Targeted Inhibitory Effect of Nasopharyngeal Carcinoma Cells by Hre2.Grp78 Chimeric Promoter Regulating Fusion Gene TK/VP3.

OBJECTIVE: To construct plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3 and elaborate the effects of overexpressed TK/VP3 on nasopharyngeal carcinoma cells. METHODS: Four plasmids were constructed, including pcDNA3.1-CMV-TK/VP3, pcDNA3.1-Hre2.TK/VP3, pcDNA3.1-Grp78.TK/VP3, and pcDNA3.1-Hre2.Grp78.TK/VP3. The human nasopharyngeal carcinoma cell line HNE1 cells were transfected with the 4 plasmids, respectively. Cell viabilities were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and apoptosis was conducted using flow cytometry analysis. The expression of TK, VP3, Grp78, and hypoxia-inducible factor 1α and apoptosis-related proteins was determined by real-time quantitative polymerase chain reaction and Western blotting. RESULTS: The recombinant plasmids that could steadily overexpress TK and VP3 were successfully constructed. Expression of TK and VP3 in cells transfected with pcDNA3.1-Hre2.TK/VP3 and pcDNA3.1-Grp78.TK/VP3 was significantly higher than pcDNA3.1-CMV-TK/VP3, and expression in cells transfected with pcDNA3.1-Hre2.Grp78.TK/VP3 was the highest. Under glucose deprivation or hypoxia condition, Grp78 or hypoxia-inducible factor 1α was overexpressed so that expression of TK and VP3 was significantly upregulated, which could further inhibit cell proliferation and enhance cell apoptosis. CONCLUSION: We successfully constructed 4 plasmids with Hre2.Grp78 chimeric promoter regulating fusion gene TK/VP3, which could significantly inhibit the proliferation as well as enhance the apoptosis of nasopharyngeal carcinoma cells under glucose deprivation or hypoxia condition.

Numb inhibits epithelial-mesenchymal transition via RBP-Jκ-dependent Notch1/PTEN/FAK signaling pathway in tongue cancer.

BACKGROUND: Oral cancer has been estimated as the sixth most frequent solid cancer all over the world, in which tongue squamous cell carcinoma (TSCC) is the most common type of oral cancers. However, the mechanism of TSCC metastasizing to lymph node and distant sites has not been completely understood. METHODS: In this study, RT-qPCR method was used to detect the mRNA level of Numb, PTEN and Notch1 genes, as well as EMT-associated genes. Western blot assay was utilized to detect protein level of these genes. In addition, we determined cell proliferation by MTT assay and employed transwell invasion assay and wound healing assay to probe the abilities of invasion and migration, respectively. To investigate the role of PTEN, its inhibitor VO-Ohpic trihydrate was used to treat SCC-4 and CAL27 cells. RESULTS: We found that Numb expression was downregulated in SCC-9 and CAL-27 cells compared to NHOK cells. Instead, Notch1 level in SCC-9 and CAL-27 cells were higher than that in NHOK cells. Furthermore, the results showed that Numb overexpression significantly suppressed proliferation, migration and invasion of SCC-9 and CAL-27 cells via regulating Notch1 signaling and EMT-related genes expression. By contrast, we observed that RBP-Jκ knockdown had an inhibitory role in proliferation, migration and invasion of SCC-9 and CAL-27 cells. In cells with Numb overexpression or RBP-Jκ knockdown, p-FAK and EMT-related genes were remarkably regulated. CONCLUSIONS: Our findings provide new mechanism of understanding the metastasis of TSCC and help develop therapeutic strategies for treating tongue cancer.

Association between RASSF1A Promoter Hypermethylation and Oncogenic HPV Infection Status in Invasive Cervical Cancer: a Meta-analysis.

