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.
Apoptosis ; Caspase 10* ; Insulin-Like Growth Factor Binding Protein 3 ; Placenta* ; Pre-Eclampsia* ; Pregnancy ; RNA, Messenger

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

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.
Apoptosis* ; Caspase 10 ; Cell Division ; Cyclin A ; Deoxyuridine ; DNA, Complementary* ; In Situ Nick-End Labeling ; Insulin-Like Growth Factor Binding Protein 3 ; Placenta* ; Pre-Eclampsia ; Trophoblasts ; Tumor Necrosis Factor-alpha

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1β, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.
Animals ; Caspase 3 ; Cell Proliferation ; Cisplatin ; Immunotherapy ; Interleukin-10 ; Interleukin-6 ; Korea ; Lung ; Mice ; Models, Animal ; Molecular Biology ; Neoplasm Metastasis ; Tumor Suppressor Protein p53 ; Vascular Endothelial Growth Factor A

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4⁺/CD8⁺ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.
Animals ; Apoptosis ; Boraginaceae ; Carcinoma, Hepatocellular ; Caspase 3 ; Cytokines ; Interleukin-10 ; Interleukin-6 ; Interleukins ; Lamiaceae ; Mice ; STAT3 Transcription Factor ; Therapeutic Uses ; Tumor Microenvironment

BACKGROUND/OBJECTIVES: Resveratrol, a natural polyphenol, has multiple functions in cellular responses including apoptosis, survival, and differentiation. It also participates in the regulation of inflammatory response and oxidative stress. MicroRNA-Let-7A (miR-Let7A), known as a tumor suppressor miRNA, was recently reported to play a crucial role in both inflammation and apoptosis. Therefore, we examined involvement of miR-Let7A in the modulation of inflammation and cell survival/apoptosis regulated by resveratrol. MATERIALS/METHODS: mRNA expression of pro-/anti-inflammatory cytokines and sirtuin 1 (SIRT1), and protein expression of apoptosis signal-regulating kinase 1 (ASK1), p-ASK1, and caspase-3 and cleaved caspase-3 were measured, and cell viability and Hoechst/PI staining for apoptosis were observed in Lipopolysaccharide (LPS)-stimulated human THP-1 macrophages with the treatment of resveratrol and/or miR-Let7A overexpression. RESULTS: Pre-treatment with resveratrol (25-200 µM) resulted in significant recovery of the reduced cell viabilities under LPS-induced inflammatory condition and in markedly increased expression of miR-Let7A in non-stimulated or LPS-stimulated cells. Increased mRNA levels of tumor necrosis factor-α and interleukin (IL)-6 induced by LPS were significantly attenuated, and decreased levels of IL-10 and brain-derived neurotrophic factor were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. Decreased expression of IL-4 mRNA by LPS stimulation was also significantly increased by miR-Let7A overexpression co-treated with resveratrol. In addition, decreased SIRT1 mRNA levels, and increased p-ASK1 levels and PI-positive cells by LPS stimulation were significantly restored by resveratrol and miR-Let7A overexpression, respectively, or in combination. CONCLUSIONS: miR-Let7A may be involved in the inflammatory response and cell survival/apoptosis modulated by resveratrol in human THP-1 macrophages.
Apoptosis ; Brain-Derived Neurotrophic Factor ; Caspase 3 ; Cell Survival ; Cytokines ; Humans ; Inflammation ; Interleukin-10 ; Interleukin-4 ; Interleukins ; Macrophages* ; MAP Kinase Kinase Kinase 5 ; MicroRNAs ; Necrosis ; Oxidative Stress ; RNA, Messenger ; Sirtuin 1

