Induction of apoptosis in human Hep3B hepatoma cells by norcantharidin through a p53 independent pathway via TRAIL/DR5 signal transduction / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory activities of norcantharidin (NCTD), a demethylated analogue of cantharidin, on Hep3B cells (a human hepatoma cell line) with deficiency of p53.</p><p><b>METHODS</b>The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined.</p><p><b>RESULTS</b>NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G(2)M phase arrest occurs at low concentration ([Symbol: see text] 25 μmol/L) but G(0)G(1) phase arrest at high concentration (50 μmol/L). The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation.</p><p><b>CONCLUSION</b>NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G(2)M or G(0)G(1) phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway.</p>

A novel microsatellite within intron 8 in caspase10 gene in Chinese of Han nationality / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate polymorphisms in the coding exons and their splicing areas of caspase10 gene (CASP10) in Chinese of Han nationality.</p><p><b>METHODS</b>Polymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC), direct sequencing and clonal sequencing were used to analyze the exon 9 and its flanking sequences.</p><p><b>RESULTS</b>In 70 blood samples of Chinese subjects researched the known single nucleotide polymorphisms (SNPs) in the exon 9 of CASP10 gene published in dbSNP of NCBI were not identified. We found a short sequence with more than ten continuous and repeated T nucleotides within the intron 8 near the exon 9 and the length of repeated sequence nucleotides T was different in samples. Clonal sequencing analysis indicated that it was a microsatellite of single nucleotide-repeated sequence. The recurrence and specificity of same result was further confirmed by PCR with high fidelity polymerase and sequencing. Furthermore all of 70 blood samples detected by DHPLC showed heterozygous profiles. We named it as IVS8-13(T)n temperately.</p><p><b>CONCLUSION</b>Our research suggested that the site be a microsatellite of single nucleotide-repeated sequence. Meanwhile, our data further showed that it was quite different from that by querying SNP database of non-Chinese population in NCBI, in the same region a SNP of type of insertion deletion existed. It could be inferred that the difference between the populations might be associated with the genetic characteristics of ethnics. The significance of the microsatellite in CASP10 gene in Chinese of Han nationality remains to be elucidated.</p>

Haplotypes of four single nucleotide polymorphisms in caspase-8, -10 genes in Han nationality of Zhejiang province in China / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-8, -10 genes in Zhejiang Han nationality in China.</p><p><b>METHODS</b>PCR, 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>The 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.</p>

Study on the Apoptosis and its Mediators in the Placentas of Preclampsia using cDNA Expression Array / 대한산부인과학회잡지

OBJECTIVE: The present study was designed to examine whether preeclampsia is associated with an increase in placental apoptosis and to detect differential expression of mediators involved in apoptosis. METHODS: Placental tissues from 10 cases of preeclampsia and 16 cases of control were analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. And cDNA expression array assay was performed for detecting mediators that were involved in placental apoptosis. RESULTS: In TUNEL staining, number of apoptotic nuclei were increased in placenta of preeclampsia. The expression of apoptotic signals was limited to trophoblasts. As a result of cDNA expression array assay, expressions of caspase-10, tumor necrosis factor receptor-1 (TNFR-1), and death domain receptor-3 (DDR-3) were increased and expression of cell division protein kinase-9 (CDK-9), cyclin A, insulin-like growth factor-1 (IGF-1), and insulin-like growth factor-binding protein-3 (IGFBP-3) were decreased in the placenta of preeclampsia. The expression of caspase-10, DDR-3, and IGFBP-3 between preeclampsia and normal revealed significantly different (p<0.05). CONCLUSION: Apoptosis was significantly increased in placenta of preeclampsia and various mediators were involved in apoptotic mechanism. Therefore, it was suggested that apoptosis might play a role in the pathophysiologic mechanisms of preeclampsia.

The Expression of Death Domain Receptor-3, Caspase-10, Insulin-Like Growth Factor Binding Protein-3 in the Placentas of Preeclampsia / 대한주산의학회잡지

OBJECTIVE: To determine that preeclampsia is associated with differential expression of death domain receptor-3(DDR-3), caspase-10, and insulin-like growth factor binding protein-3(IGFBP-3). METHODS: Placenta were collected from 10 preeclampsias and from 15 normal pregnancies and the samples were immediately frozen and stored at -70degrees C. Total mRNA was extracted from placental tissue and then analysis of the expressions of DDR-3, caspase-10, and IGFBP-3 were performed by quantitative RT-PCR. RESULTS: Quantitative RT-PCR analysis demonstrated significantly increased DDR-3 and caspase-10 expression levels and significantly decreased IGFBP-3 expression level in the placenta of preeclampsia compared with normal pregnancy. CONCLUSION: Placental apoptosis is associated with DDR-3, caspase-10, and IGFBP-3. These results may be one of possible mechanisms in the placental apoptosis of preeclampsia.