Irradiation induced epithelial-mesenchymal transition in nasopharyngeal carcinoma in vitro / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the irradiation induced epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma (NPC) in vitro.</p><p><b>METHODS</b>NPC CNE-2 cells with radioresistance (CNE-2-Rs) were established by exposure to gradiently increased dose of irradiation. CCK-8 cell viability kits, colony formation assay and fluorescence-activated cell sorting analysis were used to confirm the capacity of radioresistance of CNE-2-Rs cells. Invert microscope was used to monitor the morphological changes and western blot was applied to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin during the phase of CNE-2 exposure to irradiation.</p><p><b>RESULTS</b>Irradiation exposure successfully induced the radioresistance of CNE-2 cells. After exposed to irradiation, the survival rate in CNE-2-Rs was higher than that in CNE-2 by CCK-8 assays. No significant difference of proliferation ability was observed between the CNE-2 and CNE-2-Rs pre-radiotherapy, but a higher proliferation ability in the CNE-2-Rs post-radiotherapy. By using the colony forming assay, the parameters of CNE-2 and CNE-2-Rs in multi-target single-hit and linear quadratic model were obtained and the data demonstrated that parameters mean lethal dose (D0) , quasi-thres hold dose (Dq) , surrival fraction in 2Qy (SF2) and mean inctivation dose (MID) value increased, α and α/β value decreased (P < 0.05) . At the same time, the CNE-2-Rs cells showed higher percentage of cells in S and G2 phase (P < 0.05) . In terms of biomorphology, CNE-2-Rs cells were more narrow, long strips or fusiform shapes, stretched out tentacles, and the contacts between them were loosened. When radiation dose accumulated to 24 Gy, an over-expression of Vimentin was observed in treated cells, while E-cadherin was down-regulated (P < 0.01) .</p><p><b>CONCLUSIONS</b>NPC cells present with typical morphorlogical and biomolecular changes of EMT during exposure to irradiation, indicating the potential critical roles of EMT in the malignant behavior of radioresistance in NPC.</p>

EphA2 mediated vascular endothelial growth factor expression via the p38 MAPK signaling pathway in squamous cell carcinoma of the head and neck / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the regulatory effect of erythropoietin-producing hepatocellular receptor (EphA2) on the expression of VEGF protein, a pro-angiogenic factor, via p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in squamous cell carcinoma of the head and neck(SCCHN) in vitro.</p><p><b>METHODS</b>SCCHN Tu686 cells were transfected with EphA2 overexpression vector pEGFP-N1-EphA2. Western blot was used to detect the expression of p38 MAPK and enzyme-linked immunosorbent assay (ELISA) was applied to assay of VEGF. SB203580 as a inhibitor of p38 MAPK signaling pathway was used.</p><p><b>RESULTS</b>The expression of VEGF protein was significantly up-regulated in Tu686 cells transfected with EphA2 overexpression vector (535.31 ± 45.71) pg/ml, when compared with Tu686 cells transfected with empty vector (400.99 ± 33.50) pg/ml and Tu686 cells with no transfection (385.30 ± 33.50) pg/ml (F = 17.091, P < 0.01). The expression of phosphorylated p38 MAPK was obviously increased in Tu686 cells with EphA2 overexpression. SB203580 inhibited the expressions of VEGF and phosphorylated p38 MAPK proteins in Tu686 cells with EphA2 overexpression.</p><p><b>CONCLUSION</b>EphA2 can regulate the expression of VEGF protein and stimulate p38 MAPK signaling pathway.</p>

Expression of astrocyte elevated gene-1 protein and its clinical significance in laryngeal squamous cell carcinoma / 中华病理学杂志

