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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.

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.

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., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

Objective: To investigate the mRNA expression of collagen genes in oral squamous cell carcinoma (OSCC) and paired normal oral mucosal tissue., Methods: The differential mRNA expressions of collagen genes between 30 OSCC tissues and the paired normal oral mucosal tissues were detected by RT-PCR., Result: The relative expression level of COL1A1 mRNA in the 30 cancerous tissues was up-regulated by 2.78 folds as compared with its expression in the paired normal samples, suggesting its significant overexpression in OSCC (P<0.001). The expression levels of COL1A2, COL4A1, COL4A2, and COL5A2 mRNA in the cancerous tissues were up-regulated by 1.07, 1.15, 1.27, and 1.16 folds compared to those in paired normal samples (P>0.05)., Conclusion: COL1A1 mRNA overexpression may play an important role in the carcinogenesis of OSCC and can be a potential molecular marker of OSCC.

Aims: To investigate the expression of EMP1 in oral squamous cell carcinoma (OSCC) tissues and its correlation with available clinical parameters of patients with OSCC., Methods: The mRNA levels of EMP1 were measured in 18 paired OSCC and corresponding adjacent normal tissues using RT-PCR. Another 45 pairs of OSCC samples were selected to detect the mRNA level of EMP1 using quantitative RT-PCR (qRT-PCR). The protein levels of EMP1 were also evaluated in 60 cases of patients with OSCC using immunohistochemical staining. The correlation between EMP1 expression and clinical parameters was analysed with non-parametric analysis., Results: The results of RT-PCR and qRT-PCR showed that, compared with the paired normal tissues, the mRNA levels of EMP1 were significantly decreased in OSCC. The immunohistochemical results indicated that the EMP1 protein was also downregulated in OSCC (p=0.031). Decreased expression of EMP1 was significantly correlated with clinical stage (p=0.002) and lymph node metastasis (p=0.044) of patients with OSCC. Meanwhile, there was a significant difference between OSCCs at early and advanced stages (p=0.003), and between OSCCs with lymph node metastasis and no lymph node metastasis (p=0.045), respectively., Conclusions: The results suggest that EMP1 may be a tumour suppressor and associated with lymph node metastasis in OSCC.

Purpose: To investigate the role of NF-kappaB signal transduction pathway in apoptosis induced by shikonin in human tongue squamous cell carcinoma Tca-8113 cell line., Methods: Expression of IkappaBa, phosphatase-IkappaBa, bcl-2 and Bax proteins were detected by Western blot, NF-kappaB DNA-binding activity was detected by electrophoretic mobility shift analysis (EMSA), and activities of caspase 3, caspase 8 and caspase 9 were analyzed by enzyme linked immunosorbent assay(ELISA). The data was analyzed by one-way ANOVA test and t test using SPSS12.0 software package., Results: The expression of phosphatase-IkappaBa protein and the nuclear NF-kappaB DNA-binding activity was significantly decreased in shikonin treated cells by Western and EMSA. Bcl-2 protein expression was also decreased in the process. The activity of all the three proteases was elevated and pancaspase inhibitor Z-Asp-CH2-DCB could protect Tca8113 cells from shikonin-induced apoptosis(P=0.02)., Conclusions: Anti-tumor effects of shikonin in Tca-8113 cells act at least partially through the inactivation of NF-kappaB pathway and subsequent activation of protease caspase family. Pharmacologic inhibition of the NF-kappaB activity by shikonin might be a powerful treatment option for OSCC.

Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) and expression microarray analysis showed that the gene encoding growth differentiation factor 15 (GDF15) was significantly upregulated in this model. In this study, we confirmed that expression of GDF15 was increased both at mRNA and protein levels in a panel of OSCC lines and clinical samples from primary OSCC patients. We also observed that expression of GDF15 was positively correlated with the malignancy of the disease: a higher level of GDF15 expression indicates a higher malignant grade of OSCC. Treatment of OSCC cell line (Tca3118) with siRNA against GDF15 significantly inhibited cellular proliferation and colony formation. Based on these observations, we conclude that GDF15 is a positive gene of OSCC development and progression and GDF15 can be used as an additional marker for histopathologic evaluation of OSCC differentiation.

