Genomic alterations in oral squamous cell carcinoma cell lines detected by two-dimensional gel analysis

To initially analyze the genomic abnormalities in human oral squamous cell carcinoma, DNA extracted from each of four oral carcinoma cell lines (Ca9-22, HO-1-u-1, HSC-2, KB) was examined using restriction landmark genomic scanning (RLGS), a method especially conducive to detection of amplifications and rearrangements of genomic DNA. Isolated cell line and normal oral epithial DNAs were sequentially cleaved with specific restriction enzymes, radiolabelled and separated in two-dimensional gel electrophoreses. Thirteen distinct fragments were commonly amplified in the oral cancer cell lines, six of which were evident in all samples. These results suggest genetic alterations characteristic of oral squamous cell carcinogenesis.     

Isolation and characterization of myofibroblast cell lines from oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) invasion is followed by several stromal events such as inflammatory and immune cell infiltration, neo-vascularization, fibroblast activation and occasionally myofibroblast emergence. Our previous studies demonstrated that myofibroblasts in the stroma of OSCC are associated with a more aggressive behavior, leading to shorter patient overall survival. Therefore, we evaluated whether OSCC-associated myofibroblasts have different characteristics compared to OSCC-associated fibroblasts. OSCC myofibroblast cell lines were isolated, cultured and characterized on the basis of the expression of specific isoform α of smooth muscle actin (α-SMA) and of the excessive production of type I collagen. To assess the proliferative potential of the cell lines, growth curves were constructed, whereas the production and activity of matrix metalloproteinases (MMP) were analyzed by ELISA and enzymography, respectively. Myofibroblast clones were positive for α-SMA and vimentin, and negative for pan-cytokeratin and CD34. In long time cultures, western blotting, flow cytometry and ELISA analysis revealed constant α-SMA expression and elevated production of type I collagen. There were no differences on proliferative potential between fibroblast and myofibroblast clones, but myofibroblast cells secreted significantly higher levels of MMP-1, -2, -9 and -13. Furthermore, MMP-2 gelatinolytic activity was significantly higher in myofibroblast clones. The results of this study suggest that myofibroblasts may contribute to OSCC invasion through elevation of MMP synthesis.     

Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines

Epstein–Barr virus (EBV) has been well documented in the aetiology of nasopharyngeal carcinoma (NPC), although its role as well as the genetic basis in the genesis of NPC have not been elucidated. The p53 gene mutations are infrequently found in NPC, but the expression of p53 protein, as well as bcl-2 oncoprotein, has been reported in a high percentage of cases, and also in association with EBV. Proliferating cell nuclear antigen (PCNA) has also been shown to be increased in NPC, suggesting its association among the overexpression of p53 and bcl-2 oncoprotein. We undertook this study to evaluate the correlation among these abnormalities in the development of NPC. The expression of p53 protein, bcl-2 oncoprotein, and the level of PCNA were investigated by immunohistochemistry in 53 patients with NPC. Twenty tissue samples from these patients were studied for p53 gene mutations by single strand conformation polymorphism (SSCP) and DNA sequencing as well as EBV genomes by polymerase chain reaction. Among the 53 specimens, 42 (79%) showed expression of p53 protein and 40 (75%) gave positive result for bcl-2 oncoprotein. A significant association was found between p53 expression and bcl-2 oncoprotein (P=0.002; Fisher's exact test) with 68% of the patients showing coexpression of both markers. The PCNA labelling index in the 53 patients varied from 5% to 80%. High PCNA labelling index was frequently found in the patients with overexpression of p53 protein and bcl-2 oncoprotein. The PCNA index in patients with p53 expression was significant higher than in those without p53 expression (P=0.002). Of the 20 patients, p53 mutations were found in four cases. EBV genomes were detected in 14 cases of which 12 cases showed overexpression of both p53 and bcl-2 and one case with only p53 expression and one case with bcl-2 expression. EBV genomes were detected in two cases with p53 mutations. We conclude that EBV is the important etiologic factor in NPC which may be involved in p53 and bcl-2 overexpression. The mutant p53 protein is correlated to deregulation of PCNA. p53 mutations participate in a small proportion of the tumorigenesis.     

