OBJECTIVETo investigate single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-8, -10 genes in Zhejiang Han nationality in China.

METHODSPCR, denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the 2nd-5th exons of caspase-10 gene, the 8th-10th exons of caspase-8 and their flanking sequences. Expectation Maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium (LD) test.

RESULTS(1) Two SNPs, A2823G and A12799G, were identified in caspase-10 gene, located in exon 2 and exon 5 respectively. A12799G was newly found with low informativeness. Three SNPs were identified in caspase-8 gene; A43466G, G51484A and G52951A were located in exon 8, exon 9 and intron 9, respectively. They do not change the primary structure of the encoded protein. (2) Linkage equilibrium was observed between A2823G in caspase-10 gene and the three sites in caspase-8 gene. A43466G and G52951A, and G51484A and G52951A in caspase-8 gene were also in linkage equilibrium. Their coefficients of disequilibrium were near 0. Whereas strong linkage disequilibrium was observed between A43466G and G51484A, because its coefficient of disequilibrium was near 1. (3) A total of 11 haplotypes were estimated within A2823G in caspase-10 gene and three sites in caspase-8 gene. A-2823/A-43466/G-51484/G-52951 was the main haplotype with a frequency of 0.3811. A-2823/A-43466/G-51484/A-52951 was the second haplotype with a frequency of 0.2536. The polymorphism information content of their haplotypes was 0.7106.

CONCLUSIONThe SNPs of caspase-8, -10 genes in Han Chinese of Zhejiang could be parsed into at least three different haplotype blocks. The polymorphism information content can be improved by using haplotype analysis of several SNPs.

Alleles ; Base Sequence ; Caspase 10 ; genetics ; Caspase 8 ; genetics ; China ; ethnology ; DNA ; analysis ; Ethnic Groups ; genetics ; Gene Frequency ; Haplotypes ; Humans ; Polymorphism, Single Nucleotide

OBJECTIVEThe loss of caspase-8 expression correlates with unfavorable survival outcomes in neuroblastoma (NB). Caspase-8 gene inactivation is caused by methylation. This study aimed to explore the effect of the demethylation agent 5-azacytidine on caspase-8 expression and whether 5-azacytidine can increase the sensitivity of chemotherapy drug doxorubicin to NB cells.

METHODSCaspase-8 mRNA expression in NB cell lines (SH-SY5Y cells) was examined by RT-PCR before and after 5-azacytidine treatment. Survival rates of SH-SY5Y cells were detected using MTT analysis and compared among the doxorubicin alone treatment, 5-azacytidine along with doxorubicin treatment, and caspase-8 inhibitor+5-azacytidine+doxorubicin treatment groups.

RESULTSCaspase-8 mRNA was not expressed in untreated SH-SY5Y cell lines. Caspase-8 mRNA expression in SH-SY5Y cells was detectable 3 days after 5-azacytidine treatment, and increased significantly 5 days after 5-azacytidine treatment (P < 0.05). Survival rates of SH-SY5Y cells treated with 5-azacytidine along with different concentrations of doxorubicin (0.05, 0.1,0.25, 0.5 microg/mL) were (77.61 +/- 7.30)%, (57.35 +/- 6.64)%, (46.25 +/- 4.46)% and (35.59 +/- 5.12)%, respectively, which were significantly lower than those treated with doxorubicin alone (94.89 +/- 4.15%, 80.60 +/- 8.50%, 64.48 +/- 4.92% and 52.32 +/- 6.71%) (P < 0.01). Caspase-8 inhibitor pretreatment resulted in an increased survival rate of SH-SY5Y cells (92.95 +/- 3.48%, 78.39 +/- 4.28 %, 62.31 +/- 6.50% and 49.92 +/- 5.77%) compared with the 5-azacytidine+doxorubicin treatment group.

CONCLUSIONS5-azacytidine may enhance anti-tumor efficacy of doxorubicin to NB cell lines, possibly through an up-regulation of caspase-8 mRNA expression.

Antineoplastic Agents ; pharmacology ; Azacitidine ; pharmacology ; Caspase 8 ; genetics ; Caspase Inhibitors ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Drug Synergism ; Humans ; RNA, Messenger ; analysis

BACKGROUNDAngiopoietin-1 (Ang-1) is an endothelial-specific growth factor that can promote angiogenesis. Studies demonstrated that Ang-1 can inhibit apoptosis of umbilical endothelial cells, but so far little is known about its effects on apoptosis of microvascular endothelial cells. With the apoptotic model of murine-cerebral-derived microvascular endothelial cells (bEnd.3) induced by serum-free culture, we attempted to clarify the molecular mechanism of bEnd.3 apoptosis, particularly its relation to cytochrome C (Cyt C).

METHODSThe cultured microvascular endothelial cell strain, bEnd.3 cell, was employed. An apoptotic model of bEnd.3 was established by serum-free culture. Flow cytometry after Annexin labeling and PI staining were used to assess the apoptotic effects of Ang-1 on bEnd.3, and the expression of Bax/Bcl-2, caspase 8, caspase 3, and Cyt C were detected with Western blotting and ELISA.

