The paracaspase MALT1 is essential for the activation of NF-κB in response to T cell receptor (TCR) stimulation. It recruits downstream TRAF6 and activates the E3 ligase activity of TRAF6 to polyubiquitinate several targets, which ultimately leads to NF-κB activation. Here we identified ubiquitin-specific protease 2a (USP2a) as a MALT1-associated protein by biochemical affinity purification. Endogenous USP2a constitutively interacted with TRAF6, but dynamically interacted with MALT1 and CARMA1 in a stimulation-dependent manner. RNA interference (RNAi)-mediated silencing of USP2a attenuated TCR-induced NF-κB activation and production of interleukin-2 (IL-2). In addition, the ubiquitination of MALT1 and TRAF6 were both suppressed by USP2a knockdown. By knockdown and reconstitution assays, we found that USP2a mediated the interaction between MALT1 and TRAF6 in a catalytic activity-dependent manner. Furthermore, USP2a deSUMOylated TRAF6. Our findings implicate that USP2a plays an important role in TCR signaling by deSUMOylating TRAF6 and mediating TRAF6-MALT1 interaction.
Caspases ; metabolism ; Endopeptidases ; deficiency ; genetics ; metabolism ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Interleukin-2 ; biosynthesis ; Jurkat Cells ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; NF-kappa B ; metabolism ; Neoplasm Proteins ; metabolism ; Receptors, Antigen, T-Cell ; metabolism ; Signal Transduction ; Sumoylation ; TNF Receptor-Associated Factor 6 ; metabolism

Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities.
Amino Acid Chloromethyl Ketones ; pharmacology ; Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Caspase Inhibitors ; Caspases ; metabolism ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Cytosol ; drug effects ; metabolism ; Enediynes ; pharmacology ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; biosynthesis ; genetics

OBJECTIVETo study the expression of API2-MALT1 mRNA in mucosa-associated lymphoid tissue (MALT) lymphoma, extranodal diffuse large B-cell lymphoma (DLBCL) and Hashimoto's thyroiditis, to investigate the expression pattern of API2-MALT1 variants, and to correlate the findings with the clinicopathologic features and prognosis.

METHODSSixty-two cases of MALT lymphoma (10 from lung, 31 from stomach, 9 from intestine and 12 from thyroid), 32 cases of extranodal DLBCL (16 from stomach, 13 from intestine and 3 from thyroid), 8 cases of Hashimoto's thyroiditis and 5 cases of reactive lymph nodes hyperplasia as negative controls were collected. The expression of API2-MALT1 mRNA was studied in all cases by reverse transcriptase (RT)-polymerase chain reaction (PCR) and nested PCR. The 94 cases of lymphoma were subdivided into API2-MALT1-positive and API2-MALT1-negative groups. Among the patients, 78 were followed up for 6 to 120 months. The differences in clinicopathologic features and prognosis between the two groups were analyzed.

RESULTSAPI2-MALT1 transcripts were detected in 39 of the 94 lymphoma cases (with 28 cases being MALT lymphoma and 11 cases being extranodal DLBCL). mRNA expression was not detected in all cases of Hashimoto's thyroiditis and the negative controls. Two fusion gene variants, A1446-M1123 and A1446-M814 were found, and A1446-M1123 expression was more common. As for MALT lymphoma cases, the frequency of the fusion gene expression was lower in thyroid, when compared with that in lung, stomach and intestine. API2-MALT1-positive cases had tumors in an earlier stage with milder infiltration of cancer cells, lower relapse rate, and higher five-year survival rate.

CONCLUSIONSThe expression of API2-MALT1 mRNA can be detected in both MALT lymphoma and extranodal DLBCL, but not in Hashimoto's thyroiditis. These cases tend to show a more indolent clinical course and better survival. The frequency of t (11; 18) (q21; q21) correlates with the primary sites of MALT lymphoma. The higher incidence of breakpoint at 1123 bp of MALT1 gene in Chinese people may be due to geographical variation.