Role of interleukin-8 secreted from human oral squamous cell carcinoma cell lines

Interleukin-8 (IL-8) is an important cytokine involved in tumor growth and angiogenesis in a variety of malignancies. Furthermore, matrix metalloptoteinases (MMPs) also play important roles in the invasion and metastasis of carcinomas including oral squamous cell carcinoma (OSCC). We studied whether IL-8 and MMPs participate in tumorigenesis and metastasis of OSCC. First, we investigated the gene and protein expressions of IL-8 and IL-8 receptor (IL-8R), and the effect of IL-8 on proliferation, migration and invasion of OSCC. Second, we thus also investigated the effect of IL-8 on MMP release in OSCC cells. OSCC cell lines NA and HSC-4 constitutively expressed IL-8 mRNA and secreted its protein in vitro. The production of IL-8 was significantly enhanced by the addition of tumor necrosis factor (TNF)-alpha and IL-beta, but not interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-2. Flow cytometric analysis revealed the constitutive expression of both receptors of IL-8, IL-8RA and IL-8RB, in OSCC cell lines. The expression of IL-8 receptors in HSC-4 cells was stronger than that in NA cells. The intensity of IL-8RA expression was stronger than that of IL-8RB expression in each cell line. The expression of IL-8 receptors was not altered by the addition of cytokines such as TNF-alpha and IL-1beta. The conditioned medium containing IL-8 from OSCC cell lines induced migration and invasion of OSCC cells, but did not change cell proliferation. The differences in migrational and invasive ability between NA cells and HSC-4 cells were correlated with the expression intensity of IL-8 receptors in each cell line. Neutralizing antibodies to IL-8, IL-8RA and IL-8RB partially inhibited the chemotactic activity induced by conditioned medium. The expression of MMP-2, -7 and -9 was detected in culture supernatants from these OSCC cell lines. The expressions of MMP-7 protein and mRNA were enhanced by the addition of rIL-8, but that of other MMPs was not observed in a similar manner. These results suggest that IL-8 secreted from OSCC may contribute to the invasion of OSCC through the regulation of MMP-7 expression.     

Suicide gene therapy for human oral squamous cell carcinoma cell lines with adeno-associated virus vector

The purpose of this study was to test the possibility of gene transfer as a new therapy for oral cancer. Adeno-associated virus (AAV) has already been used in the fields of cystic fibrosis and Parkinson's disease as a potential vector for gene therapy because of its wide host range, high transduction efficiency, and lack of cytopathogenicity. Four human oral squamous cell carcinoma cell lines were transduced with an AAV vector containing the beta-galactosidase gene (AAVlacZ) in vitro. Gene transduction efficiency was from 20 to 50% at a multiplicity of infection (MOI; for the purposes of this study the number of vector genomes per target cell) of 1x10(3), and nearly 100% of each cell line were transduced at an MOI of 1x10(4). Next, four cell lines were transduced with an AAV vector containing the herpes simplex virus thymidine kinase (HSVtk) gene, which sensitizes transduced cells to ganciclovir (GCV). Subsequent administration of GCV resulted in nearly 100% tumor cell killing at an MOI of 1x10(4) and from 70 to 80% tumor cell killing at an MOI of 1x10(3). These results suggest that AAV-mediated gene transfer of HSVtk and administration of GCV has potential as a new therapy for oral squamous cell carcinoma.     

口腔舌鳞状细胞癌的临床分析

目的:总结口腔舌鳞状细胞癌的治疗经验以指导临床。方法:回顾性分析1975年1月～2000年12月初次在我院诊治的329例口腔舌鳞状细胞癌患者的临床资料。T1病变82例,T2病变190例,T3病变43例,T4病变14例。综合治疗186例,单纯手术84例,单纯放疗59例。结果:91例患者组织学检查颈部淋巴结转移,其中Ⅰ区淋巴结转移36例,Ⅱ区淋巴结转移65例,Ⅲ区淋巴结转移18例,Ⅳ区淋巴结转移10例。T1病变患者5年和10年累积生存率分别为72.23%和65.21%,T2分别为38.38%和33.11%,T3分别为10.63%和10.63%,T4分别为7.14%和7.14%(P=0.0000,Logrank=75.99)。单纯手术组患者5年和10年累积生存率分别为56.13%和47.28%,单纯放疗组分别为17.49%和17.49%,综合治疗组分别为43.51%和39.04%(P=0.0000,Logrank=42.23)。结论:口腔舌鳞状细胞癌早期病变可考虑单纯手术,而中晚期应给予综合治疗,对中晚期cN0患者可考虑扩大的肩胛舌骨肌上清扫术。     

Human papillomavirus and oral squamous cell carcinoma: A review of HPV-positive oral squamous cell carcinoma and possible strategies for future