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.

Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC.

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.

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.

Objective: To investigate the mRNA expression of matrix metalloproteinase 1 (MMP1) gene in oral squamous cell carcinoma (OSCC) and the paired normal tissues., Methods: The differential expression of MMP1 mRNA between 30 OSCC and paired normal tissues were detected with reverse transcription-PCR (RT-PCR)., Results: The relative expression level of MMP1 mRNA in the OSCC tissues showed a 3.26-fold increase in comparison with that in the paired normal tissues (4.06-/+0.52 vs 1.24-/+0.17, P<0.0001). In the 30 OSCC tissues, the relative expression level of MMP1 mRNA was higher in histological grade II/III tissues (4.31-/+0.68) than in grade I (3.87-/+0.57) tissues, higher in OSCC in advanced stages (III/IV) than in tumors in early stages (I/II) (4.18-/+0.67 vs 3.65-/+0.53), and also higher in OSCC with cervical lymph node invasion than in those without cervical lymph node invasion (4.32-/+0.71 vs 3.91-/+0.51), but these differences were not statistically significant (P>0.05)., Conclusion: MMP1 gene may play a role in local invasion of OSCC, and can serve as a potential biomarker molecule for diagnosis, treatment and prognostic evaluation of OSCC, with also clinical value for OSCC classification.

Objective: To investigate osteopontin (OPN) mRNA expression in oral squamous cell carcinoma (OSCC) and normal oral mucosa tissues., Methods: Differential OPN gene expression were detected in 30 cancerous tissues and their paired normal tissues using real-time reverse transcription-PCR (real-time RT-PCR), and the data were analyzed statistically., Results: Real-time RT-PCR results demonstrated that the relative expression level of OPN mRNA in the cancerous tissues were significantly higher than that in paired normal samples (4.17-/+0.51 vs 0.97-/+0.12, P<0.001), showing a 4.3-fold up-regulation. In the 30 OSCC specimens, OPN mRNA expression in the OSCC of histological grades I showed a 3.1-fold down-regulation, significantly lower than the expression in grade II/III tumors (2.16-/+0.17 vs 6.80-/+0.72, P<0.05); its expression was significantly lower in early stage than in advanced stage OSCCs (2.34-/+0.17 vs 4.73-/+0.35, P<0.05). In cases of cervical lymph node metastasis, the expression was significantly higher than that in cases without lymphatic metastasis (6.38-/+0.56 vs 2.89-/+0.32, P<0.05)., Conclusion: OPN mRNA overexpression may play an important role in OSCC carcinogenesis and can be a potential target for OSCC therapy.

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.

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.

Objective: To investigate cytokeratin 19 (CK19) expression in oral squamous cell carcinoma (OSCC) tissue as well as its correlation with pathologic differentiation grade and prognosis., Study Design: Cancerous and distant tissues from 33 primary OSCC patients were collected during surgery. Each sample was divided into 2 parts, one for CK19 protein detection using immunohistochemistry, and the other for CK19 mRNA-level detection of relative quantification using fluorescent real-time reverse transcriptase polymerase chain reaction (RT-PCR)., Results: The CK19 positive rate in cancerous tissue was 90.9% (30 out of 33) detected using immunohistochemistry, which was significantly higher than that in distant tissue (15.2%, 5 out of 33) by chi-square test (P < .001). The CK19 positive score in cancerous tissue was also significantly higher than that in the corresponding distant tissue by paired samples t test (P < .001). Patients with positive CK19 expression in distant tissue had higher tumor recurrence rate (P = .008) and lower survival rate (P = .015) than those with negative CK19 expression in distant tissue. Using fluorescent real-time RT-PCR, CK19 mRNA level in cancerous tissue was 2.21-fold higher than that in distant tissue (P = .020). The CK19 expression correlated significantly with pathologic differentiation grade, which was detected not only using immunohistochemistry (P = .002) but also using fluorescent real-time RT-PCR (P = .025), poorer differentiated indicating higher CK19 positive score and CK19 mRNA level., Conclusions: Increased CK19 protein expression and gene transcription in OSCC tissue correlate significantly with pathologic differentiation grade. Positive CK19 expression in distant tissue suggests a higher tumor recurrence rate and a lower survival rate.

