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Structure‐apoptotic potency evaluations of novel sterols using human leukemic cells
Author(s) -
Johnson Betty H.,
Russell Michael J.,
Krylov Alexander S.,
Medh Rheem D.,
AyalaTorres Sylvette,
Regner Justin Lee,
Thompson E. Brad
Publication year - 2000
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-000-0527-9
Subject(s) - apoptosis , tunel assay , biology , filipin , viability assay , annexin , terminal deoxynucleotidyl transferase , microbiology and biotechnology , programmed cell death , dna fragmentation , phosphatidylserine , biochemistry , phospholipid , cholesterol , membrane
Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM‐C7‐14 human leukemic cells. In addition to testing 15‐ketocholestenol (K15), 15‐ketocholestenol hydroxyethyl ether (CK15), and 7‐ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25‐hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC>K15>CK15>CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase‐mediated dUTP‐X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c‐ myc mRNA was suppressed by all three oxysterols. with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.