Aberrant methylation of DACT1 and DACT2 are associated with tumor progression and poor prognosis in esophageal squamous cell carcinoma.

BACKGROUND: The DACT (Dishevelled-associated antagonist of ß-catenin) family of scaffold proteins may play important roles in tumorigenesis. However, the epigenetic changes of DACT1, 2, 3 and their effect on esophageal squamous cell carcinoma (ESCC) have not been investigated so far. The aim of this study was to investigate the promoter methylation and expression of DACT family, in order to elucidate more information on the role of DACT with regard to the progression and prognosis of ESCC. METHODS: MSP and BGS methods were respectively applied to examine the methylation status of DACT; RT-PCR, Western blot and immunohistochemistry methods were respectively used to determine the mRNA and protein expression of DACT; MTT, Colony-formation and Wound-healing assay were performed to assess the effect of DACT1 and DACT2 on proliferation and migration of esophageal cancer cells. RESULTS: Frequent reduced expression of DACT1, DACT2 and DACT3 were found in esophageal cancer cell lines and the expression levels of DACT1 and DACT2 were reversed by 5-Aza-Dc. Decreased mRNA and protein expression of DACT1 and DACT2 were observed in ESCC tumor tissues and were associated with the methylation status of transcription start site (TSS) region. The hypermethylation of CpG islands (CGI) shore region in DACT1 was observed both in tumor and corresponding adjacent tissues but wasn't related to the transcriptional inhibition of DACT1. The methylation status of TSS region in DACT1 and DACT2 and the protein expression of DACT2 were independently associated with ESCC patients' prognosis. CONCLUSIONS: The TSS region hypermethylation may be one of the main mechanisms for reduced expression of DACT1 and DACT2 in ESCC. The simultaneous methylation of DACT1 and DACT2 may play important roles in progression of ESCC and may serve as prognostic methylation biomarkers for ESCC patients.

Aberrant CpG Island Shore Region Methylation of CAV1 Is Associated with Tumor Progression and Poor Prognosis in Gastric Cardia Adenocarcinoma.

BACKGROUND AND AIMS: Caveolin-1 (CAV1) is a multifunctional scaffolding protein and plays an important role in tumorigenesis. However, the epigenetic changes of CAV1 in gastric cardia adenocarcinoma (GCA) have not been investigated so far. The purpose of this study was to clarify the contribution of critical CpG sites in CAV1 to progression/prognosis of GCA and to further elucidate the effect of critical CpG sites on the ectopic expression of ß-catenin in GCA. METHODS: Methylation-specific polymerase chain reaction (MSP) and bisulfite genomic sequencing (BGS) methods were, respectively, applied to examine the methylation status of CAV1. RT-PCR and immunohistochemistry methods were used to determine the mRNA and protein expression of CAV1 and ß-catenin. RESULTS: Decreased mRNA and protein expression of CAV1 were observed in GCA tumor tissues and were associated with hypermethylation of CpG island shore and transcription start site (TSS) regions in CAV1. Hypermethylation of the other two regions within CpG islands in CAV1 was observed both in tumor and corresponding adjacent tissues but was not related to the transcriptional inhibition of CAV1. The methylation status of CpG island shore region in CAV1 was associated with the ectopic expression of ß-catenin and was independently associated with survival in GCA patients. CONCLUSIONS: Hypermethylation of CpG island shore and TSS regions is cancer specific and is closely associated with reduced expression of CAV1. The CpG island shore methylation of CAV1 may play an important role in progression of GCA and may serve as a prognostic methylation biomarker for GCA patients.

[Case-control studies on double bundle posterior cruciate ligament reconstruction with remnant fiber preservation].