Caspases ; biosynthesis ; genetics ; Female ; Follow-Up Studies ; Genetic Variation ; Hashimoto Disease ; metabolism ; Humans ; Lymphoma, B-Cell, Marginal Zone ; metabolism ; pathology ; Lymphoma, Large B-Cell, Diffuse ; metabolism ; pathology ; Male ; Middle Aged ; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ; Neoplasm Invasiveness ; Neoplasm Proteins ; biosynthesis ; genetics ; Neoplasm Staging ; Oncogene Proteins, Fusion ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Survival Rate

Interleukin-1beta (IL-1beta) is a pivotal proinflammatory cytokine. To investigate the mechanism of IL-1beta-induced cell death in human malignant melanoma A375-S2 cells, MTT assay, photomicroscopical observation, DNA agarose gel electrophoresis, radioimmunoassay and Western blot analysis were carried out. IL-1beta did not only induce nuclear condensation and DNA fragmentation, but also increased degradation of two substrates of caspase-3, poly ADP-ribose polymerase (PARP) and inhibitor of caspase-activated DNase (ICAD). Simultaneously, release of precursor of IL-1beta (pro-IL-1beta) and endogenous IL-1beta production were involved in the apoptotic process. IL-1beta enhanced the ratio of Bax/Bcl-2 and Bax/Bcl-xL expression and up-regulated apoptosis inducing factor (AIF) expression, which required the activation of downstream caspases. These results suggest that IL-1beta induces endogenous IL-1beta production, enhances cleavage of caspase downstream substrates and promotes mitochondria mediated apoptosis in A375-S2 cells.
Apoptosis/*drug effects ; Blotting, Western ; Caspase 1/metabolism ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Comparative Study ; DNA Fragmentation/drug effects ; Deoxyribonucleases/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation/drug effects ; Humans ; Interleukin-1/biosynthesis/*pharmacology/physiology ; Interleukin-6/pharmacology ; Lymphotoxin/pharmacology ; Melanoma/metabolism/pathology/physiopathology ; Mitochondria/*physiology ; Poly(ADP-ribose) Polymerases/metabolism ; Proto-Oncogene Proteins c-bcl-2/biosynthesis ; Time Factors

OBJECTIVETo study the mechanism of injury of cortical nerve cell in the newborn with hypoxia/ischemia brain damage (HIBD), and the neuroprotective effect of Radix Astragali (RA).

METHODSNeonatal HIBD model rats were established and divided into the sham group, the model group and the RA group. Brain of rats obtained at different time points after HIBD to conduct histopathological examination, neuron death rate count, as well as determination of caspase-3 (cysteinyl aspartate-specific proteinase-3) protein mRNA expression in cerebral cortex by immunohistochemistry, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) respectively.

RESULTSIn the model group, caspase-3 mRNA and protein showed an increase at 6 hrs, reached the peak at 24 hrs, and decreased at 48 hrs after HIBD, on the 5th and 7th day restored to baseline level. After being treated by RA, the neuron death rate of ligated side was obviously reduced, caspase-3 mRNA and protein expression peak value decreased by 45% (mRNA) and 40% - 43% (protein).

CONCLUSIONRA shows markedly neuron protection in immature brain cortex after HIBD, which is related with the inhibition on caspase-3 expression.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Astragalus membranaceus ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Cell Survival ; Cerebral Cortex ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; metabolism ; pathology ; Male ; Neurons ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the mechanisms of capsaicin-induced apoptosis of human melanoma A375-S2 cells.

METHODSMTT assay, fluorescence microscopy, DNA agarose gel electrophoresis, flow cytometry and Western blot analysis were carried out to assess the morphological and biochemical changes of A375-S2 cells after capsaicin treatment.

RESULTSCapsaicin induced A375-S2 cell death in a time- and dose-dependent manner. Sub-diploid peak was seen at 24 h after 250 micromol/L capsaicin treatment, and apoptotic bodies and DNA ladder were observed at 36 h after capsaicin treatment. The expression of inhibitor of caspase activated DNase (ICAD) was reduced with the lapse of time.

CONCLUSIONCapsaicin induces A375-S2 cell apoptosis and down-regulation of ICAD contributes to this process.

Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; biosynthesis ; Capsaicin ; pharmacology ; Caspases ; metabolism ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Melanoma ; pathology ; Signal Transduction ; Skin Neoplasms ; pathology

OBJECTIVETo investigate the effect of Astragalus injection (AI) on left ventricular remodeling and the expression of apoptotic gene caspase-3 in rats after myocardial infarction (MI).

METHODSThe MI model was established. The experimental animals were divided into 4 groups, Group I (the sham-operated group), Group II (the sham-operated plus AI group), Group III (the model group), Group IV (the model plus AI group). Animals in the II and IV group were intraperitoneally injected with 2 ml AI once a day after operation, while animals in the I and III group were treated with normal saline of equal volume. After treated for 4 weeks successively, the structural change of left ventricle and the level of oxyproline in myocardium were observed, and expression of caspase-3 was determined by immunohistochemical method.

