Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracellular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/ 10% FBS at 37(i)E, in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-kappaB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respectively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-kappaB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis- associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-kappaB activation in mouse spleen cells.
bcl-2-Associated X Protein/metabolism ; Toxoplasma/*physiology ; RNA, Messenger/metabolism ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors ; NF-kappa B/*metabolism ; Mice ; Gene Expression Regulation ; Flow Cytometry ; DNA Fragmentation ; Cells, Cultured ; Caspase 3/*antagonists & inhibitors ; Apoptosis/*physiology ; Animals

Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracellular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/ 10% FBS at 37(i)E, in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-kappaB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respectively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-kappaB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis- associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-kappaB activation in mouse spleen cells.
bcl-2-Associated X Protein/metabolism ; Toxoplasma/*physiology ; RNA, Messenger/metabolism ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors ; NF-kappa B/*metabolism ; Mice ; Gene Expression Regulation ; Flow Cytometry ; DNA Fragmentation ; Cells, Cultured ; Caspase 3/*antagonists & inhibitors ; Apoptosis/*physiology ; Animals

BACKGROUNDBrain dysfunction is a frequent complication of sepsis, usually defined as sepsis-associated encephalopathy (SAE). Although the Notch signaling pathway has been proven to be involved in both ischemia and neuronal proliferation, its role in SAE is still unknown. Here, the effect of the Notch signaling pathway involved γ-secretase inhibitor DAPT on SAE in septic rats was investigated in a cecal ligation and puncture (CLP) model.

METHODSFifty-nine Sprague-Dawley rats were randomly divided into four groups, with the septic group receiving the CLP operation. Twenty-four hours after CLP or sham treatment, rats were sacrificed and their hippocampus was harvested for Western blot analysis. TNF-α expression was determined using an enzyme-linked immunosorbent assay (ELISA) kit. Neuronal apoptosis was assessed by TUNEL staining, and neuronal cell death was detected by H&E staining. Finally, a novel object recognition experiment was used to evaluate memory impairment.

RESULTSOur data showed that sepsis can increase the expression of hippocampal Notch receptor intracellular domain (NICD) and poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1), as well as the inflammatory response, neuronal apoptosis, neuronal death, and memory dysfunction in rats. The γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT) can significantly decrease the level of NICD and PARP-1, reduce hippocampal neuronal apoptosis and death, attenuate TNF-α release and rescue cognitive impairment caused by CLP.

CONCLUSIONThe neuroprotective effect of DAPT on neuronal death and memory impairment in septic rats, which could be a new therapeutic approach for treating SAE in the future.

Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Animals ; Apoptosis ; drug effects ; Dipeptides ; therapeutic use ; Hippocampus ; drug effects ; metabolism ; Male ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Notch ; metabolism ; Sepsis ; complications ; Sepsis-Associated Encephalopathy ; drug therapy ; enzymology ; Signal Transduction ; drug effects

Antineoplastic Agents, Alkylating ; pharmacology ; Apoptosis ; drug effects ; Benzimidazoles ; pharmacology ; DNA Repair ; Dacarbazine ; analogs & derivatives ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; physiology ; Humans ; Membrane Proteins ; metabolism ; O(6)-Methylguanine-DNA Methyltransferase ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerase Inhibitors ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; metabolism ; Purines ; pharmacology ; Pyrimidines ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the protective mechanism of neuroactive steroid allopregnanolone on N-methyl-D-aspartate (NMDA) induced toxicity in primary mouse cortical neurons.

METHODSPrimary cultured mouse cortical neurons were subjected to allopregnanolone, the expression of beta-aminobutyric acid receptor beta2 subunit (beta2-GABA-R) mRNAs was detected by RT-PCR and Akt phosphorylation was assayed by Western blot using Akt-phosphoserine 473-specific antibody. After the cultured mouse cortical neurons were pretreated with or without allopregnanolone prior to treatment with NMDA , DNA isolated was analyzed by agarose gel electrophoresis and proteins collected were analyzed by Western blot with anti-cleaved-PARP, anti-cleaved caspase-3, and anti-cleaved caspase-9 antibodies.

