Induction of apoptosis by monosaccharide butyrate stable derivatives in chronic lymphocytic leukemia cells

Background and Objectives. Different therapeutic approaches are needed to r estore apoptotic mechanisms in CLL cells, as present ones are not successfu l. We assessed the apoptotic effects of stable butyrate derivatives on CLL lymphocytes: in these molecules a mannose molecule is bound as an ester to 1-5 butyrate moieties, conferring pharmacologic stability to the pro-drugs which are able to induce apoptosis in primary AML blasts. Design and Methods. Peripheral blood samples obtained from 17 patients with typical B-CLL were cultured in the presence of 0.51mM D1 (O-n-butanoyl-2,3 -O-isopropylidene-alpha-D-mannofuranoside), F1 (1-0-n-butanoyl-2,3-O-isopro pylidene-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 fra gmentation, annexin V exposure on cell membrane, and cell cycle parameters. Bcl2, bar, and fas oncogene expression were also evaluated by the APAAP me thod. Results. The addition to cell cultures of D1 or F1 or G1 butyrate monosacch arides as well as sodium butyrate 0.5 and 1 mM determined different increas es in the percentage of apoptotic cells in all CLL samples, depending on th e method and butyrate molecule added to the culture. Heterogeneity in CLL c ell 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 accounted f or 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 n ot altered by addition of butyrates, but expression of Annexin V was greatl y enhanced. In a limited number of CLL cases fas, bcl2/bax ratio was analyz ed and found unmodified. Interpretation and Conclusions. Monosaccharide butyrate stable derivatives are potent inducers of primary CLL cell apoptosis, both in untreated and al kylating agent pre-treated cases. Our results suggest that the apoptotic pa thways elicited by butyrate in CLL lymphocytes are direct, specific and mos t probably do not involve bcl2/bax. Pro-apoptotic agents like the stable mo nosaccharide butyrate derivatives here studied could give more insights int o CLL biology and resistance to apoptosis, and possibly give rise alternati ve treatments for CLL. (C)1999, Ferrata Storti Foundation.