OBJECTIVE: To study the clinical efficacy of double bundle posterior cruciate ligament (PCL) reconstruction with remnant preservation. METHODS: From January 2007 to November 2011, 50 patients with PCL rupture met the inclusion criteria were divided into two groups: remnant preservation group (RP group) and remnant resection group (RR group). There were 19 males and 7 females in the RP group, ranging in age from 18 to 55 years, with a mean of (32.250 +/- 11.085) years old. The duration from injury to operation ranged from 2 to 66 months, with an average of (17.481 +/- 3.568) months. Among the RR group, 17 patients were male and 7 patients were female, ranging in age from 20 to 54 years old, with an average of (31.458 +/- 9.569) years. The duration from injury to operation ranged from 3 to 72 months, with a mean of (19.354 +/- 3.950) months. The patients in both groups suffered from instability of knee joint, got a positive result of posterior drawer test. In the RP group, the intercondylar notch remnant fiber, scar tissue and synovial were preserved in operation, only the free ligament in the intercondylar notch was resected. In the RR group, the remnant fiber, scar tissue and synovial tissue of adhesive parts were resected. In both groups, autologous semitendinosus and gracilis tendon double-bundle PCL reconstruction were carried out, the tibia was fixed with an absorbable interference screw with post-tie fixation, and the femur side was compositely fixed with absorbable interference screws and suspending fixation. Each patient received both subjective assessment (IKDC subjective evaluation, Lysholm scoring and Cincinnati rating) and objective clinical assessment (IKDC objective evaluation and Kneelax 3 tibia backward measurement) before operation and two years after operation. RESULTS: IKDC subjective evaluation: 92.167 +/- 4.177 in the RP group,which was higher than 87.542 +/- 5.687 in the RR group (P = 0.010). Lysholm scores: 90.917 +/- 4.413 in the RP group, which was higher than 87.083 +/- 5.149 in the RR group (P = 0.027). Cincinnati knee scores: 92.125 +/- 4.003 in the RP group, which was higher than 87.791 +/- 6.665 in the RR group (P = 0.027). IKDC objective evaluation:no significant statistical differences between RP group and RR group. Kneelax 3 assessment : tibia backward test with Kneelax 3 under 132 N showed no significant statistical difference between RP group and RR group, which were (3.958 +/- 0.693) mm and (4.029 +/- 0.846) mm respectively (P = 0.795). CONCLUSION: The study shows a significant advantage of remnant fiber preservation than remnant fiber resection in double-bundle PCL construction in terms of subjective knee function recovery after operation. There is no significant difference in postoperative knee stability.

A cholesterol tag at the N terminus of the relatively broad-spectrum fusion inhibitory peptide targets an earlier stage of fusion glycoprotein activation and increases the peptide's antiviral potency in vivo.

In previous work, we designed peptides that showed potent inhibition of Newcastle disease virus (NDV) and infectious bronchitis virus (IBV) infections in chicken embryos. In this study, we demonstrate that peptides modified with cholesterol or 3 U of polyethylene glycol (PEG3) conjugated to the peptides' N termini showed even more promising antiviral activities when tested in animal models. Both cholesterol- and cholesterol-PEG3-tagged peptides were able to protect chicken embryos from infection with different serotypes of NDV and IBV when administered 12 h prior to virus inoculation. In comparison, the untagged peptides required intervention closer to the time of viral inoculation to achieve a similar level of protection. Intramuscular injection of cholesterol-tagged peptide at 1.6 mg/kg 1 day before virus infection and then three times at 3-day intervals after viral inoculation protected 70% of the chickens from NDV infection. We further demonstrate that the cholesterol-tagged peptide has an in vivo half-life greater than that of untagged peptides. It also has the potential to cross the blood-brain barrier to enter the avian central nervous system (CNS). Finally, we show that the cholesterol-tagged peptide could play a role before the viral fusion peptide's insertion into the host cell and thereby target an earlier stage of fusion glycoprotein activation. Our findings are of importance for the further development of antivirals with broad-spectrum protective effects.

[Correlation of hypermethylation of TSP1 gene with TGF-beta1 level and T cell immunity in gastric cardia adenocarcinoma].