Objective: To select and identify the target genes related to oral squamous cell carcinoma (OSCC) and provide target genes for designing oligo-nucleotide functional microarray of OSCC., Methods: Genes possibly related to oral squamous cell carcinoma were selected from the 5 years' published data of differently expressed profiles with microarray testing in OSCC. Then mRNA expression of selected genes were evaluated by real time quantitative polymerase chain reaction (RT-PCR) in 22 cases of OSCC, including tumor tissues and paried normal mucosas and quantified according to an internal control GAPDH., Results: Eight genes were tested. The overexpression of SPARC, PDGF-A, SERPINE1, TGF-beta(1) and VEGF-C genes were measured in 16, 18, 16, 20, 18 cases of tumor specimens, respectively. The expression of CK15 gene was lower than that of its normal tissue. There were overexpression of CCND1, BIRC3 in tumor tissues, but there was no significant difference of CCND1 and BIRC3 expression between tumor tissue and normal tissue (P > 0.05)., Conclusions: SPARC, PDGF-A, SERPINE1, TGF-beta(1), VEGF-C and CK15 genes were closely related to tumor progress of OSCC. They can be used as the target genes for designing oligo-nucleotide functional microarray of OSCC.

Objective: To clarify the relationship between cyclin D1 and cisplatin resistance of Tca8113/cis diamminedichloroplatinum (CDDP) in vitro and in vivo., Methods: We applied the transfection method with plasmids pcDNA3.1-antisense-cyclin D1 by Lipofectamine 2000. Tca8113/CDDP cells were used as control. MTT assay was used to identify the proliferation and sensibility of those cells to cisplatin. Subsequently, 18 nude mice were subcutaneously injected by those cells and divided into 3 groups with 6 mice in each group. Every mouse was treated by cisplatin with 5 mg . kg(-1) . d(-1) for 5 days. The sizes of tumor were measured every other day and were described with the growth curves. After 20 days, tumors were anatomized and weighed. The tumor inhibition ratios were calculated and the HE slides were observed to determine the cell sensibility to cisplatin., Results: The transfected cells with pcDNA3.1-antisense-cyclin D1grew more slowly than other cells and showed higher sensibility to cisplatin in vitro. The tumors developed by cells with pcDNA3.1-antisense-cyclin D1 were smaller than the The tumor inhibition ratio was 74% (P < 0.05). The necrosis area was larger in the tumors developed by the transfected cells with pcDNA3.1-antisense-cyclin D1 than other groups., Conclusions: Antisense oligonucleotides of cyclin D1 can improve the sensibility of Tca8113/CDDP cells to cisplatin and inhibit the growth of tumors.

Purposes: To transform HPV E6/E7 immortalized human oral epithelial cell line HIOEC cells by benzo(a)pyrene B(a)P and tetradecanoyl phorbol acetate (TPA) in vitro, and establish a carcinogenesis model of oral squamous cell carcinoma., Methods: HIOEC cells were treated with 0.1 microg/ml -1.2 microg/ml B(a)P for 6 months. Some of these cells were treated with 0.1 microg/ml TPA 24 hours at 4th passage and 10th passage, respectively. The cells were cloned at 18th passage, and then were cultured with DMEM medium contain 10% FBS at 21st passage. The cells were cultured in vitro for 1 year and developed into a malignant cell line HIOEC-B(a)P-TPA. The morphological changes of the cells were observed with differential interference contrast microscope and HE staining. The expression of cytokeratin and vimentin was identified with immunohistochemical staining. The soft agar colonies forming ability and tumorigenesity of the cells were identified to confirm the malignant characteristics of HIOEC-B(a)P-TPA cells., Results: (1) After HIOEC cells were treated with B(a)P plus TPA for 6 months, HIOEC-B(a)P-TPA cells grew well in DMEM medium containing with physical concentration of calcium and 10% FBS. (2) During HIOEC cells were treated with chemical carcinogens, the morphology of the cells was changed. HIOEC-B(a)P-TPA cells showed as fibroblast-like cells with many atypical mitosis. (3) The expression of cytokeratin decreased in the cells while that of vimentin increased in the cells. (4) HIOEC-B(a)P-TPA cells had strong soft agar colony formation ability and the colony formation ratio was 24.5%. (5) HIOEC-B(a)P-TPA cells have no tumorigenisity till now., Conclusions: We established a biological factors and chemical carcinogens induced malignant cell line-HIOEC-B(a)P-TPA after a long period. It will provide a good multiple factors, multistage carcinogenesis model of OSCC for further research.