Oral squamous cell carcinoma (OSCC) is a common cancer worldwide. Besides tobacco use and alcohol consumption, human papillomavirus (HPV) infection has also been identified as a risk factor for OSCC recently. The OSCC incidence has increased in recent years, especially among younger women. The purpose of this article is to review clinical and epidemiological studies on the association between HPV infection and OSCCs, and the efficacy of HPV vaccine, so as to provide possible policy implications for preventing HPV-positive OSCC. It is necessary to review the present related body of knowledge to determine whether the association between HPV infection and OSCC has been thoroughly studied. The study was based on literature review. Studies were identified using electronic databases including MEDLINE, PubMed, EMBASE, etc. The inclusion and exclusion criteria were based on consultation from a panel of experts in this area and carefully designed. Based on a systematic review of literatures, HPV infection is a possible cause for the incidence of HPV-positive OSCCs. The prevalence of HPV infection possibly contributed to the increasing trends of HPV-positive OSCCs. Oral HPV infection is a form of HPV transmission. Oral sex behaviors and open-mouthed kissing are probably reasons for oral HPV infection. We also have some epidemiological evidences proving that HPV vaccine provides a possible solution for preventing oral HPV infection. Increased awareness of HPV-positive OSCCs is essential due to the severity of this problem. Biological and epidemiological data regarding the link between sexual behavior and HPV-associated cancers indicate a probable connection, although definitive data are needed. Future studies are needed to investigate the mechanisms of how HPV infection causes HPV-positive OSCCs, whether HPV vaccine provides a prevention for OSCCs, and other important issues.     

The role of tamoxifen in combination with cisplatin on oral squamous cell carcinoma cell lines

The purpose of this study was to evaluate the effect of tamoxifen (TAM) when used in combination with cisplatin on oral squamous cell carcinoma (OSCC). For this, the relation between estrogen receptor (ER) expression level and the cytotoxic effect of TAM, the apoptotic effect and its molecular mechanisms of TAM were investigated using OSCC cell lines. Combination treatment demonstrated a superior cytotoxic and apoptotic effect on OSCC cell lines. Considerable amount of ER was detected in some OSCC cell lines, but there were no significant differences of cytotoxic effect of TAM. TAM inhibited PKC activity and up-regulated TGF-beta1 secretion.     

Apoptotic effect of CKD-602 (Camtobell) on oral squamous cell carcinoma cell lines

The purposes of this study were to measure the cytotoxic effect of CKD-602 on oral squamous cell carcinoma (OSCC) cell lines, to evaluate the apoptotic aspect of dead cells, and to identify the signaling molecules involved in apoptosis. The human OSCC cell lines A253, HSC-3 and KB were treated with CKD-602. The apoptotic proportion of the cells was analyzed using flow cytometry. The expression of Bax, Bcl-2, and p53 were detected by western blotting analysis. CKD-602 showed excellent cytotoxicity to the OSCC cell lines. Most cell death was attributed to apoptosis rather than necrosis. CKD-602 induced the down-regulation of Bcl-2 in A253 and HSC-3 cells, and p53 was expressed in the KB cell line after treatment with CKD-602.     

Anti-tumor activity of dehydroxymethylepoxyquinomicin against human oral squamous cell carcinoma cell lines in vitro and in vivo

Several reports have indicated that nuclear factor-kappa B (NF-κB) is constitutively activated in a variety of cancer cells including human oral squamous carcinoma cells, and play a key role in their growth and survival. Recent studies report that NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), inhibits proliferation and induces apoptosis in prostate cancer cell lines. However this anti-tumor effects are still unknown in end human oral squamous carcinoma cells. In the present study, we investigated the effects of DHMEQ on oral squamous carcinoma cell (OSCC) lines in vitro and in vivo. Human OSCC cell lines (HSC-3, SAS) were treated with DHMEQ and examined for cell viability by MTT assay, cell cycle distribution by flow-cytometry, apoptosis by TUNEL assay, and protein expression by western blotting, respectively. In vivo activities were also investigated in a mouse xenograft model. DHMEQ inhibited growth of two OSCC cell lines in a dose-dependent manner measured by MTT assay. A flow cytometric analysis demonstrated that treatment with DHMEQ induced accumulation in sub-G1 phase. TUNEL assay showed that DHMEQ induced DNA fragmentation. Protein expression by western blotting analysis revealed that DHMEQ induced nuclear down regulation of Survivin, cIAP-1, and cIAP-2. In nude mice, DHMEQ inhibited growth of OSCC without major toxic side effects. The present results demonstrated that administration of DHMEQ is suggested to be a novel anti-tumor approach to the treatment of OSCC.     