BACKGROUND AND OBJECTIVE: Thrombospondin-1(TSP1) is an inhibitor of angiogenesis and its promoter hypermethylation has been found resulting in gene silencing in some primary human carcinomas. This study was to investigate the promoter methylation of TSP1 and its correlation with TGF-beta1 level and T cell immunity in gastric cardia adenocarcinoma (GCA). METHODS: Methylation specific polymerase chain reaction (MSP) approach and immunohistochemistry method were used to examine the methylation status of the 5' CpG island and expression of TSP1 protein, respectively. The level of TGF-beta1 was measured by ELISA and T cell immunity of GCA by flow cytometry analysis. RESULTS: TSP1 methylation frequency was significantly higher in tumor specimens than in corresponding normal tissues (35.4% vs. 3.1%, P<0.001) and significantly higher in Stages III and IV tumor tissues than in Stages I and II tumor tissues (P<0.05). TSP1 protein expression was significantly lower in the tumor tissues than in corresponding normal tissues (P<0.05) and statistically correlated with its methylation status (P<0.01). The total level of TGF-beta1 was significantly higher in the GCA patients than in healthy controls(P<0.05) and significantly higher in Stages III and IV GCA patients than in Stages I and II GCA patients (P<0.05). The level of active TGF-beta1 was significantly higher in the GCA patients with hypermethylation of TSP1 than in the GCA patients without methylation of TSP1(P<0.05), but there was no statistical difference(P>0.05). The function of T cell immunity was significantly different between the GCA patients with hypermethylation of TSP1 and those without methylation of TSP1 (P<0.05). CONCLUSION: Promoter hypermethylation of TSP1 may play an important role in the development of GCA and reflect the biological behaviours of GCA.

[Association of single nucleotide polymorphism of MnSOD gene with carcinogenesis and development of esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the association of single nucleotide polymorphism (SNP) of manganese superoxide dismutase (MnSOD) gene with carcinogenesis and progression of esophageal squamous cell carcinoma. METHODS: The MnSOD9 T-->C SNP was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 103 patients with esophageal squamous cell carcinoma and 195 healthy controls. RESULTS: A significant difference was observed in the MnSOD allelotype distribution among esophageal squamous cell carcinomas and healthy controls (chi(2) = 4.645, P < 0.05). Individuals with the 9 C allele had a significantly higher risk to develop esophageal squamous cell carcinoma compared with those with the TT allele. The frequency of C allelotype among patients with lesions of different lengths ( 5 cm) was 16.3% and 36.7%, respectively. A significant difference was observed in the MnSOD allelotype distribution between patients with lesions of different lengths (chi(2) = 5.147, P < 0.05). No significant association of the MnSOD polymorphism at 9 T-->C with the tumor site, maximal length and clinical staging was found in esophageal squamous cell carcinoma. CONCLUSION: Single nucleotide polymorphism (SNP) of MnSOD gene may be correlated with the susceptibility and disease progression of esophageal squamous cell carcinoma, and may become a tumor marker for prediction of this cancer.

[Polymorphisms of thymidylate synthase gene and correlation of its protein expression to lymph node metastasis of esophageal squamous cell carcinoma].

BACKGROUND & OBJECTIVE: Thymidylate synthase (TS) is a key enzyme in DNA synthesis. The 28-bp tandem repeat in the 5'-untranslated region (UTR) of TS gene and the G/C single nucleotide polymorphism (SNP) of TS gene may modify the expression and activity of TS protein, therefore, may change the susceptibility and prognosis of tumors. This study was to explore the correlations of TS 5'-UTR polymorphism to lymph node metastasis of esophageal squamous cell carcinoma (ESCC) and the expression of TS protein. METHODS: Peripheral leucocyte DNA was extracted from 232 ESCC patients and 348 age-and gender-matched healthy controls. TS 5'-UTR tandem repeat and the G/C SNP genotype was detected by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP), respectively. TS expression in 51 specimens of ESCC was detected by SP immunohistochemistry. RESULTS: The frequencies of 3G/3G, 3G/3C, 3C/3C, 2R/3G, 2R/3C, 2R/2R, and other genotypes were 17.5%, 17.3%, 29.3%, 12.9%, 17.8%, 3.7%, and 1.5% in the healthy controls, and 16.0%, 16.0%, 29.3%, 13.8%, 17.6%, 4.3%, and 3.0% in the ESCC patients; whereas the frequencies of 3G, 3C, 2R, and other alleles were 32.8%, 47.0%, 19.5%, and 0.7% in the healthy controls, and 31.2%, 46.8%, 20.5%, and 1.5% in the ESCC patients, respectively. Compared with 3G/3G genotype, 2R/3G genotype significantly increased the risk of lymph node metastasis of ESCC [age and gender adjusted odds ratio (OR), 11.53; 95% confidence interval (CI), 2.67-49.74]. TS protein expression was significantly related to TS 5'-UTR genotype (P<0.05), but was not related to gender, age, lymph node metastasis and clinicopathologic stage. CONCLUSION: TS 5'-UTR tandem repeat and G/C SNP genotype, but not TS expression, might be a candidate molecular marker to predict lymph node metastasis of ESCC.