RESULTSWhen cultured mouse cortical neurons were exposed to allopregnanolone both the expression of beta2-GABA-R mRNAs and Akt phosphorylation increased. Allopregnanolone inhibited the NMDA-induced apoptosis and decreased the level of active-PARP, active-caspase-3 and active-caspase-9 notably at a final concentration of 5 x 10(6) mol/L.

CONCLUSIONPretreatment with allopregnanolone may be neuroprotective on NMDA-induced neuronal cells apoptosis by increasing beta2-GABA-R expression and Akt phosphorylation.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cerebral Cortex ; cytology ; Mice ; N-Methylaspartate ; antagonists & inhibitors ; toxicity ; Neurons ; cytology ; Neuroprotective Agents ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Pregnanolone ; pharmacology ; Primary Cell Culture ; RNA, Messenger ; genetics ; metabolism ; Receptors, GABA-B ; genetics ; metabolism

Pancreatic ductal adenocarcinoma is one of the most dreaded malignancies and the 5th leading cause of cancer-related death in Korea. Late diagnosis and unfavorable response to both chemotherapy and radiotherapy result in exceptionally poor prognosis. Recently, the rapid advances of molecular biology allowed an in-depth understanding of pancreatic carcinogenesis, and there are many attempts to modulate signal pathway using specific targeted agent. However, the most of them have so far failed to improve survival significantly except erlotinib. The real challenge is now how these impressive advances of molecular biology could be successfully integrated into better clinical implications. Herein, we summarize the latest insights into the carcinogenesis, and their repercussions for novel targeted agents for pancreatic cancer, and provide a review of recent clinical trials using molecular targeted therapy.
Antineoplastic Agents/*therapeutic use ; Epigenesis, Genetic ; Humans ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/metabolism/pathology ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

Pancreatic ductal adenocarcinoma is one of the most dreaded malignancies and the 5th leading cause of cancer-related death in Korea. Late diagnosis and unfavorable response to both chemotherapy and radiotherapy result in exceptionally poor prognosis. Recently, the rapid advances of molecular biology allowed an in-depth understanding of pancreatic carcinogenesis, and there are many attempts to modulate signal pathway using specific targeted agent. However, the most of them have so far failed to improve survival significantly except erlotinib. The real challenge is now how these impressive advances of molecular biology could be successfully integrated into better clinical implications. Herein, we summarize the latest insights into the carcinogenesis, and their repercussions for novel targeted agents for pancreatic cancer, and provide a review of recent clinical trials using molecular targeted therapy.
Antineoplastic Agents/*therapeutic use ; Epigenesis, Genetic ; Humans ; Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism ; Molecular Targeted Therapy ; Pancreatic Neoplasms/*drug therapy/metabolism/pathology ; Poly(ADP-ribose) Polymerases/antagonists & inhibitors/metabolism ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptor, IGF Type 1/antagonists & inhibitors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

The role of mast cells in tumor growth is still controversial. In this study we analyzed the effects of both histamine and pre-formed mediators spontaneously released by mast cells on the growth of two human hepatocellular carcinoma cell lines, HA22T/VGH and HuH-6, with different characteristics of differentiation, biological behavior and genetic defects. We showed that total mast cell releasate, exocytosed granules (granule remnants) and histamine reduced cell viability and proliferation in HuH-6 cells. In contrast, in HA22T/VGH cells granule remnants and histamine induced a weak but significant increase in cell growth. We showed that both cell lines expressed histamine receptors H1 and H2 and that the selective H1 antagonist terfenadine reverted the histamine-induced inhibition of HuH-6 cell growth, whereas the selective H2 antagonist ranitidine inhibited the histamine-induced cell growth of HA22T/VGH cells. We demonstrated that histamine down-regulated the expression of beta-catenin, COX-2 and survivin in HuH-6 cells and that this was associated with caspase-3 activation and PARP cleavage. On the contrary, in HA22T/VGH cells expression of survivin and beta-catenin increased after treatment with granule remnants and histamine. Overall, our results suggest that mediators stored in mast cell granules and histamine may affect the growth of liver cancer cells. However, mast cells and histamine may play different roles depending on the tumor cell features. Finally, these data suggest that histamine and histamine receptor agonists/antagonists might be considered as "new therapeutic" drugs to inhibit liver tumor growth.
Animals ; Apoptosis ; Carcinoma, Hepatocellular/*metabolism/*pathology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cyclooxygenase 2/metabolism ; Enzyme Activation ; Exocytosis ; Female ; Histamine/*pharmacology ; Histamine Antagonists/pharmacology ; Humans ; Liver Neoplasms/*metabolism/*pathology ; Mast Cells/*physiology ; Microtubule-Associated Proteins/metabolism ; Neoplasm Proteins/metabolism ; Poly(ADP-ribose) Polymerases/metabolism ; Ranitidine/pharmacology ; Rats ; Rats, Wistar ; Receptors, Histamine/metabolism ; Terfenadine/pharmacology ; beta Catenin/metabolism

