ABSTRACT
The in vitro phosphorylated and non-phosphorylated Hsp27 forms concentrations and Bcl-2 proteins affected by Hsp27 inhibition were studied in Jurkat-line tumor cells and healthy donor mononuclear lymphocytes by Western blotting technique. The Hsp27 inhibition causes the increase of intracellular Bax protein concentration and the decrease of Bcl-2 level leading to an increase of apoptotic changes in Jurkat line cells.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , HSP27 Heat-Shock Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2-Associated X Protein/metabolism , bcl-Associated Death Protein/metabolism , Apoptosis/drug effects , Blotting, Western , Humans , Jurkat Cells/drug effects , Jurkat Cells/metabolism , Molecular Chaperones/metabolism , Proto-Oncogene Proteins c-bcl-2/drug effects , bcl-2-Associated X Protein/drug effects , bcl-Associated Death Protein/drug effectsABSTRACT
The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.
Subject(s)
Anisoles/pharmacology , Apoptosis/physiology , HSP27 Heat-Shock Proteins/metabolism , Isoxazoles/pharmacology , Leukemia/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Adolescent , Adult , Annexin A5/metabolism , Apoptosis Regulatory Proteins , Cell Line, Tumor , Female , HSP27 Heat-Shock Proteins/antagonists & inhibitors , Humans , Leukocytes, Mononuclear , Male , Middle Aged , Molecular Chaperones/metabolism , Monocyte-Macrophage Precursor Cells/pathology , Young AdultABSTRACT
rTNFalpha-induced programmed death of Jurkat tumor cells cultured with 17-AAG, a selective inhibitor of heat shock protein (Hsp90), was studied by fluorescent microscopy with the use of FITC-labeled annexin V and propidium iodide. Caspase-3 and -8 activities were determined by spectrophotometry using a caspase- 3 and -8 colorimetric assay kit. It was shown that inhibition of Hsp90 leads to activation of Jurkat cell apoptosis while Hsp90 itself suppresses this process. 17-AAG enhances rTNFa-induced apoptosis of tumor cells.
Subject(s)
Apoptosis/drug effects , Benzoquinones/metabolism , Caspase 3 , Caspase 8 , HSP90 Heat-Shock Proteins/metabolism , Lactams, Macrocyclic/metabolism , Tumor Necrosis Factor-alpha/metabolism , Annexin A5/metabolism , Apoptosis/physiology , Caspase 3/analysis , Caspase 3/metabolism , Caspase 8/analysis , Caspase 8/metabolism , Enzyme Inhibitors/metabolism , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Humans , Jurkat Cells , Microscopy, Fluorescence , Propidium , SpectrophotometryABSTRACT
Programmed death of Jurkat tumor cells was studied under conditions of culturing with 17-AAG selective inhibitor of heat shock protein with a molecular weight of 90 kDa and etoposide. Apoptosis realization was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Activity of caspase-3 was evaluated spectrophotometrically. Inhibition of heat shock protein with a molecular weight of 90 kDa activated the apoptotic program in Jurkat tumor cells and etoposide-induced apoptosis. The heat shock protein with a molecular weight of 90 kDa acted as apoptosis inhibitor in tumor cells.
Subject(s)
Apoptosis/drug effects , Apoptosis/physiology , HSP90 Heat-Shock Proteins/metabolism , Neoplasms/metabolism , Benzoquinones/pharmacology , Caspase 3/biosynthesis , Caspase 3/metabolism , Etoposide/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Jurkat Cells , Lactams, Macrocyclic/pharmacologyABSTRACT
We studied the in vitro apoptosis-inducing effect of recombinant TNF-α (rTNF-α) on blood lymphocytes from healthy donors. rTNF-α-induced apoptosis was accompanied by an increase in the number of cells with low mitochondrial transmembrane potential, increased intracellular content of reactive oxygen species, reduced content of Bcl-2, Bcl-xL, and Bax proteins, and elevated Bad content. The molecular mechanisms of these changes are discussed.
