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

The biflavonoid isochamaejasmin is mainly distributed in the root of Stellera chamaejasme L. (Thymelaeaceae) that is used in traditional Chinese medicine (TCM) to treat tumors, tuberculosis, and psoriasis. Herein, isochamaejasmin was found to show similar bioactivity against Bcl-2 family proteins to the reference Bcl-2 ligand (-)-gossypol through 3D similarity search. It selectively bound to Bcl-xl and Mcl-1 with Ki values being 1.93 ± 0.13 μmol·L(-1) and 9.98 ± 0.21 μmol·L(-1), respectively. In addition, isochamaejasmin showed slight growth inhibitory activity against HL-60 with IC50 value being 50.40 ± 1.21 μmol·L(-1) and moderate growth inhibitory activity against K562 cells with IC50 value being 24.51 ± 1.62 μmol·L(-1). Furthermore, isochamaejasmin induced apoptosis of K562 cells by increasing the intracellular expression levels of proteins of the cleavage of caspase-9, caspase-3, and PARP which involved in the Bcl-2-induced apoptosis pathway. These results indicated that isochamaejasmin induces apoptosis in leukemia cells by inhibiting the activity of Bcl-2 family proteins, providing evidence for further studying the underlying anti-cancer mechanism of S. chamaejasme L.
Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Apoptosis ; drug effects ; Biflavonoids ; pharmacology ; therapeutic use ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Gossypol ; pharmacology ; HL-60 Cells ; Humans ; Inhibitory Concentration 50 ; K562 Cells ; Leukemia ; drug therapy ; metabolism ; Myeloid Cell Leukemia Sequence 1 Protein ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Thymelaeaceae ; chemistry ; bcl-2-Associated X Protein ; metabolism

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

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

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

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

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

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

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