Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1.

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence ß-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Proteomic identification of alpha-2-HS-glycoprotein as a plasma biomarker of hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) has very poor prognosis compared with other head and neck squamous cell carcinomas. Late-stage diagnosis of HSCC increases mortality. Therefore, more effective biomarkers for early diagnosis of HSCC are necessary. Unfortunately, appropriate biomarkers for clinical diagnosis and prognosis have not been identified yet. However, recent progresses in quantitative proteomics have offered opportunities to identify plasma proteins as biomarkers for HSCC. In the present study, plasma samples were analyzed by two-dimensional differential gel electrophoresis (2D-DIGE), and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). A total of 26 proteins representing 12 unique gene products were identified. The up-regulation proteins were alpha-2-HS-glycoprotein (AHSG), complement C4-B, haptoglobin, C-reactive protein, and ceruloplasmin, whereas the down-regulation proteins were serum albumin, angiotensinogen, alpha-1-antichymotrypsin, Ig gamma-3 chain C region, fibrinogen gamma chain, apolipoprotein A-I, and Ig kappa chain C region. Among all the differentially expressed proteins, AHSG was validated by western blot and ELISA. The results were consistent with the data from 2D-DIGE, further suggesting that AHSG may be employed as a potential biomarker for the early diagnosis of HSCC. In summary, this study was the first to use 2D-DIGE and MALDI-TOF/TOF platform to identify the potential plasma biomarkers for HSCC. The plasma AHSG showed great potential for HSCC screening.

Levels of plasma Epstein-Barr virus DNA prior and subsequent to treatment predicts the prognosis of nasopharyngeal carcinoma.

The level of Epstein-Barr virus DNA (EBV-DNA) in the plasma prior and subsequent to treatment is a reliable biomarker for the screening, diagnosis, monitoring and prognosis of nasopharyngeal carcinoma (NPC). The present retrospective study aimed to determine whether pre- and post-treatment levels of plasma EBV-DNA were predictive of survival in a large sample of patients with NPC. The level of plasma EBV-DNA in 637 NPC patients prior and subsequent to treatment was determined by quantitative polymerase chain reaction. The value of pre- and post-treatment plasma EBV-DNA in predicting the survival of NPC patients was then analyzed. The results revealed that pre-treatment plasma EBV-DNA loads were significantly higher in patients with NPC than those in healthy controls (P<0.001). The percentage of patients with positive plasma EBV-DNA was markedly higher prior to treatment (70.64%; median, 1150 copies/ml; range, 0-9.75×106 copies/ml) than following treatment (25.99%; median, 0 copies/ml; range, 0-3.83×106 copies/ml) (P<0.001). Patients with a high plasma EBV-DNA load presented with a higher clinical tumor classification, lymph node status, metastatic status and overall cancer stage. The risk of NPC relapse and mortality was higher in patients with pre-treatment plasma EBV-DNA levels of ≥1,500 copies/ml than that in patients with <1,500 copies/ml. Furthermore, the risk of relapse and mortality was higher in patients with positive post-treatment plasma EBV-DNA than in patients with negative post-treatment plasma EBV-DNA. Detectable post-treatment plasma EBV-DNA was the most significant prognostic factor to affect relapse-free survival, whilst metastasis was the prognostic factor with the greatest effect on overall survival. These data indicated that pre- and post-treatment levels of plasma EBV-DNA were able to predict the prognosis of NPC. This finding may provide novel references for research and clinical practice.

EZH2 promotes angiogenesis through inhibition of miR-1/Endothelin-1 axis in nasopharyngeal carcinoma.

Emerging evidence clearly indicates that EZH2 plays a crucial role in tumor angiogenesis. However, the role of EZH2 in angiogenesis is still unknown in nasopharyngeal carcinoma (NPC). We here showed that the elevated EZH2 level was closely associated with an aggressive and poor prognostic phenotype, and was positively correlated with microvessel density (MVD) in NPC tissues. Functional studies showed that EZH2 upregulation promoted cell proliferation, migration and tubule formation of endothelial cells, and knockdown of EZH2 suppressed tumor growth, metastasis and angiogenesis in vivo. Mechanistic investigations revealed that EZH2 inhibited miR-1 transcription via promoter binding activity, leading to enhanced expression of Endothelin-1 (ET-1) which is suppressed by miR-1 targeting of ET-1 3'UTR. Furthermore, knockdown of EZH2 or overexpression of miR-1 exerted anti-angiogenic effect on NPC cells. More importantly, the neutralizing antibody against ET-1 significantly abrogated the pro-angiogenic effect of EZH2, and forced expression of ET-1 rescued the anti-angiogenic effect induced by EZH2 knockdown. In clinical specimens, ET-1 was widely overexpressed and associated with clinical stage and MVD. Taken together, our results identify a novel signaling pathway involved in NPC angiogenesis, and also suggest that EZH2-miR-1-ET-1 axis represents multiple potential therapeutic targets for NPC.