Enhanced susceptibility of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5-fluorouracil

Our study was conducted to investigate whether anticancer drugs, cisplatin (CDDP) and/or 5-fluorouracil (5-FU), can modulate Fas-mediated apoptosis in oral squamous cell carcinoma (OSCC) cell lines. When OSCC cell lines, NA and HSC-4, were treated with CDDP and/or 5-FU, Fas and its mRNA expression on the plasma membrane were enhanced. An increase in caspase-3 and -8 activities was then observed by the addition of agonistic anti-Fas antibody, CH-11. Apoptosis of OSCC cells treated with anticancer drugs were significantly enhanced by CH-11, whereas untreated cells were nearly resistant to apoptosis. Moreover, the combination of CDDP and 5-FU resulted in an increasing susceptibility to apoptosis. Caspase-3 and -8 inhibitors, but not caspase-9 inhibitor, reduced Fas-mediated apoptosis enhanced by the anticancer drugs. Furthermore, OSCC cells treated with anticancer drugs exhibited decreased cellular FADD-like interleukin 1-converting enzyme-inhibitory protein (c-FLIP) levels, whereas neither the Fas-associated death domain-containing protein (FADD) nor procaspase-8 changed the expression. Moreover, antisense oligonucleotide to c-FLIP confirmed that down-regulation of c-FLIP induced sensitization to Fas-mediated apoptosis. These results suggest that CDDP and 5-FU may enhance the susceptibility to Fas-mediated apoptosis through down-regulation of c-FLIP. From these findings, a new potential strategy may be developed to improve the efficacy of anticancer drugs.     

Molecular analysis of anoikis resistance in oral cavity squamous cell carcinoma

Oral cavity squamous cell carcinoma (OSCC) is one of the leading causes of cancer deaths worldwide and most of these deaths result from local-regional recurrence and metastases. Evasion of apoptosis is an important hallmark of cancer development and progression, and previous studies have shown that evasion of anoikis, or detachment-induced apoptosis, correlates with a more aggressive phenotype of carcinoma cells in OSCC. To elucidate the cytogenetic and molecular characteristics of anoikis resistance, we generated several cell lines and clones that displayed this cellular phenotype. To test the hypothesis that chromosomal alterations may underlie this phenotypic transformation, we used karyotype analysis to observe changes in the chromosomal structure of anoikis-sensitive and anoikis-resistant cell lines. We further hypothesized that a unique pattern of gene expression was induced by cell-detachment of anoikis-resistant cell lines, and cDNA microarray analysis was performed using a panel of anoikis-resistant oral cancer cell lines grown under attached and detached growth conditions. We identified S100P, KLK6 and CTNNAL1 as genes whose expression levels were differentially regulated in the anoikis-resistant cell lines compared to the anoikis-sensitive cells under detached conditions. These results were verified using real-time RT-PCR. The anoikis-resistant phenotype of squamous cell carcinoma has a distinct genetic expression pattern that is marked by chromosomal alterations that may contribute to differential expression of genes involved in diverse cellular functions. Therapies targeting these potential mediators of anoikis resistance may prove to be beneficial in the treatment of metastatic squamous cell carcinoma.     

Outcome analysis of patients with well-differentiated oral cavity squamous cell carcinoma

The prognosis of well-differentiated oral cavity squamous cell carcinoma (OSCC) is better than less-well-differentiated neoplasms. The aim of this retrospective study was to identify prognostic factors in patients with well-differentiated OSCC. The 5-year outcomes of 467 patients with well-differentiated OSCC who underwent radical surgery and neck dissection were analyzed. In the entire cohort, the presence of pathological node metastases (pN+ vs. pN0) was an independent predictor of 5-year outcomes. In pN0 patients, tumor depth (?8 mm) was the only independently prognostic factor for 5-year survival rates on multivariable analysis (disease-free survival [DFS], P = 0.001, hazard ratio [HR] = 2.634, 95% confidence interval [95% CI] = 1.496–4.636; disease-specific survival [DSS], P < 0.001, HR = 6.794, 95% CI = 2.364–19.525). In pN+ patients, level IV/V neck nodal metastases (DFS, P < 0.001, HR = 47.483, 95% CI = 8.942–252.122; DSS, P < 0.001, HR = 14.301, 95% CI = 5.337–38.323), and ?3 positive nodes (DFS, P = 0.037, HR = 2.107, 95% CI = 1.047–4.242; DSS, P = 0.044, HR = 2.093, 95% CI = 1.020–4.295) were independently associated with 5-year outcomes. Our results suggest that a tailored treatment approach in well-differentiated OSCC patients should take into account the presence of either pN0 or pN+ disease.     