Cervical carcinoma is the main cause of cancer-related mortality in women and is correlated with more than 15 risk cofactors, including infection of cervical cells with high-risk types of HPV (hrHPV). Indeed, both aberrant methylation of the RASSF1A promoter and hrHPV infection are often observed in cervical carcinomas. The purpose of our meta-analysis was to evaluate the role of RASSF1A promoter methylation and hrHPV infection in cervical cancer. Our meta-analysis involved 895 cervical cancer patients and 454 control patients from 15 studies. Our results suggested that RASSF1A promoter hypermethylation increased the risk of cervical cancer (OR=9.77, 95%CI=[3.06, 31.26], P=0.0001, I2=78%). By grouping cases according to cancer subtypes, we found that HPV infection was higher in cervical squamous cell carcinomas (SCCs) than in cervical adenocarcinomas/ adenosquamous cancers (ACs/ASCs) (OR=4.00, 95%CI=[1.41, 11.30], P=0.009, I2=55%). Interestingly, HPV infection tended to occur in cervical cancers with relatively low levels of RASSF1A promoter methylation (OR=0.59, 95%CI=[0.36, 0.99], P=0.05, I2=0%). Our study provides evidence of a possible interaction between HPV infection and RASSF1A promoter methylation in the development of cervical cancers.

[Surgical management for residual or recurrent diseases in the neck after radiotherapy in nasopharyngeal carcinoma].

OBJECTIVE: To investigate the surgical managements for residual or recurrent diseases in the neck after radiotherapy in nasopharyngeal carcinoma. METHODS: Seventy-eight cases of neck masses (39 cases for unilateral residual diseases, 9 for bilateral residual diseases and 30 for recurrent diseases) after radiotherapy in nasopharyngeal carcinoma who were treated surgically between January 1990 and December 2005 were retrospectively analyzed. There were 56 males and 22 females. Their ages ranged from 28 to 65 years (median 41 years). There were 27 patients with skin involvement. After preoperative imaging assessment, 17 patients whose common carotid arteries or internal carotid arteries were difficult to separate routinely attained the carotid balloon occlusion test which confirmed that 15 cases of cerebral arterial circle open to compensation, however, two cases of poor compensation. Surgical procedures included expanded neck mass resection (21 cases), unilateral radical neck dissection (49 cases) and bilateral radical neck dissection (8 cases). Of them 5 patients were with unilateral internal carotid artery resection. Neck defects were repaired with pectoralis major muscle flaps (15 cases), free anterolateral femoral skin flaps (9 cases) and trapezius muscle flaps (3 cases). Of the 78 patients, 13 with microscopic positive diseases and 2 with residual diseases in internal carotid artery walls underwent postoperative radiotherapy, with the doses of 45 to 50 Gy. RESULTS: All patients were closely followed-up more than 5 years. Three- and five-year survival rates were 46.2% and 28.3% respectively. Neck defects were successfully repaired with skin flaps immediately after resecting diseases in 27 cases, only one patient with delayed healing. Of 51 patients without skin flap repair, neck wounds healed successfully in 45 patients and with delayed healing in 6 patients. Pathological positive rates of lymph nodes located in the level I, II, III, IV and V were 10.5%, 61.4%, 10.5%, 1.8% and 28.1% respectively. CONCLUSIONS: Preoperative balloon occlusion test is required to assess the function of Willis' ring before determining ligation or resection of internal carotid artery. Residual or recurrent diseases commonly exist in level II, VA and III, which should be included in neck dissection. Pectoralis major muscle flap and free anterolateral femoral skin flap are recommended for the repair of neck defect.

[Management of thyroid goiters invading mediastinum and thoracic cavity].

OBJECTIVE: To investigate the diagnosis and treatments of thyroid goiters invading mediastinum and thoracic cavity. METHODS: Seventy-eight cases of thyroid goiters invading mediastinum and thoracic cavity and undergoing surgery from 1995 to 2005 were reviewed. There were 22 males and 56 females and their age ranged from 45 years to 78 years with a median age of 59 years. According to the classification of intrathoracic thyroid goiters, there were 50 cases in Class I, 20 cases in Class II and 8 cases in Class III. In these patients, 38 cases suffered from dyspnea at degree I and 20 cases at degree II. Of the patients, 71 underwent thyroidectomy through neck approach and 7 underwent thyroidectomy by a combined approach of neck incision plus sternotomy or lateral thoracotomy. Tracheal defects in 4 cases and esophageal defects in 3 cases were repaired. Postoperative residual diseases were found in tracheoesophageal wall (5 cases) and mediastinum (6 cases). Eleven patients received postoperative radiotherapy and 18 underwent (131)I treatment. No case died of operation and no case with wound infection. RESULTS: The time of follow-up was 60 - 180 months with a median of 110 months. Three patients lost follow-up. Dyspnea in 58 cases were improved after operation. Three of 49 patients with nodular goiters died from cardiocerebrovascular diseases. Of 29 patients with thyroid papillary carcinoma, 2 died from lung metastasis and 3 died from neck relapse. Five-year survival rate was 75.0% in the patients with thyroid cancer. CONCLUSIONS: Most of thyroid goiters invading mediastinum and thoracic cavity can be completely resected via neck approach, but a combined approach of neck incision plus sternotomy or lateral thoracotomy may be used in some cases with malignant goiters to dissect the diseases completely. Postoperative external beam radiotherapy are required for the residual diseases. (131)I may be considered in high-risk differentiated thyroid carcinoma cases.