RESULTSAs compared with Group Ill, the ultrastructure of myocardium and indexes of left ventricular remodeling were improved, the myocardial content of oxyproline decreased (P < 0.05), the caspase-3 positive cells reduced and caspase-3 mRNA expression significantly down-regulated (P < 0.05).

CONCLUSIONAI can improve left ventricular remodeling, inhibit apoptosis by down-regulate the expression of apoptotic gene caspase-3 in rats after MI.

Animals ; Astragalus membranaceus ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Injections ; Male ; Myocardial Infarction ; metabolism ; pathology ; physiopathology ; Myocardium ; ultrastructure ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Ventricular Remodeling ; drug effects

OBJECTIVETo explore the inhibitory effect of Fuzheng Yiliu Granule (FYG) on growth and apoptosis of H22 tumor cell, and expressions of p53 and Caspase-3 gene.

METHODSThe tumor inhibitory rate of FYG on H22 tumor cell line was observed in vivo, cell apoptosis rate and cell cycle were determined by flow cytometry (FCM), and expressions of wild type p53 and Capase-3 mRNA were determined by RT-PCR.

RESULTSFYG could inhibit the growth of H22 tumor cell at the dose of 12g/kg, 24g/kg, the maximal inhibitory rate up to 51.24% (P < 0.01). By FCM, it was shown that FYG could significantly enhance apoptosis rate of cell line H22, with the peak reached 15.84% (P < 0.01), and cause the tumor cell cycle being blocked at G0/G1 phase, with decrease of cells in S phase. RT-PCR illustrated that FYG could significantly up-regulate the level of p53 and Caspase-3 mRNA expression.

CONCLUSIONFYG can significantly inhibit the growth of tumor cell in mice, its anti-tumor mechanisms may relate to the cell apoptosis induction, cell cycle regulation and wild type p53 and capase-3 gene expression enhancing.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Drugs, Chinese Herbal ; pharmacology ; Liver Neoplasms ; metabolism ; pathology ; Male ; Mice ; Neoplasm Transplantation ; Random Allocation ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVE: To determine the effects of sodiun aescinate on Bcl-2 and Caspase-3 protein expression and neuronal apoptosis after focal cerebral ischemia reperfusion injury in rats. METHODS: One hundred male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) and reperfusion. The rats were divided randomly into 4 groups: sham-operated group and MCAO and reperfusion model groups which were randomly divided into control group, saline group, and sodium aescinate group. The immunohistochemistry staining and microscope image were used to observe the dynamic Bcl-2 and Caspase-3 protein expression in the ischemic penumbra after the reperfusion. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining was performed for the detection of apoptosis. RESULTS: Bcl-2 protein expression in the sodium aescinate group was significantly higher than that of the saline group and control group (P < 0.05). While Caspase-3 protein expression in the sodium aescinate group was then compared with those of the saline group and control group, and showed the difference was significant (P < 0.05). Compared with the saline group and control group, the number of apoptotic cells in the sodium aescinate group was significantly reduced (P < 0.01). CONCLUSION Sodium aescinate increases Bcl-2 protein expression and decreases Caspase-3 protein expression,through which it can protect the ischemia brain on reperfusion injury.
Alginates ; pharmacology ; Animals ; Apoptosis ; drug effects ; Brain Ischemia ; metabolism ; pathology ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Glucuronic Acid ; pharmacology ; Hexuronic Acids ; pharmacology ; Male ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; pathology ; Saponins ; pharmacology

OBJECTIVE: To explore the effect of simvastatin on the cell cycle and caspase-3 expression in human omental preadipocytes. METHODS: The preadipocytes were randomly divided into a blank group, a 10(-5) mol/L simvastatin group, and a 10(-4) mol/L simvastatin group. Each group was incubated with different concentrations of simvastatin for 48 hours. MTT method was used to analyze the effect of simvastatin on the proliferation. Distribution of the cell cycle was measured by flow cytometric. Caspase-3 expression was examined by cyto- immunochemistry. RESULTS: After being induced to differentiate for 16 days the human omental preadipocytes developed to mature adipocyte penotypes with lipid droplet. Simvastatin 10(-4) mol/L had significant anti-proliferation effect. Flow cytometric analysis showed the cell cycle was blocked in G0/G1 phase and caspase-3 positive cells increased dramatically. CONCLUSION Simvastatin may block the cells in G0/G1 phase, and induce caspase-3 expression, which may trigger apoptosis.
Adipocytes ; cytology ; drug effects ; Anticholesteremic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cell Separation ; Cells, Cultured ; Humans ; Omentum ; cytology ; Simvastatin ; pharmacology