Objective: To transform HPV E6/E7 immortalized human oral epithelial cell (HIOEC) line cells by benzo(a)pyrene [B(a)P] in vitro, and to establish a carcinogenesis model of oral squamous cell carcinoma., Methods: HIOEC cells were treated with 0.1 mg/L-1.2 mg/L B(a)P for 6 months. The cells were cloned at 18th passage, and then the culture medium was changed into DMEM containing 10% FBS at 21th passage. Cells were cultured in vitro for half and one year and the cell line was named HIOEC-B(a)P. The morphological changes of the cells were observed with differential interference contrast microscope and HE staining. The soft agar colony forming ability and tumorigenicity of the cells in nude mice were identified to confirm the malignant characteristics of HIOEC-B(a)P cells., Results: (1) After HIOEC cells were treated with B(a)P for 6 months, HIOEC-B(a)P cells could grow well in DMEM medium containing 10% FBS and physical concentration of calcium. (2) When HIOEC cells were treated with chemical carcinogens, the morphology of the cells was changed. Cells showed the character of polygon epithelial cells with much atypical mitosis. (3) The 93th passage of HIOEC-B(a)P cells had soft agar colony formation ability. (4) The 55th passage of HIOEC-B(a)P cells could develop parakeratosis mass. The 69th passage of HIOEC-B(a)P cells could develop typical well-differentiated squamous cell carcinoma. The 74th and the 96th HIOEC-B(a)P cells developed I-II grade squamous cell carcinoma-like clinical lesions in nude mice., Conclusions: B(a)P may induce HIOEC cells to be oral squamous cell carcinoma (OSCC) carcinogenetic cells. It will provide a multiple factors, multistage carcinogenesis model of OSCC for the further research.

Purpose: To investigate the expression and its significance of cytokeratin 13 gene in oral squamous cell carcinoma (OSCC)., Methods: Differential CK13 gene expression between 30 cases of paired normal and cancerous tissues were detected by reverse transcription-PCR (RT-PCR), and immunohistochemistry was conducted using monoclonal antibody against CK13 in them. The data were analyzed with SAS6.12 software for Fisher's exact test., Results: RT-PCR results demonstrated that CK13 gene mRNA was upregulated in 27 cases of normal tissues than in paired cancerous samples, by 4.2-fold in total; and immunostain results revealed that expression of CK13 protein between normal oral squamous cell and OSCC, high differentiated OSCC and poor differentiated ones were significantly decreased (P<0.05)., Conclusions: CK13 gene might play an important role in OSCC carcinogenesis and be regulated at post-transcriptional levels. Further research will contribute to confirm its role and mechanism.

Objective: To screen and clone the novel genes related to cellular proliferation of oral squamous cell carcinoma., Methods: We selected the Rb gene as the bait protein gene to construct the fusion bait plasmid of yeast two-hybrid. The whole code sequence of Rb gene was acquired by digestion with restricted enzyme EcoRI and BamH1 and reclaimed from its original vector pGBT9-pRb. After being confirmed by electrophoresis, the Rb gene was cloned into the MCS of the plasmid pGBKT7 to construct a recombined plasmid pGBKT7-pRb and the sequence of the recombined plasmid was detected in company. According to the protocol of yeast two hybrid system III, the competent Y187 yeast was prepared, and transformed with recombined plasmid pGBKT7-pRb. Following that, the toxicity and transcriptional activation of this recombined plasmid pGBKT7-pRb in Y187 yeast were tested., Results: The sequence of the recombined plasmid was correct compared with the sequence provided in Genbank. The protein could be correctly synthesized in vitro, and no self-activating transcriptional activation and toxicity was observed in Y187 yeast., Conclusions: The construction of the recombined plasmid was capable to be used as the fusion bait plasmid in yeast two-hybrid system III, and the recombined Rb-protein could be used as the bait protein successfully.