<p><b>OBJECTIVE</b>To assess the protein expression of astrocyte elevated gene-1 (AEG-1) in tissue specimens of laryngeal squamous cell carcinoma (LSCC), and to correlate its expression with clinicopathological parameters and prognosis in patients with LSCC.</p><p><b>METHODS</b>RT-PCR was used to assay the expression of AEG-1 mRNA in 13 pairs of LSCC tissues and their corresponding noncarcinoma epithelia. Immunohistochemistry was performed on paraffin-embedded tissue specimens to investigate the protein expression of AEG-1 in 88 cases of LSCC specimens and 15 cases of adjacent epithelial samples.</p><p><b>RESULTS</b>The expression of AEG-1 mRNA was significantly increased in LSCC tissues compared to adjacent noncarcinoma epithelial tissues (0.81 ± 0.17 vs. 0.23 ± 0.10;t = 10.337, P < 0.001). Meantime, the positive rate of AEG-1 protein in 88 cases of LSCC was 87.5% (77/88). However, 15 cases of adjacent noncarcinoma epithelial merely demonstrated negative or mild expression of AEG-1 protein. AEG-1 overexpression was closely correlated with T stage (χ(2) = 6.289, P = 0.018), clinical stage (χ(2) = 11.049, P < 0.01), metastasis (χ(2) = 20.859, P < 0.01) and recurrence(χ(2) = 13.459, P < 0.01). The overall survival rates of patients with AEG-1 overexpression and low expression were 35.9% and 86.4%, respectively (χ(2) = 23.409, P < 0.01). Multivariate Cox regression analysis revealed that AEG-1 expression was an independent prognostic factor (P = 0.016).</p><p><b>CONCLUSION</b>AEG-1 protein may play a critical role in the initiation and progression of LSCC, implicating its predictive value in prognosis.</p>

EGCG regulates TGF-β1-induced epithelial mesenchymal transition in squamous cell carcinoma of head and neck / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the effect and molecular mechanism of epigallocatechin-3-gallate (EGCG) on epithelial-mesenchymal transition (EMT) in vitro induced by human recombinant TGF-β1 protein in squamous cell carcinoma of the head and neck.</p><p><b>METHODS</b>EMT morphological changes of Tu686 cells were observed after sequential treatment of 5 ng/ml TGF-β1 and 20 µmol/L EGCG. Tu686 cells were collected after the treatment of 5 ng/ml TGF-β1 for 24 h and EGCG with different concentrations (0, 10, 20, 30 µmol/L) for another 24 h or 20 µmol/L EGCG treatment for different time phase (6, 12, 24 h). Then RT-PCR and Western-blot were applied to detect mRNA and protein expression level of epithelial cell marker E-cadherin, mesenchymal cell marker Vimentin and Smad7, an inhibit molecule of TGF-β1 mediated pathway in Tu686 cells.</p><p><b>RESULTS</b>TGF-β1 successfully induced characterized EMT morphological and molecular changes in Tu686 cells, in which expression of E-cadherin decreased, Vimentin increased and Smad7 declined. However, EGCG could reverse the TGF-β1 mediated process of EMT by downregulating the expression of Vimentin and upregulating the expression of E-cadherin and Smad7.</p><p><b>CONCLUSION</b>EGCG significantly inhibits TGF-β1-mediated EMT inTu686 cell lines of SCCHN, which maybe associated with the upregulated-expression of Smad7, an inhibitor in TGF-β1 signaling pathway.</p>

Expressions and clinical significance of high mobility group box-1 mRNA and protein in laryngeal squamous cell carcinoma tissues and serum / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expressions of high mobility group box-1 (HMGB1) in the tumor tissues and sera of patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance.</p><p><b>METHODS</b>Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detected the expressions of HMGB1 mRNA and protein in the tumors and adjacent normal epithelial tissues in 30 patients with LSCC. Serum HMGB1 protein levels in the patients with LSCC and in 10 healthy volunteers were detected with enzyme-linked immunosorbent adsorption experiment (ELISA).</p><p><b>RESULTS</b>RT-PCR demonstrated that the mean relative mRNA expression levels of HMGB1 (HMGB1/GAPDH) in LSCC tissues and in adjacent normal epithelial tissues were 1.25 ± 0.12 and 0.32 ± 0.04, respectively (t = 40.27, P < 0.05). Western blot revealed that the mean relative protein expression levels of HMGB1 (HMGB1/β-actin) were 1.29 ± 0.10 and 0.34 ± 0.03 (t = 49.84, P < 0.05), respectively. Both mRNA and protein expression levels of HMGB1 were associated with T stage, clinical stage, lymph node metastasis status and smoking (all P < 0.05), but no significant correlation with age, alcohol consumption and primary tumor grade and location (all P > 0.05). Mean serum HMGB1 protein levels in patients with LSCC and healthy volunteers were (24.80 ± 14.08) ng/ml and (23.58 ± 14.69) ng/ml (t = 0.37, P > 0.05).</p><p><b>CONCLUSIONS</b>Both mRNA and protein expressions of HMGB1 were obviously elevated in LSCC, which were associated closely with T stage, clinical stage and lymph node metastasis.</p>

