Inhibition of hepatitis B virus by a novel L-nucleoside,β-L-D4A and related analogues

AIM: To explore the inhibition of β-L-D4A on hepatitis B virus (HBV) in 2.2.15 cells derived from HepG2 cells transfected with HBV genome.METHODS: 2.2.15 cells were plated at a density of 5×10^4 per well in 12-well tissue culture plates, and treated with various concentrations of β-L-D4A for 6 days. In the end,5μl of medium was used for the estimation of HBsAg and HBeAg, the other medium was processed to obtain virions by a polyethlene glycol precipitation method. At the same time, intracellular DNA was also extracted and digested with HindⅢ. Both DNAs were subjected to Southern blot,hybridized with a ^32P-labeled HBV probe and autoradiophed.Intensity of the autoradiographic bands was quantitated by densitometric scans of computer and ED50 was calculated. Then Hybond-N membrane was washed and rehybridized with a ^32P-labeled mtDNA-specific probe, and effect of β-L-D4A on mitochondrial DNA was studied. 2.2.15 cells were also seeded in 24-well tissue culture plates,and cytotoxicity with different concentrations was examined by MTT method. ID50 was calculated. Structure-activity relationships between D2A and D4A were also studied as above.RESULTS: Autoradiographic bands were similar between supernatant and intracellular HBV DNA. Episomal HBV DNAwas inhibited in a dose-dependent manner. ED50 was 0.2μM. HBsAg or HBeAg was not apparently decreased, and inhibition of mitochondrial DNA was not obvious. The experiment of cytotoxicity gained ID50 at 200 μM.CONCLUSION: β-L-D4A possesses potent inhibitory effects on the replication of HBV in vitro with little cytotoxidty and mitochondrial toxicity, TI value is 1000. It is expected to be developed as a new clinically anti-HBV drug.

Significance of vascular endothelial growth factor expression and its correlation with inducible nitric oxide synthase in gastric cancer

AIM:To investigate the clinical significance of the expression of VEGF165mRNA and the correlation with vascular endothelial growth factor(VEGF) protein and inducible nitric oxide synthase (iNO) in human gastric cancer.METHODS:We tested VEGF165mRNA expression in 31 cases of resected gastric cancer specimens and normal paired gastric mucosae by RT-PCR.Total RNA was extracted with TRIzol reagents,transcribed into cDNA with gligo (dT15) priming,inner controlled with β-actin expression and agarose gel isolated after PCR.VEGF expression was quantitated with IS1000 imaging system.Meanwhile we also examined expression levels of VEGF protein and iNOS in 85 cases of gastric cancer.All paraffin-embedded samples were immunohistochimically stained by streptavidin-peroxidase method (SP).RESULTS:The mean expression of VEGF165mRNA in gastric cancer was 1.125±0.356,significantly higher than that of normal paired mucosea,which was 0.760±0.278.The data indicated that the expression level of VEGF165mRNA was well related to lymph node metastasis and TNM stages of UICC.The expression levels in patients with lymph node metastasis and without lhmph node metastasis were 1.219±0.377 and 0.927±0.205 respectively (p<0.05),The expression in stages Ⅰ,Ⅱ,Ⅲ,Ⅳ was 0.934±0.194,1.262±0.386 respectively (p<0.01).Further analysis showed the lymph node metastasis rate in the group with over-expression of VEGF was higher than that in the group with low expression of VEGF(83.3% vs 46.2%),and the ratio of stage Ⅲ+Ⅳ in the group with over-expression of VEGF was also higher than that in the group with low expression with VEGF (77.8% vs 33.8%)(p<0.05).The positive rates of expression of VEGF protein and iNOS in 85 cases of gastric cancer were 75.4% and 58.8% respectively,and 50.1% of the patients showed positive staining both for iNOS and VEGF,the correlation with the two tactors was significant (p=0.018).But more intensive analysis showed the immunoreactive grades of VEGF were not associated with that of iNOS.CONCLUSIONS:The expression of VEGF165mRNA is well related with lymph node metastasis and TNM stages of UICC in gastric cancer of gastric cancer.The relationship can be observed between the expression of VEGF and iNOS in gastric cancer.

Expression of tumor related gene NAG6 in gastric cancer and restriction fragment length polymorphism analysis

AIM: NAG6 gene is a novel tumor related gene identified recently. This study was designed to examine the expression of this gene in gastric cancer and corresponding normal tissues, and to investigate its role in the occurrence and development of gastric cancer, also to study if the genetic structure of NAG6 was altered in gastric cancer.METHODS: Reverse transcription-polymerase chain reaction (RT-PCR), Northern blot analysis and dot hybridization were used to compare the expression level of NAG6 gene in 42 cases of gastric cancer tissues with their corresponding normal tissues of the same patients respectively. In addition,restriction fragment length polymorphism (RFLP) analysis was adopted to study if the genetic structure of NAG6 was altered in gastric carcinomas.RESULTS: The expression of NAG6 in 57.1% gastric cancer tissues (25/42) was absent by RT-PCR analysis. The down-regulation rate of NAG6 in gastric cancer tissues was significantly higher than that in corresponding normal tissues(P<0.01). However no correlation between the down-regulation of NAG6 and lymph-node and/or distance metastasis was found in this study (P>0.05). Dot hybridization confirmed the results of RT-PCR. Furthermore,the results of EcoRI RFLP analysis of NAG6 gene demonstrated that 3 of 7 cases of gastric cancer showed loss of 5 kb fragment in comparison with their corresponding normal tissues.CONCLUSION: NAG6 gene is significantly down regulated in gastric cancer. The loss of genetic materials may be the cause of down-regulation of NAG6 expression. This seems to suggest that NAG6 may represent a candidate of putative tumor suppressor gene at 7q31-32 loci associated with gastric carcinoma. The down-regulation of this gene may play a role in occurrence and development of this disease, however it may not be associated with lymph node and/or distance metastasis.