Interleukin (IL)-20, a proinflammatory cytokine of the IL-10 family, is involved in acute and chronic renal failure. The aim of this study was to elucidate the role of IL-20 during diabetic nephropathy development. We found that IL-20 and its receptor IL-20R1 were upregulated in the kidneys of mice and rats with STZ-induced diabetes. In vitro, IL-20 induced MMP-9, MCP-1, TGF-β1 and VEGF expression in podocytes. IL-20 was upregulated by hydrogen peroxide, high-dose glucose and TGF-β1. In addition, IL-20 induced apoptosis in podocytes by activating caspase-8. In STZ-induced early diabetic nephropathy, IL-20R1-deficient mice had lower blood glucose and serum BUN levels and a smaller glomerular area than did wild-type controls. Anti-IL-20 monoclonal antibody (7E) treatment reduced blood glucose and the glomerular area and improved renal functions in mice in the early stage of STZ-induced diabetic nephropathy. ELISA showed that the serum IL-20 level was higher in patients with diabetes mellitus than in healthy controls. The findings of this study suggest that IL-20 induces cell apoptosis of podocytes and plays a role in the pathogenesis of early diabetic nephropathy.
Animals ; Apoptosis ; Blood Glucose ; Caspase 8 ; Diabetes Mellitus ; Diabetic Nephropathies* ; Enzyme-Linked Immunosorbent Assay ; Glucose ; Humans ; Hydrogen Peroxide ; In Vitro Techniques ; Interleukin-10 ; Interleukins ; Kidney ; Kidney Failure, Chronic ; Mice ; Podocytes* ; Rats ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate polymorphisms in the coding exons and their splicing areas of caspase10 gene (CASP10) in Chinese of Han nationality.

METHODSPolymerase 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.

RESULTSIn 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.

CONCLUSIONOur 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.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Caspase 10 ; Caspases ; genetics ; China ; ethnology ; Ethnic Groups ; Genetics, Population ; Humans ; Introns ; genetics ; Microsatellite Repeats ; genetics ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVE: To investigate the effects of Shenfu Injection (, SFI) on endothelial damage in a porcine model of hemorrhagic shock (HS). METHODS: After being bled to a mean arterial pressure of 40±3 mm Hg and held for 60 min, 32 pigs were treated with a venous injection of either shed blood (transfusion group), shed blood and saline (saline group), shed blood and SFI (SFI group) or without resuscitation (sham group). Venous blood samples were collected and analyzed at baseline and 0, 1, 2, 4, and 6 h after HS. Tumor necrosis factor-α (TNF-α), serum interleuking (IL)-6, and IL-10 levels were measured by enzyme-linked immunosorbent assay (ELISA); expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule 1 (ICAM -1), von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1) and Bcl-2, Bax, and caspase-3 proteins were determined by Western blot. RESULTS: The serum level of TNF-α in the SFI group was significantly lower than in the other groups at 0, 1, and 2 h after HS, while the level of IL-6 was lower at 4 and 6 h compared with the saline group (P<0.01 or P<0.05). The concentration of serum IL-10 was significantly higher in the SFI group than in the other groups at 0, 1, 4, and 6 h after HS (P<0.01). Western blot and immunohistochemistry of vascular tissue showed that the expression of caspase-3 was downregulated, and that of Bcl-2 and Bax was upregulated in the SFI group compared to other groups (P<0.05). CONCLUSION SFI attenuated endothelial injury in the porcine model of HS by inhibiting cell apoptosis, suppressing the formation of proinflammatory cytokines, and reducing endothelial activation.
Animals ; Caspase 3/metabolism* ; Drugs, Chinese Herbal ; Interleukin-10 ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Shock, Hemorrhagic/drug therapy* ; Swine ; Tumor Necrosis Factor-alpha/metabolism* ; bcl-2-Associated X Protein/metabolism*

OBJECTIVETo investigate the inhibitory activities of norcantharidin (NCTD), a demethylated analogue of cantharidin, on Hep3B cells (a human hepatoma cell line) with deficiency of p53.

METHODSThe 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.

RESULTSNCTD 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.

CONCLUSIONNCTD 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.

Antibodies, Neoplasm ; pharmacology ; Antibodies, Neutralizing ; pharmacology ; Apoptosis ; drug effects ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Caspase 10 ; metabolism ; Caspase 3 ; metabolism ; Caspase Inhibitors ; pharmacology ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Fragmentation ; drug effects ; Humans ; Immunohistochemistry ; Liver Neoplasms ; enzymology ; pathology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Signal Transduction ; drug effects ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Tumor Suppressor Protein p53 ; metabolism