Epstein-Barr virus nuclear antigen 1 (EBNA1) protein induction of epithelial-mesenchymal transition in nasopharyngeal carcinoma cells.

BACKGROUND: The Epstein-Barr virus (EBV)-encoded EB nuclear antigen 1 (EBNA1) protein is required for maintenance and transmission of the viral episome in EBV-infected cells. The objective of this study was to investigate the role of EBNA1 protein in nasopharyngeal carcinoma (NPC). METHODS: Tissue samples from 48 patients with NPC and 12 patients with chronic nasopharyngitis were subjected to immunohistochemical analysis of EBNA1 expression. EBNA1 combinational DNA was used to overexpress EBNA1 protein in NPC cell lines to assess tumor cell epithelial-mesenchymal transition (EMT), colony formation, migration and invasion, and gene expression. RESULTS: EBNA1 protein was highly expressed in NPC tissue specimens, and its expression was associated with NPC lymph node metastasis. EBNA1 expression affected NPC cell morphology and the expression of EMT markers in vitro. Furthermore, overexpression of EBNA1 inhibited the expression of microRNA 200a (miR-200a) and miR-200b and, in turn, up-regulated expression of their target genes, zinc finger E-box binding homeobox 1 ( ZEB1) and ZEB2, which are well known mediators of EMT. In addition, EBNA1-regulated miR-200a and miR-200b expression was mediated by transforming growth factor-ß1. CONCLUSIONS: The current findings provided novel insight into the vital role of EBNA1 in manipulating a molecular switch of EMT in EBV-positive NPC cells.

Diagnostic and prognostic value of plasma microRNA deregulation in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is uncommon worldwide but often highly invasive in late stages. Due to its special location and lack of specific symptoms, NPC is hardly detected in regular medical examination at the beginning. Development of sensitive and specific biomarkers should help to save lives against this type of disease. In the present report, we investigated the value of plasma miRNAs for diagnosis and prognosis of NPC. Using candidate approach, we selected 21 miRNAs from literature to compare their expression levels in the plasma of NPC patients and controls. As a result, 5 miRNAs showed diagnostic potentials (P<0.01). Among them, miR-16, -21, -24, and -155 had increased levels in NPC patients, whereas the level of miR-378 was decreased. There was a negative correlation between plasma miRNA expression and cancer progression, where miR-21 was statistically significant in T and N staging and miR-16 and 24 were significant in N staging only. Combination of miR-16, -21, -24, -155, and -378 gives 87.7% of sensitivity and 82.0% of specificity for NPC diagnosis. Without miR-16, combination of the rest 4 miRNAs gives the same sensitivity but a slightly reduced specificity. After treatment, all 5 miRNAs were somewhat back to normal levels in patients without cancer recurrence but the prognostic value was not statistically significant. In conclusion, plasma miRNA expression is a useful biomarker for NPC diagnosis but not for its prognosis. More importantly, it is simple, effective, and non-invasive. Combination of several plasma miRNAs can increase both NPC diagnostic sensitivity and specificity.

miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2.

Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic type of cancer that is widely prevalent in Southern China. Studies have shown that several microRNAs (miRNAs) are implicated in NPC metastasis. Our previous studies have demonstrated that miRNA miR-26a inhibits cell growth and tumorigenesis of NPC through the repression of enhancer of zeste homolog 2 (EZH2). However, the role of miR-26a in NPC metastasis remains unknown. In this study, we showed that ectopic expression of miR-26a inhibited the migratory and invasive capacities of NPC cells in vitro. Additionally, we used a murine model to investigate the role of miR-26a in NPC metastasis and results showed that miR-26a overexpression suppresses the metastatic behavior of NPC cells in vivo. Furthermore, the data demonstrated that miR-26a decreased the expression levels of EZH2 in vitro and in vivo, suggesting that the antimetastatic effect of miR-26a in NPC was mediated by regulating EZH2. Therefore, these findings indicate that miR-26a functions as an antimetastatic miRNA in NPC and that its antimetastatic effects are mediated mainly by repressing EZH2 expression.

