The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.
Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Blotting, Western ; Cathepsin D ; metabolism ; Cytochromes c ; metabolism ; Fluorescent Antibody Technique ; Glucosamine ; pharmacology ; Humans ; K562 Cells ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism ; physiology

A vast portion of human disease results when the process of apoptosis is defective. Disorders resulting from inappropriate cell death range from autoimmune and neurodegenerative conditions to heart disease. Conversely, prevention of apoptosis is the hallmark of cancer and confounds the efficacy of cancer therapeutics. In the search for optimal targets that would enable the control of apoptosis, members of the BCL-2 family of anti- and pro-apoptotic factors have figured prominently. Development of BCL-2 antisense approaches, small molecules, and BH3 peptidomimetics has met with both success and failure. Success-because BCL-2 proteins play essential roles in apoptosis. Failure-because single targets for drug development have limited scope. By examining the activity of the BCL-2 proteins in relation to the mitochondrial landscape and drawing attention to the significant mitochondrial membrane alterations that ensue during apoptosis, we demonstrate the need for a broader based multi-disciplinary approach for the design of novel apoptosis-modulating compounds in the treatment of human disease.
Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

Apoptosis/*drug effects/physiology ; BH3 Interacting Domain Death Agonist Protein/physiology ; Drug Design ; Genes, bcl-2 ; Humans ; Mitochondria/physiology/ultrastructure ; Mitochondrial Membranes/*metabolism/physiology ; Multigene Family ; Proto-Oncogene Proteins c-bcl-2/*antagonists & inhibitors ; Signal Transduction

OBJECTIVETo explore the role of Bid protein in the mitochondria and endoplasmic reticulum (ER) associated apoptotic pathway.

METHODSApoptosis of MUTZ-1 cells induced by homoharringtonine (HHT) was measured by FACS. Mitochondria and ER associated apoptotic pathway was detected by RT-PCR and Western blotting. And the translocation of Bid protein was measured by laser scanning confocal microscope (LSCM).

RESULTSAfter exposure of MUTZ-1 to HHT at 0.05 microg/ml for 24 h, typical ER-stress phenomenon induced apoptotic cells and release of Ca2+ from the cytosolic Ca2+ storage and the loss of mitochondrial membrane potential were observed. RT-PCR analysis revealed that mRNAs for ER stress-associated proapoptotic factor were markedly increased at 4 h after 0.05 microg/ml HHT treatment and peaked at 12 h, then decreased steady. Activation of caspase protein was also observed at 8 h. The translocation of Bid protein from ER to mitochondria was observed at 12 h after HHT treatment.

CONCLUSIONHHT can induce MUTZ-1 cells apoptosis. The cell death may be likely mediated by the ER stress pathway as well as mitochondrial pathway and Bid protein may be the cross talk of the two apoptotic pathways.

Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; physiology ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Caspases, Initiator ; metabolism ; Cell Line ; DNA-Binding Proteins ; metabolism ; Endoplasmic Reticulum ; metabolism ; physiology ; Harringtonines ; pharmacology ; Humans ; Mitochondria ; metabolism ; physiology ; Regulatory Factor X Transcription Factors ; Transcription Factors ; metabolism

Pore-formation and protein-protein interactions are considered to play critical roles in the regulation of apoptosis by Bcl-2 family proteins. During the initiation of apoptosis, the anti-apoptotic Bcl-2 and the pro-apoptotic Bax form different pores to regulate the permeability of mitochondrial outer membrane, playing their opposite functions. Overexpression of Bcl-2 has been found in various cancer cells, therefore it is gaining widespread interest to discover small molecules to compromise Bcl-2 function for anti-cancer treatment. Since Bax binds to Bcl-2's hydrophobic groove via its BH3 domain (composed of helices 2 and 3), by which their functions are inhibited each other, the H2-H3 peptide that contains the functional BH3 domain of Bax has been considered as a potential Bcl-2 antagonist. We recently reported that Bax peptide H2-H3 promotes cell death by inducing Bax-mediated cytochrome c release and by antagonizing Bcl-2's inhibitory effect on Bax. However, the mechanism of how H2-H3 inhibits the anti-apoptotic activity of Bcl-2 remains poorly understood. To address this question, we reconstituted the Bcl-2 or Bax pore-forming process in vitro. We found that H2-H3 inhibited Bcl-2's pore formation and neutralized Bcl-2's inhibitory effect on Bax pore formation in the membrane, whereas the mutant H2-H3 peptide that does not induce apoptosis in cells was shown to have no effect on Bcl-2's activities. Thus, inhibiting Bcl-2's pore-forming and anti-Bax activities in the membrane is strongly correlated with H2-H3's pro-apoptosis function in cells.
Apoptosis ; physiology ; BH3 Interacting Domain Death Agonist Protein ; chemistry ; Humans ; Membrane Proteins ; chemistry ; metabolism ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Membranes ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; chemistry ; bcl-2 Homologous Antagonist-Killer Protein ; chemistry ; bcl-2-Associated X Protein ; chemistry

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.
Antibiotics, Antineoplastic/pharmacology ; Apoptosis/*drug effects ; Apoptotic Protease-Activating Factor 1 ; BH3 Interacting Domain Death Agonist Protein ; Blotting, Western ; Bronchi/metabolism/*pathology ; Carcinogens/*pharmacology ; Carrier Proteins/metabolism ; Caspases/metabolism ; Cytochromes c/metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells/metabolism/*pathology ; Eukaryotic Initiation Factor-4E/metabolism ; Flow Cytometry ; Humans ; Nitrosamines/*pharmacology ; Protein Biosynthesis ; Proteins/metabolism ; Proto-Oncogene Proteins c-bcl-2/metabolism ; RNA Cap-Binding Proteins/*physiology ; Sirolimus/pharmacology ; Time Factors ; bcl-2-Associated X Protein