OBJECTIVETo investigate the apoptotic effects of Hsp90 selective inhibitor 17-AAG on human leukemia HL-60 and NB4 cells and analyse its possible mechanism.

METHODSCCK-8 assay was used to quantify the growth inhibition of cells after exposure to 17-AAG for 24 hours. Flow cytometrve with annexin V/propidium iodide staining was used to detect apoptosis of leukemia cells. Then Western blot was used to detect the activation of apoptosis related protein caspase-3 and PARP level. Gene expression profile of NB4 cells treated with 17-AAG was analyzed with real-time PCR arrays.

RESULTSThe inhibition of leukemia cell proliferation displayed a dose-dependent manner. Annexin V assay, cell cycle analysis and activation of PARP demonstrate that 17-AAG induced apoptosis leukemia cells. Real-time PCR array analysis showed that expression of 56 genes significantly up-regulated and expression of 23 genes were significantly down-regulated after 17-AAG treatment.

CONCLUSIONThe 17-AAG can inhibit the proliferation and induce the apoptosis of leukemia cells. After leukemia cells are treated with 17-AAG, the significant changes of apoptosis-related genes occured, and the cell apoptosis occurs via activating apoptosis related signaling pathway.

Apoptosis ; Benzoquinones ; pharmacology ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; HL-60 Cells ; HSP90 Heat-Shock Proteins ; antagonists & inhibitors ; Humans ; Lactams, Macrocyclic ; pharmacology ; Leukemia ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Transcriptome

OBJECTIVETo study the effect of ABT-737 combined with cisplatin on apoptosis of breast cancer cell line T47D cells.

METHODST47D cells cultured in vitro was used for this experiment. Cell proliferation was measured by MTT assay. The expression of apoptosis-related protein was determined by Western blot. Morphological changes of apoptotic cells were observed by fluorescence microscopy. The apoptosis rate was examined by flow cytometry.

RESULTSThe MTT assay showed that ABT-737 significantly decreased the IC(50) of cisplatin in T47D cells [(26.00 ± 1.41) µmol/L of single cisplatin vs. (13.00 ± 1.11) µmol/L of combination (ABT-737 + cisplatin)]. As a single agent, ABT-737 did not inhibit the proliferation of T47D cells, but enhanced the inhibitory effect of cisplatin in a dose-dependent manner. The detection of the cleavage of PARP showed that ABT-737 lowered the doses of cisplatin to induce apoptosis and shortened the induction time of apoptosis in T47D cells. Compared with the single use of cisplatin, the combination of ABT-737 and cisplatin accelerated the cleavage of PARP and caspase3, but did not alter the expression levels of Bcl-2, Bcl-X(L), and Bax. Both flow cytometry and fluorescence microscopy showed that ABT-737 combined with cisplatin significantly increased the apoptosis induction in T47D cells (2.3% ± 0.1 % in the control, 30.0% ± 0.8% in the cisplatin alone, and 49.0% ± 0.5% in the cisplatin + ABT-737 groups, P < 0.05).

CONCLUSIONThe Bcl-2 inhibitor ABT-737 can significantly enhance cisplatin-induced apoptosis in human breast cancer T47D cells in vitro.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Biphenyl Compounds ; administration & dosage ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Female ; Humans ; Nitrophenols ; administration & dosage ; pharmacology ; Piperazines ; administration & dosage ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; metabolism ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism