title: Induction of apoptosis by monosaccharide butyrate stable derivatives in chronic lymphocytic leukemia cells.
abstract: OBJECTIVE
  Different therapeutic approaches are needed to restore apoptotic mechanisms in CLL cells, as present ones are not successful. We assessed the apoptotic effects of stable butyrate derivatives on CLL lymphocytes: in these molecules a mannose molecule is bound as ester to one-five butyrate moieties, conferring pharmacological stability to the pro-drugs which are able to induce apoptosis in primary AML blasts.
  
  
  METHODS
  Peripheral blood samples obtained from 17 patients with typical B-CLL were cultured in the presence of 0.5-1mM D1 (O-n-butanoyl-2, 3-O-isopropylidene-a-D-mannofuranoside), F1 (1-O-n-butanoyl-2, 3-O-isopropylidene-D,L-xylitol) and G1 (1-O-n-butanoyl-D,L-xylitol) derivatives for 4 days and equimolar sodium butyrate as comparison. After culture, apoptosis was evaluated by cell morphology, cellular DNA content, pattern of DNA fragmentation, annexin V exposure on cell membrane, and cell cycle parameters. Bcl2, bax, and fas oncogene expression were also evaluated by the APAAP method.
  
  
  RESULTS
  The addition to cell cultures of D1 or F1 or G1 butyrate monosaccharides as well as sodium butyrate 0.5 and 1 mM determined to different extents an increase in the percentage of apoptotic cells in all CLL samples, relatively to the method and butyrate molecule added in culture. Heterogeneity in CLL cell sensitivity to the three butyrates was observed. Up to 60-68% apoptotic bodies were present in treated cultures after exposure to D1 0.5-1 mM, 60-72% after F1 0.5-1 mM and 48-60% after G1 0.5-1 mM. Comparison of untreated versus treated cultures yielded important significance (p< 0.001). At DNA content analysis, analyzed by flow cytometry, apoptotic events were accounting for up to 70-77% of D1-treated and 68-74% of F1-treated CLL cells at 0.5 and 1 mM concentrations (p= 0. 0001, vs controls 0-39%), and for 72-81% of G1 (0.5-1 mM) treated cells (overall, p=0.005). Cell cycle parameters were not altered by addition of butyrates, but expression of Annexin V was greatly enhanced. In a limited number of CLL cases fas, bcl2/bax ratio was analyzed and found unmodified.
  
  
  CONCLUSIONS
  Monosaccharide butyrate stable derivatives are potent inducers of primary CLL cell apoptosis, both in untreated and alkylating agent pre-treated cases. Our results suggest that the apoptotic pathways elicited by butyrate in CLL lymphocytes are direct, specific and most probably do not involve bcl2/bax. Pro-apoptotic agents like the stable monosaccharide butyrate derivatives here studied could bring more insights into CLL biology and resistance to apoptosis, and possibly originate alternative treatments for CLL.
authors.affiliation: Dept. of Hematology, Policlinico di Careggi, viale Morgagni 85, 50134 Florence, Italy. santini@unifi.it
authors.forename: V
authors.initials: V
authors.lastname: Santini
authors.forename: A
authors.initials: A
authors.lastname: Gozzini
authors.forename: B
authors.initials: B
authors.lastname: Scappini
authors.forename: R
authors.initials: R
authors.lastname: Caporale
authors.forename: A
authors.initials: A
authors.lastname: Zoccolante
authors.forename: L
authors.initials: L
authors.lastname: Rigacci
authors.forename: E
authors.initials: E
authors.lastname: Gelardi
authors.forename: A
authors.initials: A
authors.lastname: Grossi
authors.forename: R
authors.initials: R
authors.lastname: Alterini
authors.forename: P R
authors.initials: PR
authors.lastname: Ferrini
mesh_terms: D000368:Aged; D017304:Annexin A5 / Q000096:biosynthesis / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects*; D001773:Blood Cells / Q000187:drug effects / Q000473:pathology; D002087:Butyrates / Q000494:pharmacology*; D018929:Cell Culture Techniques; D048429:Cell Size / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D053222:Fas Ligand Protein; D005260:Female; D006801:Humans; D007399:Interphase / Q000187:drug effects; D007700:Kinetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000187:drug effects; D008875:Middle Aged; D009005:Monosaccharides / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000187:drug effects; D051028:bcl-2-Associated X Protein
chemical_list: D017304:Annexin A5; C494826:BAX protein, human; D002087:Butyrates; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D009005:Monosaccharides; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein