28-Deacetylbelamcandal, a Tumor-Promoting Triterpenoid from Iris tectorum

Abstract

A spiroiridal-type triterpenoid, 28-deacetylbelamcandal (1), was isolated from the rhizomes of Iris tectorum as an active principle that stimulated differentiation of human promyelocytic leukemia (HL-60) cells, a short-term screening method for 12-O-tetradecanoylphorbol 13-acetate (TPA)-type tumor promoters. In the same manner as TPA, compound 1 bound to protein kinase C (PKC) and activated PKC, and induced tumor necrosis factor-alpha release from HL-60 cells. In an in vivo study, groups treated with 100 microg 7, 12-dimethylbenz[a]anthracene plus 400 nmol of 1 showed 64.3% tumor incidence by week 20. It has thus been demonstrated that 1 represents a new structural class of mouse skin-tumor promoters.

Protein Kinase C Activation by Iridal Type Triterpenoids.

“…Extraction and Isolation Through an analogous procedure to that described in preceding papers, 11,13) compounds 1-3 were isolated. Briefly, each methanolic extract of dried rhizomes of I. tectorum (500 g) and B. chinensis (500 g) was partitioned between water and CHCl 3 .…”

“…11,13) Both compounds 10 and 11 exist as an equilibrium mixture of epimers at the hemiacetal carbon (C-26) in the presence of water. Partial PKC purification was done by DEAE cellulose (DE-52) and phenyl-Sepharose column chromatography steps as described previously.…”

“…Assay of Protein Kinase C Activation PKC activity was determined by measuring the incorporation of 32 P into histone III-S from [g-32 P]ATP, as described previously. 13) Briefly, a standard reaction mixture (0.1 ml) containing 40 mM Tris/HCl (pH 7.5), 10 mM [g-32 P]ATP (200 dpm/pmol), 10 mM MgCl 2 , 0.5 mg/ml histone III-S, 2.5 mg phosphatidylserine, 20 mM CaCl 2 and 5 ml PKC was incubated for 10 min at 30°C. After incubation, ice-cold 25% trichloroacetic acid was added to the reaction mixture, and the acid-precipitable materials were collected on a GF/C filter (Whatman, Maidstone, U.K.).…”

“…13) Briefly, binding was carried out in a standard reaction mixture (0.2 ml) in 20 mM Tris-malate (pH 6.8), 100 mM KCl, 0.2 mM CaCl 2 , 0.1 mg/ml phosphatidylser- ine, 30 nM [ 3 H]PDBu (370 GBq/mmol), 0.5% DMSO, and 10 ml PKC. After incubation for 3 h in ice-cold water, 4 ml of ice-cold 0.5% DMSO was added to the reaction mixture.…”

“…(Iridaceae), 11) and that 28-deacetylbelamcandal (4) 12) from I. tectorum is a PKC activator as well as a new class of tumor promoter for mouse skin two-stage carcinogenesis. 13) For further study of iridal type triterpenoids, eleven iridal type triterpenoids from I. tectorum and B. chinensis were examined with respect to effect on PKC enzyme activity and inhibitory effect against [ 3 H] phorbol 12,13-dibutyrate (PDBu) binding to PKC. This paper describes characterization of iridal type triterpenoids as PKC activators, and structure-activity relationships are also discussed.…”