RESULTSThe apoptotic rate of bEnd.3 cells after stimulation with Ang-1 (100 ng/L) in serum-free medium was significantly higher than that in control group. Ang-1 inhibited early-stage apoptosis more than late-stage apoptosis provided by propidium iodide (PI) and AnnexinV double staining. The inhibition of Ang-1 on bEnd.3 cell apoptosis was strengthened with the increase in concentration (0 - 400 ng/ml). Ang-1 could decrease the expression of Bax, caspase3 and 8, and increase that of Bcl-2. The results of ELISA indicated that Ang-1 significantly decreased CytC content in cytoplasm and increase that in mitochondria.

CONCLUSIONSAng-1 could inhibit bEnd.3 apoptosis induced by serum-free medium culture. The apoptosis was associated with decreased Bax expression, increased Bcl-2 expression, which result in Cyt C transferring from mitochondria to cytoplasm, and then caspases activation are reduced and cell apoptosis is suppressed.

Angiopoietin-1 ; pharmacology ; Animals ; Annexin A5 ; analysis ; Apoptosis ; drug effects ; Caspase 3 ; Caspase 8 ; Caspases ; analysis ; Cells, Cultured ; Cytochromes c ; physiology ; Endothelial Cells ; cytology ; drug effects ; Mice ; Microcirculation ; drug effects ; bcl-2-Associated X Protein ; analysis

OBJECTIVETo explore the expression of CASP8 and its clinical significance in nasopharyngeal carcinoma (NPC).

METHODSThe differentially expressed genes between pooled NPC tissues and non-cancerous nasopharyngeal (NP) tissues were screened using 8 microarrays. Real-time PCR and immunohistochemistry were used to validate the detection results of CASP8 expression in NPC, and the correlation of CASP8 expression to the clinical characteristics was analyzed in the NPC cases.

RESULTSReal-time PCR confirmed a reduced expression of CASP8 mRNA level in NPC tissues (P<0.0001), which was consistent with the microarray data. Immunohistochemistry indicated that CASP8 protein expression was also significantly down-regulated in NPC tissue compared to that in the non-cancerous nasopharyngeal tissues (P=0.02). The reduction of CASP8 expression was inversely correlated to lymph node metastasis (P=0.002) and the clinical stages (P=0.026) of NPC.

CONCLUSIONDecreased CASP8 expression is an unfavorable factor that promotes the development and progression of NPC.

Caspase 8 ; genetics ; metabolism ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis

OBJECTIVETo study the effects of diallyl disulfide (DADS) on apoptosis of human leukemia K562 cells and possible mechanisms.

METHODSThe morphologic changes of leukemia K562 cells after DADS treatment were observed by Hoechst 33258 staining. Cell apoptosis rates after different concentrations and different durations of DADS treatment were determined by flow cytometry. Fas, FasL and caspase-8 mRNA expression was estimated by reverse transcription-polymerase chain reaction (RT-PCR) 48 hrs after DADS treatment.

RESULTSThe characteristics of apoptosis in K562 cells induced by DADS were observed. After 24 hrs of DADS treatment, the apoptosis rate of K562 cells increased from (11.60 ± 0.83)% at the concentration of 10 mg/L to (37.94 ± 0.87)% at the concentration of 40 mg/L. The apoptosis rate of K562 cells increased after 40 mg/L DADS with the increasing time from (37.94 ± 0.87)% (24 hrs) to (47.02 ± 0.66)% (72 hrs). Expression of Fas and caspase-8 mRNA increased, while FasL mRNA expression decreased significantly 48 hrs after DADS treatment compared with the control group (P<0.05).

CONCLUSIONSDADS can induce apoptosis of human leukemia K562 cells in a time- and concentration-dependent manner, possibly through increasing Fas and caspase-8 expression and decreasing FasL expression.

Allyl Compounds ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bisbenzimidazole ; Caspase 8 ; genetics ; Disulfides ; pharmacology ; Fas Ligand Protein ; genetics ; Flow Cytometry ; Humans ; K562 Cells ; RNA, Messenger ; analysis ; fas Receptor ; genetics

OBJECTIVE: To provide the evidence of the relationship between brain injury and the time of injury. METHODS: Rats were contused on brain by fluid percussion, then were killed after injury for 15 min, 30 min, 1,3,6,12 h, and 1,4,7,14 d respectively. The expression of caspase-8 were detected by immunohistochemical technology on rat brain section and the results were assessed by image analysis system in the cerebral cortex, thalamus, and hippocampus. RESULTS: The expression of caspase-8 in cortex and hippocampus could be detected in 30 min after injury, increased significantly in 3h, reached apex in 1d after injury, remained 4d before decreased. In addition, the expression of caspase-8 can be detected in 1h after injury and reached apex in 1d after injury, and remained 4d then reduced. CONCLUSION It seems that the expression of caspase-8 should be a useful target for diagnosis of early brain injury.
Animals ; Brain Injuries/pathology* ; Caspase 8/analysis* ; Cerebral Cortex/pathology* ; Disease Models, Animal ; Head Injuries, Closed/pathology* ; Hippocampus/pathology* ; Image Processing, Computer-Assisted ; Immunohistochemistry ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Staining and Labeling ; Thalamus/pathology* ; Time Factors