Diagnosis and therapy of oral squamous cell carcinoma

Oral squamous cell carcinoma ranks among the top ten most common cancers worldwide. Despite the success in diagnosis and therapy during the past 30 years, oral squamous cell carcinoma still belongs to the tumor types with a very unfavorable prognosis. In an effort to identify genomic alterations with prognostic relevance, we applied the comparative genomic hybridization technique on oral squamous cell carcinoma. The tumors exhibited from five up to 47 DNA copy number alterations, indicating a considerable degree of genomic imbalance. Out of 35 tumors, 19 showed a gain of chromosome band 7p12. Genomic imbalances were investigated by hierarchical cluster analysis and clustered image mapping to investigate whether genomic profiles correlate with clinical data. Results of the present investigation show that profiling of genomic imbalances in general, and especially of the epidermal growth factor receptor (EGFR) on 7p12, may be suitable as prognostic factors. In order to identify small-molecule inhibitors for EGFR, we established a database of 531 natural compounds derived from medicinal plants used in traditional Chinese medicine. Candidate compounds were identified by correlation analysis using the Kendall tau-test of IC50 values of tumor cell lines and microarray-based EGFR mRNA expression. Further validation was performed by molecular docking studies using the AutoDock program with the crystal structure of EGFR tyrosine kinase domain as docking template. We estimate these results will be a further step toward the ultimate goal of individualized, patient-adapted tumor treatment based on tumor molecular profiling.     

Cytokeratin alteration in oral leukoplakia and oral squamous cell carcinoma

Intermediate filaments are involved in cell migration and intracellular signal transduction pathways. In a variety of organs, the expression of distinct intermediary filaments are further associated with distinct steps of malignant transformation. In this study, we seeked to define the cytokeratin (Ck) expression pattern in oral leukoplakia and oral squamous cell carcinoma (OSCC). One hundred and ninety-two patients with OSCC, 117 patients with oral leukoplakia without dysplasia (OL) and 23 with oral leukoplakia with dysplasia (squamous intraepithelial neoplasia) (OLD) of the oral cavity were investigated for the immunohistochemical expression of Ck 5-6, Ck 8/18, Ck 1 Ck 10, Ck 14, Ck 19 using the tissue microarray technique. Correlations between clinical features and the expression of cytokeratins were evaluated statistically by chi2 tests. The expression of Ck 8/18, Ck 19 and Ck 1 was seen in 3.1% (Ck 8/18), 12.5% (Ck 19), 75.4% (Ck 1) of all leukoplakias, 1.0% (Ck 8/18), 9.4% (Ck 19), 76.8% (Ck 1) in OL, 13.0% (Ck 8/18), 27.3% (Ck 19), 68.4% (Ck 1) in OLD and was significantly associated with the degree of dysplasia (Ck 8/18 p<0.01; Ck 19 p<0.01; Ck 1 p<0.01) and the acquisition of invasive growth properties. The highest frequencies were observed in invasive squamous cell carcinomas. The expression of Ck 8/18 and Ck 19 in transformed oral lesions can be regarded as an early feature in the pathogenesis of invasive OSCC. However, the aberrant expression of Ck 8/18 and Ck 19 in an even higher frequency in invasive carcinomas characterizes the expression of typical glandular cytokeratins as a general progression marker in squamous cell carcinomas. These results can be interpreted as first hints that oral leukoplakias with an expression of Ck 8/18 or 19 independent of dysplasia, should be resected totally since they might indicate an increased progression potential.     

Epigenetic alterations in methylation in oral squamous cell carcinoma cell lines detected by two-dimensional gel electrophoresis

It has been described that DNA alterations in human oral squamous cell carcinomas were successfully demonstrated as 6 spots being commonly reduced in more than 50% of 10 lesions using the restriction landmark genomic scanning (RLGS) method. In the present study, the question of whether the DNA alterations involve not only genetic but also epigenetic change was examined with the use of cell lines Ca9-22, HO-1-u-1, HSC-2 and KB and treatment with a demethylating agent 5-aza-2'-deoxycytidine (5-AzaCDR). Intensities of two of the reduced spots were amplified in the cell lines by the 5-AzaCDR treatment, showing that they were due to altered DNA methylation. Our study provides clear evidence that epigenetic changes, like the methylation are related to carcinogenesis in human oral squamous cell epithelium.