[Effects of enhancer of zeste homolog (EZH2) downregulation on the proliferation and invasion of nasopharyngeal carcinoma cell and the possible mechanisms].

OBJECTIVE: To investigate the effects of the enhancer of zeste homolog 2 (EZH2) gene on cell growth and invasion of the nasopharyngeal carcinoma (NPC). METHODS: Recombinant lentivirus vector for shRNA delivery of EZH2 was constructed and transfected into 293FT cells. After collecting the viral particles, the NPC cell line 5-8F cells were transfected. The effects of EZH2 silence on cell proliferation and cell cycle were detected using MTT assay, plate colony formation assay and flow cytometry. The migration and invasion of 5-8F cells were determined by wound healing assay and matrigel invasion assay, respectively. The expressions of EZH2 and epithelial-mesenchymal transition (EMT)-related markers at mRNA and protein levels were examined by real-time PCR and Western blot respectively. RESULTS: The expressions of EZH2 mRNA and protein in the transfected 5-8F cells were obviously reduced. MTT assay showed that EZH2 downregulation significantly inhibited the growth of 5-8F/shEZH2 cells (P < 0.001). Colony formation rate (84.44%) of 5-8F/shEZH2 cells was lower than control (31.56%, P = 0.001). Cell cycle analysis showed that most 5-8F/shEZH2 cells were arrested in G0/G1 phase, with a very low ratio of cells in S phase. Wound healing assay indicated that the migration ability of cells silencing EZH2 decreased significantly, and the 48-hour relative migration distance of 5-8F/ShEZH2 cells and control cells was 0.58 ± 0.05, and 0.81 ± 0.02, respectively (P < 0.000). Matrigel invasion assay, showed the invasive capacity of cells silencing EZH2 was significantly inhibited, with less penetrating cells (72.23 ± 4.08) compared to control (150.95 ± 16.27), P < 0.000. The mRNA expressions of epithelial markers E-cadherin and Keratin 18 in the cells silencing EZH2 increased by 177% and 158% respectively, and the mRNA expressions of mesenchymal markers ß-catenin and N-cadherin decreased by 18.04% and 41.18% respectively. Similar results also were obtained with Western blot analysis. CONCLUSION: EZH2 significantly enhanced the proliferation and invasion of nasopharyngeal carcinoma cells in vitro, which might be mediated by inducing EMT.

[Enhancement of target gene expression by recombinant adeno-associated virus combined with recombinant adenovirus in vivo].

OBJECTIVE: To study the changes in the intensity and temporal pattern of target gene expression in the tumor tissue of nude mice bearing human nasopharyngeal carcinoma (NPC) following injection of recombinant adeno-associated virus (rAAV) and recombinant adenovirus (AdV) in vivo. METHODS: EBV-positive human NPC cell line C666-1 was inoculated subcutaneouly in nude mice. After the tumor mass reached 3 mm in diameter, 1.5 × 10(11) v.g (virus genome) rAAV-EGFP, 2.5 × 10(8) pfu rAdV-EGFP or their balanced mixture was injected intratumorally. At 5 and 10 days after the injection, the tumor tissues were harvested for immunohistochemical staining of GFP, and the ratio of the GFP-positive cells and the intensity of GFP expression was determined. RESULTS: Immunohistochemistry for GFP showed that 5 days after the injection, GFP expression was detected (1.70 ∓ 0.48) in the tumor tissue in rAAV group, and the peak expression levels was seen in rAdV group (6.00∓1.94); the expression level was comparable between the combination group (6.90 ∓ 1.92) and rAdV group. At 10 days, GFP expression was considerably lowered to 2.00 ∓ 0.67 in rAdV group but increased to 8.00∓1.15 in rAAV group. The expression in the combination group maintained a high level at 10 days (10.10∓1.63), which was significantly higher than that in rAAV group (P%0.001). CONCLUSION: Transfection with rAAV combined with rAdV allows instant, sustained and significantly enhanced expression of the target gene in the tumor tissue. This approach takes advantages of the two viruses and can be ideal for exogenous gene delivery into the tumor tissues.

[Application of SELDI-TOF-MS in establishing a model for predicting radiotherapy response of hypopharyngeal cancers].