The quorum-sensing system AvsR-AvsI regulates both long-chain and short-chain acyl-homoserine lactones in Agrobacterium vitis E26.

Agrobacterium vitis strain E26 is a nonpathogenic bacterium isolated from grape crown gall. In this study, the identification of a luxR-luxI type quorum-sensing system in strain E26 is reported. This system is involved in the induction of hypersensitive response on tobacco, but not its biocontrol activity. The deduced components AvsI(E26) and AvsR(E26) show the greatest similarity to AvsI(F2/5) and AvsR(F2/5), respectively from A. vitis strain F2/5. The mutant in AvsI(E26) abolished the production of both long-chain and short-chain acyl-homoserine lactones signals as well as the ability to cause hypersensitive response on tobacco. Complementaion of avsI (E26) and avsR (E26) genes restored the lost phenotypes to the level of wild type E26. In pot trial, no significant difference on biocontrol efficiency against grapevine crown gall was found between the wide type E26 and its quorum sensing negative mutants.

Expression and hypermethylation of p27 kip1 in hepatocarcinogenesis.

AIM: To study the expressions of p27 kip1 protein and p27mRNA, the hypermethylation of p27 kip1 and the relation between them in various stages of hepatocarcinogenesis. METHODS: p27 protein and p27mRNA were detected by immunohistochemical staining and in situ hybridization respectively in 68 cases of normal liver, liver cirrhosis, pericancerous cirrhosis and hepatocellular carcinoma (HCC). The hypermethylation of p27 kip1 was detected by methylation-specific PCR (MSP) in 44 cases of normal liver, liver cirrhosis, and HCC. RESULTS: The positive rate of p27 protein was 66.7% (4/6) in normal liver, 60.0% (6/10) in liver cirrhosis, 50.0% (12/24) in pericancerous cirrhosis and 21.4% (6/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 protein significantly decreased in HCC compared to that in the other groups (P = 0.006, chi2 = 7.664). The positive rate of p27 kip1 mRNA was 83.3% (5/6) in normal liver, 70.0% (7/10) in liver cirrhosis, 75.0% (18/24) in pericancerous cirrhosis and 25.0% (7/28) in HCC. There were no statistical differences in normal liver, liver cirrhosis and pericancerous cirrhosis, but the positive rate of p27 kip1 mRNA also significantly decreased in HCC compared to that in the other groups (P = 0.000, chi2 = 16.600). In addition, there was a significant correlation between the expression of p27 protein and p27mRNA in the integrated group of normal liver and liver cirrhosis. However, no significant correlation was found between pericancerous cirrhosis and HCC. Using MSP, we found that 1 HCC in 44 cases (including 6 cases of normal liver, 10 cases of liver cirrhosis and 28 cases of HCC) was methylated, whose p27 protein and p27mRNA were negative. CONCLUSION: The reduction or loss of p27 protein and p27mRNA are potentially involved in hepatocarcinogenesis. The hypermethylation of p27 might lead to the loss of p27mRNA transcription.

[Targeted inhibition of vascular endothelial growth factor (VEGF) expression in human osteosarcoma cell line by antisense VEGF165 cDNA promoted by hypoxia reaction element].

