based on GFP expression. RT-PCR showed that MSCs had low transcription of BMP-4 that was enhanced by the gene transfer. Morphological feature of MSCs transfected with pEGFP-hBMP-4 changed but growth curves did not show much difference among the groups. In pEGFP-hBMP-4 group, ALP positive stain area and the number of calcium nodules were increased, as well as the expression of OC. CONCLUSIONS: A high transfer efficiency of MSCs was achieved under optimized conditions. The gene transfer technique strengthened the transcription of BMP-4 and promoted differentiation from MSCs to osteoblasts. hBMP-4 transferred MSCs may serve as an ideal cell source for tissue-engineered bone.

Tricholemmal carcinoma of the head and neck region: A report of 15 cases.

Tricholemmal carcinoma is an extremely rare malignancy of the skin, and its biological behavior and management is controversial. The objective of the present study was to investigate the clinicopathological characteristics and management of tricholemmal carcinoma of the head and neck region. The study analyzed 15 patients with tricholemmal carcinoma. Demographic and clinical data were collected, and features associated with the management and prognosis of tricholemmal carcinoma were analyzed. Two of the 15 patients were lost to follow-up. The results showed that, during the follow-up period, 5 of the 13 available patients succumbed to the causes of recurrence (n=3), neck lymph node metastasis (n=1) and Parkinson's disease (n=1). No patients developed distant metastasis. The disease-free survival (DFS) and overall survival (OS) were 31.1±7.8 and 32.9±7.4 months (mean ± SE), respectively, and the DFS and OS rates were 69.2 and 61.5%, respectively. In conclusion, the biological behavior of tricholemmal carcinoma is locoregionally aggressive. The recommended management for head and neck tricholemmal carcinoma is radical resection and neck dissection, and post-operative radiotherapy may be considered for high-risk patients.

[Screening of the gene mutation in D-loop region of mitochondrial DNA in oral squamous cell carcinoma].

OBJECTIVE: To investigate the gene mutation in D-loop region of mitochondrial DNA (mtDNA) in oral squamous cell carcinoma (OSCC) tissue and to explore the role of the gene mutation in D-loop region in the OSCC tumorigenesis. METHODS: mtDNA was obtained from cancer, paracancerous and normal mucosa tissues of thirty patients with OSCC. The D-loop regions of mtDNA were amplified with PCR, sequencing and then analyzed by Chromas software and BLAST to identify the mutation site. RESULTS: Mutation in the D-loop region was found in eight cases, with the mutation rate of 27%. There were nine mutations totally, including one point mutation, two base deletions, three insertion mutations, three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations had no same mutation form, while the three insertion mutations were same, the insertion of base C. One case had T/A heterozygous mutation and base C insertion at the same time. CONCLUSIONS: There were mutations in mtDNA D-loop in OSCC, but the relationship between occurrence of OSCC and mutation of mtDNA needs further study.