EphA2 promotes angiogenesis and metastasis of head and neck squamous cell carcinoma in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of EphA2 on the angiogenesis and cervical lymph node metastasis of squamous cell carcinoma of the head and neck (SCCHN) in vivo.</p><p><b>METHODS</b>EphA2 short hairpin (shRNA) lentiviral particles were used to knockdown the expression of EphA2 in SCCHN cell line M2 with high lymph nodes metastasis rate. Stable clones, obtained by puromycin screening, were assayed by RT-PCR and Western blot to validate the gene silencing efficiency and were used to establish SCCHN metastatic xenograft mouse model. Hematoxylin-eosin staining was applied to identify cervical lymph node metastasis of SCCHN in xenografted tumors. Immunohistochemistry was used to observe microvessel density. Western blot was used to investigate the protein expressions of EphA2 and vascular endothelial, growth factor (VEGF).</p><p><b>RESULTS</b>EphA2 shRNA lentiviral particles efficiently decreased the mRNA and protein expressions of EphA2 in SCCHN cell line M2, which were further successfully utilized to establish SCCHN metastatic xenograft mouse model. Compared with xenografted tumors in control group, xenografted tumors in M2EphA2RNAi(+) group decreased significantly tumor volume [(430.7 ± 190.0) mm(3) (x(-) ± s) vs (1179.0 ± 289.4) mm(3)] and weight [(0.26 ± 0.10) g vs (0.54 ± 0.12) g] (both P < 0.05). More importantly, bilateral cervical lymph node metastasis rate in M2EphA2RNAi(+) was also greatly declined (Mann-Whitney U = 10.0, P < 0.05). Decreased protein expressions of EphA2 and VEGF and microvessel density were observed in M2EphA2RNAi(+) group (t = 26.751, P < 0.01).</p><p><b>CONCLUSIONS</b>Knockdown of EphA2 expression led to the inhibition of tumor growth and metastasis in SCCHN nude mouse model. More importantly, SCCHN angiogenesis was also impeded, which might be associated with the decreased expression of VEGF.</p>

Expression of HMGB1 protein in laryngeal squamous cell carcinoma and its clinical significance / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the expression of HMGB1 protein in tissue specimens of laryngeal squamous cell carcinoma (LSCC) and adjacent normal mucosa, and explore the correlation of HMGB1 protein expression with clinicopathologic features and prognosis in LSCC.</p><p><b>METHODS</b>Ninty-three cases of LSCC and 5 cases of adjcent mucosal tissue samples were included in this study. Immunohistochemical staining was performed on paraffin-embedded tissue specimens to examine the HMGB1 protein expression. The data were futher correlated with the clinicopathological features and prognosis of the LSCC patients.</p><p><b>RESULTS</b>The positive rates of HMGB1 expression in LSCC specimens was 87.1%, significantly higher than that in the adjcent normal mucosa samples (46.7%, P = 0.001), and its overexpresion was closely correlated with T stage (Chi2 = 10.878, P = 0.004), clinical stage (Chi2 = 21.115, P < 0.01), metastasis (Chi2 = 28.298, P < 0.01) and recurrence (Chi2 = 14. 923, P = 0.001) in patients with LSCC. Patients with HMGB1 overexpression had both poorer disease-free survival and poorer overall survival compared with that in patients with low HMGB1 expression (Chi2 = 13.815, Chi2 = 11.912; Both P < 0.01). Univariate and multivariate Cox regression analyses revealed that HMGBI expression is an independent prognostic factor for patients with LSCC.</p><p><b>CONCLUSIONS</b>The results of this study demonstrate that HMGB1 protein expression is significantly increased in LSCC tissues, and HMGB1 protein overexpression is associated with a poorer prognosis in patients with LSCC. These results suggest that HMGB1 may play a critical role in the initiation and progression of LSCC, implicating HMGB1 may become a valuable marker for the prediction of prognosis in patients with LSCC.</p>

Correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in laryngeal squamous cell carcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To evaluate the expression of EphA2 protein in tissue specimens and cell lines of laryngeal squamous cell carcinoma (LSCC), and to further study the correlation of EphA2 protein expression with clinicopathological characteristics and prognosis in LSCC.</p><p><b>METHODS</b>Western blot was applied to assess the EphA2 protein expression in LSCC cell line Hep-2 cells and the head and neck immortalized epithelial cell line NP-69 cells. Immunohistochemical staining was performed on paraffin sections of 88 cases of LSCC specimens and 16 cases of adjcent normal tissue samples to investigate the EphA2 protein expression, and to futher elucidate its correlation with clinicopathological characteristics.</p><p><b>RESULTS</b>Compared with the NP-69 cells, EphA2 expression in LSCC cell line Hep-2 cells was upregulated. The positive rates of EphA2 expression in LSCC and adjcent normal tissues samples were 80.7% and 43.8%, respectively, with a significant difference between the two groups (P < 0.001). EphA2 overexpresion was closely correlated with clinical stage (I + II/III + IV, P = 0.005), metastasis (P = 0.025) and recurrence (P = 0.021) in LSCC. Furthermore, patients with EphA2 overexpression had poorer tumor-free survival and 5-year overall survival compared with that in patients with low EphA2 expression (33.3% vs. 63.2%, P = 0.003; 46.7% vs. 81.6%, P = 0.002). EphA2 expression combined with clinical stage provided a better predictive value in prognosis. Univariate and multivariate Cox regression analysis revealed that EphA2 expression is an independent prognostic factor for patients with LSCC (P = 0.019).</p><p><b>CONCLUSIONS</b>The results of this study demonstrate that EphA2 protein expression is significantly increased in LSCC tissues and cell lines, and EphA2 protein overexpression is associated with tumor recurrence, metastasis and poorer prognosis in LSCC patients. These results suggest that EphA2 may play a critical role in the initiation and progression of LSCC, implicating EphA2 as a valuable marker for the prediction of recurrence, metastasis and prognosis in LSCC.</p>

Tumor-targeted human telomerase reverse transcriptase promoter/tk gene therapy against human nasopharyngeal carcinoma cells in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the targeted killing effect of human telomerase reverse transcriptase promoter (hTERTp)/tk gene on human nasopharyngeal carcinoma (NPC) cells.</p><p><b>METHODS</b>The recombinant plasmid hTERTp/tk/pGL3 was transfected into human NPC HNE1 cells and the expressions of TK and telomerase were investigated. The targeted killing effect induced by hTERTp/tk on HNE1 cells was assessed using RT-PCR and MTT assay.</p><p><b>RESULTS</b>TK gene expression was detected in HNE1 cells transfected by hTERTp/tk/pGL3, and the cells showed reduced telomerase and hTERT expression as compared with the control cells. hTERTp/tk/pGL3 resulted in target killing of HNE1 cells but not of the normal control cells. The tumor cell-killing effect of hTERTp/tk/pGL3 was slightly milder than that of the positive control CMV/tk/pGL3 that produced nonselective cell killing.</p><p><b>CONCLUSION</b>hTERTp/tk, a tumor-specific expression system, allows targeted tumor cell killing and reduces the activity of telomerase in NPC cells in vitro.</p>

Killing effect of VP3 on human nasopharyngeal carcinoma cell line CNE-2 cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the killing effects of VP(3) on nasopharyngeal carcinoma cell line CNE-2.@*METHODS@#Plasmid expression vector pcDNA3.1(-) CMV.VP(3)-His was constructed and identified by Kpn I/EcoR I endonuclease analysis, and then sequenced to verify successful insertion in the sense direction of VP(3) gene. pcDNA3.1(-) CMV.VP(3)-His and pcDNA3.1(-)-His expression plasmid was transiently transfected into nasopharyngeal carcinoma cell line CNE-2 . VP(3) protein expression was detected by Western blotting. MTT assay was used to determine the killing effects of VP(3) gene on nasopharyngeal carcinoma cell line CNE-2.@*RESULTS@#Endonuclease analysis and sequencing confirmed the recombinant plasmid contained the complete VP(3) CDS sequence. Western blotting detected a 14.03 kD protein expression from the transfected cells, which was the expecting band of VP(3) gene. The growth of CNE-2 cells that expressed VP(3) gene was inhibited,while the growth of CNE-2 cells that did not express VP(3) gene was not inhibited.@*CONCLUSION@#VP(3) gene can kill nasopharyngeal carcinoma cell CNE-2.

