Synergistic antileukemic activity of carnosic acid-rich rosemary extract and the 19-nor Gemini vitamin D analogue in a mouse model of systemic acute myeloid leukemia.
OBJECTIVE
Differentiation therapy with the hormonal form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25D(3)), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D(3) induces hypercalcemia at pharmacologically active doses. We investigated the in vitro and in vivoantileukemic efficacy of combined treatment with non-toxic doses of a low-calcemic 1,25D(3) analogue, 1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol (19-nor-Gemini; Ro27-5646), and rosemary plant agents in a mouse model of AML.


METHODS
Proliferation and differentiation of WEHI-3B D- (WEHI) murine myelomonocytic leukemia cellsin vitro were determined by standard assays. Reactive oxygen species, glutathione and protein expression levels were measured by flow cytometry, enzymatic assay and Western blotting, respectively. Systemic AML was developed by intravenous injection of WEHI cells in syngeneic Balb/c mice.


RESULTS
19-nor-Gemini had a higher potency than its parent compounds, Gemini (Ro27-2310) and 1,25D(3), in the induction of differentiation (EC(50) = 0.059 +/- 0.011, 0.275 +/- 0.093 and 0.652 +/- 0.085 nM, respectively) and growth arrest (IC(50) = 0.072 +/- 0.018, 0.165 +/- 0.061 and 0.895 +/- 0.144 nM, respectively) in WEHI cells in vitro, and lower in vivo toxicity. Combined treatment of leukemia-bearing mice with 19-nor-Gemini (injected intraperitoneally) and standardized rosemary extract (mixed with food) resulted in a synergistic increase in survival (from 42.2 +/- 2.5 days in untreated mice to 66.5 +/- 4.2 days, n = 3) and normalization of white blood cell and differential counts. This was consistent with strong cooperative antiproliferative and differentiation effects of low concentrations of 19-nor-Gemini or 1,25D(3) combined with rosemary extract or its major polyphenolic component, carnosic acid, as well as with the antioxidant action of rosemary agents and vitamin D derivatives in WEHI cell cultures.


CONCLUSIONS
Combined effectiveness of 1,25D(3) analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.
Shabtay
Ayelet
A
Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Sharabani
Hagar
H
Barvish
Zeev
Z
Kafka
Michael
M
Amichay
Doron
D
Levy
Joseph
J
Sharoni
Yoav
Y
Uskokovic
Milan R
MR
Studzinski
George P
GP
Danilenko
Michael
M
D045784:Abietanes / Q000627:therapeutic use*; D000818:Animals; D000975:Antioxidants / Q000627:therapeutic use; D001853:Bone Marrow / Q000187:drug effects; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000627:therapeutic use; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation; D002762:Cholecalciferol / Q000031:analogs & derivatives / Q000627:therapeutic use; D004195:Disease Models, Animal; D004357:Drug Synergism; D006801:Humans; D015151:Immunoblotting; D007942:Leukemia, Experimental / Q000188:drug therapy*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D010936:Plant Extracts / Q000627:therapeutic use*; D017382:Reactive Oxygen Species / Q000378:metabolism; D027542:Rosmarinus / Q000737:chemistry; D015996:Survival Rate; D014407:Tumor Cells, Cultured
C116777:1,25-dihydroxy-16,23-diene vitamin D3; C411462:1,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D(3); D045784:Abietanes; D000975:Antioxidants; D010936:Plant Extracts; D017382:Reactive Oxygen Species; D002762:Cholecalciferol; D002117:Calcitriol; C018381:salvin