OBJECTIVE: Utilizing the hypoxia inducible factor 1/hypoxia reaction element (HIF-1/ HRE) gene regulation system to construct antisense vascular endothelial growth factor (VEGF165) cDNA eukaryotic expression vector promoted by HRE, and investigate its targeted inhibiting VEGF expression of osteosarcoma cells in hypoxia environment. METHODS: Eukaryotic expression plasmid with HRE promoter was constructed containing luciferase reporter gene and antisense VEGF165 cDNA by using PCR and recombinant DNA techniques. The recombinant vectors were transfected into osteosarcoma cells with lipofectin method. Hypoxia-inducible reporter gene expression was determined by liquid scintillation analyzer and the expression of VEGF protein was detected by ELISA method. RESULTS: The eukaryotic expression plasmid containing antisense VEGF165 and luciferase promoted by HRE was constructed successfully. After being transferred into MG63 cells, luciferase expression was increased 3.5 x 10(2) times and VEGF protein expression decreased 45% under hypoxia condition. CONCLUSION: Antisense VEGF165 cDNA expression, efficiently realized by HRE promoter under hypoxia condition, provides an experimental basis for targeted antiangiogenesis of tumors.

Detection of HBV, PCNA and GST-pi in hepatocellular carcinoma and chronic liver diseases.

AIM: To investigate the change of HBV DNA, PCNA and GST-pi in chronic liver disease and hepatocellular carcinoma (HCC). METHODS: Hepatitis B surface antigen (HBsAg), proliferating cell nuclear antigen (PCNA) and glutathione S-transferases (GST-pi) were detected by immunohistochemical staining and HBV DNA was detected by in situ hybridization (ISH) in formalin-fixed and paraffin-embedded sections with a total of 111 specimens of chronic hepatitis, liver cirrhosis, paratumorous tissue, HCC and normal liver tissue. RESULTS: The positive rates of HBsAg and HBVDNA were 62.5 %(15/24) and 75.0 %(12/16) in chronic hepatitis, 64.0 %(16/25) and 83.3 %(15/18) in liver cirrhosis, 72.7 %(16/22) and 85.7 %(12/14) in the paratumorous tissu and 45.0 %(14/31) and 64.3 %(9/14) in HCC. The positive HBVDNA granules in chronic hepatitis, liver cirrhosis and the paratumorous tissue were more intense than that in HCC. The positive rates of PCNA and GST-pi were 34.8 %(8/23) and 25.0 %(4/16) in chronic hepatitis, 73.7 %(14/19) and 17.6 %(3/17) in liver cirrhosis, 86.7 %(13/15) and 53.3 %(8/15) in the paratumorous tissue, 100 %(15/15) and 60.0 %(9/15) in HCC, respectively, and the positive rate of GST-pi in the paratumorous tissue was significantly higher than that in the liver cirrhosis without tumor (P<0.05), but same as that in HCC (P>0.05). CONCLUSION: The HBV infection may increase expression of PCNA and GST-pi. The paratumor cirrhosis may be a sequential lesion of precancerous cirrhosis around HCC.

[Expression and clinical significance of Fas-FasL of pleomorphic adenoma and adenoid cystic carcinoma in parotid gland].

OBJECTIVE: To determine the expression and clinical significance of Fas and FasL in cases of occurrence and development of pleomorphic adenoma and adenoid cystic carcinoma(ACC) in parotid gland. METHODS: Using Immunohistochemistry techniques and quantitative analysis under computerized system, we investigated Fas-FasL expression in 21 cases of normal parotid gland, 35 cases of pleomorphic adenoma in parotid gland and 32 cases of ACC in parotid gland. RESULTS: All cases of ACC of parotid gland showed Fas and FasL negative expression, no correlation was found between the expression of Fas-FasL and the histological type and the pathological grades of ACC. Although both normal parotid gland and pleomorphic adenoma of parotid gland had distinct expression of Fas and FasL, the positive expression of Fas FasL in pleomorphic adenoma of parotid gland cases were both significantly lowered (P<0.01) than that in normal parotid gland. CONCLUSION: Expression of Fas-FasL and Fas FasL mediated cell apoptosis disturbance had significant correlations with the occurrence and malignant development of pleomorphic adenoma and ACC in parotid gland.