Objective: To compare the clinical efficacy and toxicity of teniposide (VM26) of higher dose with those of lower dose, both combined with cisplatin (CDDP) and pingyangmycin (PYM), in the treatment of patients with squamous cell carcinoma of oral and maxillofacial region (SCCOMR)., Methods: Sixty-five patients with SCCOMR entered into this study prospectively. Thirty-three patients were treated with higher dose of VM26 (total dose was 320 mg) combined with CDDP and PYM (PTP1), the other thirty-two patients were treated with lower dose (total dose was 158 mg) of VM26 combined with CDDP and PYM (PTP2)., Results: Thirty-three patients received a total of 38 cycles of PTP1. The overall response rate was 81.82% (27/33). Thirty-two patients received a total of 36 cycles of PTP2 and showed overall response rate by 81.25% (26/32). There was no significant difference between PTP1 and PTP2 groups in response rate (P > 0.05). But the blood toxicity was more severe in PTP1 group than in PTP2 group (P < 0.01). Bone marrow depression rate (1-4 stage) was 48.48% in PTP1 group versus 25.00% in the other group., Conclusions: A high response rate of 81.25% and relatively slighter adverse events could be obtained for lower dose of VM26 combined with CDDP and PYM (PTP2). So, the chemotherapy schedule, PTP2, a novel teniposide based regimen in SCCOMR could be employed and spread in clinical practice.

Purpose: To investigate the immunological function for patients with oral cancer undergoing operation., Methods: Peripheral blood was taken from patients with oral cancer before and after operation. Proportions of CD3(+) total T, CD4(+) Th, CD8(+) Tc/Ts, CD19(+) total B, CD16(+)/CD56(+)NK were detected by flow cytometry (FCM). Detections of serum tumor necrosis factor alpha (TNF-alpha) and soluble interleukin-2 receptor (sIL-2R) were performed with enzyme-linked immunosorbent assay(ELISA). Paired t test was used for statistical analysis., Results: The proportions of CD8(+)Tc/Ts, CD19(+) total B and CD4(+)/CD8(+) increased significantly after surgery. Post operative proportion of CD16(+)/CD56(+)NK decreased more significantly. The concentration of TNF-alpha was less than that before operation(P<0.05)., Conclusions: Surgery could change immunological function and alleviate immunosuppression of oral cancer patients. Detections for immune conditions of oral cancer patients might have important clinical values in prognosis evaluation and guiding treatment, especially immunotherapy, after operation.

Objective: To investigate the antitumor effectiveness of teniposide in oral squamous cell carcinomas (OSCC) and to find evidence for using teniposide for treatment of patients with OSCC., Methods: Seventy-five patients with OSCC from the School of Stomatology, Shanghai Second medical University during 1999 to 2001 were evaluated. Tumors were diagnosed pathologically, and drug sensitivity tested. The antitumor drugs tested were cisplatin (CDDP) and teniposide (VM-26). Fresh drug was diluted in complete medium at fold of five times of peak plasma concentration (PPC x 5) achieved by intravenous administration of clinical doses. The concentrations were VM-26 60 mg/L, CDDP 15 mg/L., Results: The MTT assay was performed in 75 of 81 patients (success rate 92.6%). The clinical stages of the 75 patients according to the UICC TNM classification of malignant tumors were 28 with stage IV, 34 with stage III, 11 with stage II and 2 with stage I. The pathological grades of the 75 patients according to three step classification were 18 with Grade I, 37 with Grade I approximately II and 20 with Grade III. At a drug concentration of PPC x 5, the inhibition rates of tumor cells for VM-26 and CDDP were 63.34% and 24.08%, respectively. The inhibition rates of tumor cells for VM-26 were significantly higher than those for CDDP (P < 0.01)., Conclusions: The inhibition rates of OSCC cells for VM-26 are significantly higher than for CDDP. VM-26 may be the first selected drug for treating patients with OSCC.