Enhancement of All-trans Retinoic Acid-Induced HL-60 Cell Differentiation by Thalidomide and Its Metabolites

Abstract

Thalidomide (Thal: 1) and its two metabolites, 5-hydroxythalidomide (5-HT: 2) and N-hydroxythalidomide (N-HT: 3), showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation. 5-HT and N-HT showed tubulin polymerization-inhibiting activity, but thalidomide did not.Key words thalidomide; hydroxythalidomide; retinoic acid; differentiation; tubulin Thalidomide (Thal: 1), a sedative drug which was once withdrawn from the market because of its teratogenicity, has been established to be effective for the treatment of various diseases, including leprosy, multiple myeloma (MM), AIDS, and various cancers.1-5) The anti-MM activity of Thal (1) is of particular interest, because Thal (1) overcomes the resistance of human MM cells to conventional drug therapy.6) Although various pharmacological effects elicited by Thal (1), including tumor necrosis factor (TNF)-a production-regulating activity, anti-angiogenic activity and cyclooxygenase (COX)-inhibiting activity, [1][2][3][4][5]7,8) have been reported, the mechanism of its anti-MM activity is unclear.During our structural development studies of Thal (1), we found that one of its major metabolites, 5-hydroxythalidomide (5-HT: 2), possesses tubulin polymerization-inhibiting activity, resulting in G1 arrest and apoptosis of myeloma cells. 9) We also previously reported that tubulin polymerization inhibitors, including colchicine, vinblastine, rhizoxin, maytansine, and ustiloxin, and tubulin depolymerization inhibitors, including taxol, enhance the chemically induced differentiation of human leukemia cell lines.10) Though the relationship between cell differentiation induction and tubulin polymerization/depolymerization inhibition is not clear, enhancement of chemically induced cell differentiation seems to be one of general features of tubulin disruptors.10) These findings led us to suspect that the enhancing effect of Thal (1) and/or its metabolites plays a role in the anti-MM activity of the drug, at least in part. Thal (1) is known to be metabolically unstable, and many metabolites have been identified, including 5-HT (2) and N-hydroxythalidomide (N-HT: 3). In this paper, we deal with the enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation and the tubulin polymerization-inhibiting activity of Thal (1), 5-HT (2) and N-HT (3).Thal (1) and 5-HT (2) were prepared as described previously.9,11) N-HT (3) was prepared by the method described by Robin et al. 12) Briefly, N-phthaloylglutamic acid anhydride was condensed with O-benzylhydroxylamine in the presence of dicyclohexylcarbodiimde (DCC) to give N-benzyloxythalidomide, which was deprotected by hydrogenation (H 2 gas, Pd/C) to afford N-HT (3). The structures of the synthesized compounds were confirmed by NMR and mass spectroscopy and elemental analysis. First, we investigated the effect of the compounds on tubulin polymerization using the method previously described. 9)Briefly, microtubulin was prepared from porcine brain and tubulin polymerization was followe...

Enhancement of Chemically-Induced HL-60 Cell Differentiation by 3,3'-Diindolylmethane Derivatives

“…Chemistry DIM (1), its derivatives (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), and isomers of DIM [2,2Ј-DIM (22) and 2,3Ј-DIM (25)] were synthesized using the methods reported by Nagarajan and Perumal 11) and usual organic synthetic methods as shown in Charts 1 and 2. Briefly, DIM (1) and its derivatives (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were obtained by the reaction of indole or methylindole with various aldehydes or ketones (Chart 1).…”

“…Briefly, DIM (1) and its derivatives (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) were obtained by the reaction of indole or methylindole with various aldehydes or ketones (Chart 1). Bis(1-benzenesulfonyl-1H-indol-2-yl)methanol (20) and (1-benzenesulfonyl-1H-indol-2-yl)(1-benzenesulfonyl-1H-indol-3-yl)methanol (24) was obtained by the reaction of 2-(19) or 3-formyl-1-benzenesulfonyl-1H-indole (23) with 1-benzenesulfonyl-1H-indole (18), followed by reduction and desulfonylation to give 2,2Ј-DIM (22) or 2,3Ј-DIM (25) (Chart 2).…”

“…HL-60 cell differentiation was assayed by the use of nitroblue tetrazolium (NBT) reduction assay, as described previously. 12) Of course, the population of NBT positive cells (%) deviates from experiments and experiments. However, the deviation of the value in one experiment (performed in duplicate or triplicate) is less than 5%, and the order of the activity of the test compounds was reproducible (repeated at least three times).…”

Thalidomide as a Multi‐Template for Development of Biologically Active Compounds

“…4) effects of thalidomide 1 and its metabolites (9 and 10) on all-trans retinoic acid (ATRA)-induced granulocytic differentiation of human leukemia HL-60 cells were found (Fig. 4) [40].…”

“…activity in the presence of 2 nM ATRA could be observed at a concentration as low as 1 lM thalidomide 1, 5-OHthalidomide 9, or N-OH-thalidomide 10. Cell morphological analysis and studies using flow cytometry suggested that the three compounds enhanced ATRA-induced HL-60 cell differentiation [40].…”