Clinical analysis of 170 cases head and neck lymphoma / 中南大学学报(医学版)

OBJECTIVE@#To study the characteristics of head and neck lymphoma in order to improve its diagnose rate.@*METHODS@#Review and analysis 170 patients with head and neck lymphoma in department of otolaryngology of Xiangya hospital from 1997 to 2005.@*RESULTS@#Nasal cavity and nasal sinuses, neck, tonsil were the common place of the origin of head and neck lymphoma. There are 9 cases Hodgkin disease and 161 cases non-Hodgkin lymphoma (NHL). T cellularity, B cellularity lymphoma, the mixed pattern and nullityping accounted for 60.9%, 36.0% and 3.1% of these patients with NHL, respectively. CHOP and radiotherapy were the main treatment method.@*CONCLUSION@#The clinical and imageology manifestation of head and neck lymphoma were of diversification and no specificity, whose final diagnosis depended on immunohistochemistry.

Primary functional analysis of CK13 gene 5' flanking region / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of cytokeratin 13 (CK13) gene expression control and the effects of different motifs of CK13 gene 5' flanking region on its transcriptional activity.</p><p><b>METHODS</b>The molecular clone technique and reporter gene analysis were used to assay the effects of different motifs of 513 bp of CK13 gene 5' flanking region on its transcriptional activity. The pCAT enhancer vectors with different motifs of CK13 gene 5' flanking region were constructed and transferred to HeLa cells with the help of lipofectin. The instant CAT expression of different clones was detected and the effects of different motifs of the CK13 gene 5' flanking region on its transcriptional activity were evaluated.</p><p><b>RESULTS</b>119 bp from -nt.325 to -nt.207 upstream of the first ATG of CK13 gene 5' flanking region included a silent element. 113 bp region from -nt.206 to -nt.94 included an enhanced element.</p><p><b>CONCLUSION</b>513 bp of CK13 gene 5' flanking region includes a silent element and an enhanced element. Further locating these cis elements and detecting the related trans reaction factors may unveil some important clues to the details of the mechanisms for the CK13 gene expression and tissue-specific expression.</p>

Fermentative production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) by recombinant Aeromonas hydrophila 4AK4 (pTG01) / 生物工程学报

Copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (PHBHHx), a new type of biodegradable material, are receiving considerable attentions recently. The material properties are strongly related to the 3HHx fraction of PHBHHx. As the 3HHx fraction increase, crystallinity and melting point of PHBHHx decrease, flexibility and tractility increase. PHBHHx of different 3HHx fraction can meet different demands of commercial application and research. Aeromonas are the best studied PHBHHx-producing strains. Recent studies have been focused on optimizations of fermentative culture media and culture conditions for low-cost and efficient fermentative production. Aliphatic substrates such as long-chain fatty acid and soybean oil were used in the PHBHHx fermentation as the sole carbon source and energy source. Two-stage fermentation method was also developed for more efficient PHBHHx production. While studies on Aeromonas hydrophila revealed that the monomer composition of PHBHHx could not easily be regulated by fermentative process engineering methods such as changing substrates and fermentative conditions because precursors involved in the PHBHHx synthesis were all from the beta-oxidation pathway. In this study, phbA gene encoding beta-ketothiolase and phbB gene encoding acetoacetyl-CoA reductase were introduced into a PHBHHx-producing strain Aeromonas hydrophila 4AK4 so as to provide a new 3HB precursors synthesis way. phbA gene encodes beta-ketothiolase which can catalyze two acetyl-CoA to form acetoacetyl-CoA; phbB gene encodes acetoacetyl-CoA reductase catalyzing acetoacetly-CoA into 3HB-CoA which is the precursor of 3HB. The introduced novel 3-hydroxybutyrate precursor synthesis pathway allowed the recombinant strain to use unrelated carbon source such as gluconate to provide 3HB precursors for PHBHHx synthesis. Shake-flask experiments were carried out to produce PHBHHx of controllable monomer composition and fermentations in 5 L fermentor were also proceeded for confirmation of these result in large-scale culture. In flask culture, it was possible to reduce the 3HHx mol fraction in PHBHHx from 15 % in the wild type to 3% - 12% in the recombinant by simply changing the ratio of gluconate to lauric acid in the culture media. When lauric acid was used as the sole carbon source, 51.5 g/L Cell Dry Weight (CDW) containing 62 % PHBHHx with 9.7 % 3HHx mol fraction was obtained in 56 hours of fermentation in a 5 liter fermentor. When co-substrates of sodium gluconate and lauric acid (1:1) were used as carbon sources, 32.8 g/L CDW containing 52 % PHBHHx with 6.7% 3HHx mol fraction was obtained in 48 hours of fermentation. These results showed the possibility for fermentative production of PHBHHx with controllable monomer composition.