OBJECTIVE: To detect the serum proteomic fingerprints in patients with hypopharyngeal squamous cell carcinoma (HPSCC) by surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) protein chip array technique. METHODS: The serum samples were obtained from 58 HPSCC patients for protein expression analysis using SELDI-TOF Protein Chip technique and cation-exchange (CM10) protein array. All the spectra were compared and the qualified mass peaks with mass-to-charge ratios (m/z) between 1 and 70 kD were autotimatically detected. The tree analysis pattern was generated using Biomarker Patterns Software. RESULTS: The protein profiles of HPSCC serum were analyzed according to the clinical and pathological features of the patients and their treatment response. No significant difference was noted in the serum proteins between HPSCC patients with different statuses of cervical lympha node metastasis (P>0.05), and the difference between well differentiated and poorly differentiated HPSCC was only minor. No significant difference was found in the serum proteins between chemotherapy-sensitive patients and the insensitive patients (P>0.05), but 5 proteins were identified to be overexpressed in the sensitive patients (P < / = 0.05). Radiotherapy-sensitive HPSCC patients were segregated from the insensitive group with a sensitivity of 86.67% and specificity of 100%. CONCLUSION: The serum protein at the m/z value of 6115.74 is overexpressed in radiotherapy-sensitive HPSCC patients. Serum protein profiling allows the prediction of radiotherapy response in HPSCC patients, and the identified proteins may serve as candidate biomarkers for predicting the radiotherapy sensitivity of HPSCC.

[Analysis of clinical characteristics of chronic rhinosinusitis in patients with postirradiated nasopharyngeal carcinoma].

OBJECTIVE: To study the characters of chronic rhinosinusitis in patients with irradiated nasopharyngeal carcinoma. METHODS: There were 65 cases of chronic rhinosinusitis after irradiated nasopharyngeal carcinoma (NPC, experimental group) and 65 cases of common chronic rhinosinusitis (CRS, control group) in the study. The visual analogue scale (VAS) was used to evaluate the intensity of subjective symptoms. Endoscopic finding was recorded and CT results were evaluated by Lund-Mackay scoring system. RESULTS: As to the VAS, nasal secretion was significantly more severe in experimental group (7.86+/-1.62), compared with control group (5.12+/-1.32, t=10.541, P<0.01). As to endoscopic finding, middle nasal meatus were clean in 35 (53.8%) cases in experimental group, and 23 cases (35.4%) in control group (chi2=4.483, P<0.05). CT score was (7.03+/-4.63) in experiment group, and (11.42+/-3.32) in control group (t=-6.207, P<0.05). The main reason lays in lower CT score and lower involved rate of ostiomeatal complex, frontal sinus, maxillary sinus, anterior ethmoid sinus. CONCLUSIONS: The characters of chronic rhinosinusitis in patients with irradiated nasopharyngeal carcinoma is quite different from the common CRS and different therapeutic measures should be taken.

[Treatment and prognosis of stage III-IV laryngeal squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Laryngeal squamous cell carcinoma (LSCC) is a common malignancy of the head and neck. Stage I-II LSCC patients have a favorable prognosis after operation or radiotherapy, but the curative effect and prognosis of stage III-IV LSCC are not satisfying, and its treatment is also controversial. This study was to summarize our experience in treating stage III-IV LSCC patients, evaluate the treatment results, and seek more reasonable therapeutic modality. METHODS: Records of 202 stage III-IV LSCC patients, treated in Cancer Center of Sun Yat-sen University from Jan. 1, 1991 to Jan. 1, 2000, were retrospectively analyzed. Of the 202 patients, 64 received surgery alone, 83 received surgery and preoperative or postoperative radiotherapy, 41 received radiotherapy, and 14 received chemotherapy. The overall survival rate was estimated using life table method by SPSS10.0 software. Survival curves were compared with Wilcoxon method; relapse statuses were compared with Chi(2) test. RESULTS: The 5- and 10-year overall survival rates of the 202 patients was (42.12+/-3.62)% and (33.20+/-4.32)%, and the median survival time was 48.5 months. The 5-year survival rates were 61.07% in glottic carcinoma group and 26.07% in supraglottic carcinoma group, and were 53.41% in surgery alone group, 51.04% in surgery plus radiotherapy group, 18.33% in radiotherapy group and 7.14% in chemotherapy group. There was no significant difference between surgery alone group and surgery plus radiotherapy group (P>0.05). Of the 147 patients received surgery, the 5-year survival rates were 56.15% for the 31 patients received partial laryngectomy and 52.08% for the 116 patients received total laryngectomy. Eleven patients had tumor relapsed after total laryngectomy within 5 years. CONCLUSIONS: Surgery, especially total laryngectomy, is the major treatment modality for stage III-IV LSCC. Partial laryngectomy may be performed with strict selection. Postoperative radiotherapy may be preformed on the patients with suspect of tumor residue or positive margin.