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

Abstract

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.

Conclusion: 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.

Fig. 1.

Structures of the rosemary polyphenol, carnosic acid and deltanoids used in this study. Carnosic acid, the polyphenolic diterpene with antioxidant properties derived from the plant R. officinalis L. (rosemary). 1,25D₃ = Parent vitamin D compound, 1α,25-dihydroxyvitamin D₃; Gemini (Ro27-2310) = the vitamin D analogue which contains two side chains, combining a C-20-normal with a C-20-epi side chain [1,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)-cholecalciferol]; 19-nor Gemini (Ro27-5646) = its derivative with deletion of C-19 [1,25-dihydroxy-21(3-hydroxy-3-methyl-butyl)-19-nor-cholecalciferol].

Fig. 2.

19-nor-Gemini is a more potent antiproliferative and differentiation agent than 1,25D₃ or Ro27-2310, with lower calcemic activity. a, b WEHI cells (5 × 10³/ml) were incubated with 1,25D₃ (D₃), Gemini or 19-nor-Gemini (19-nor) for 96 h. Means ± SE of at least three independent experiments performed in duplicate. a Viable cell counts. The data are expressed as percent of cell numbers in control cultures incubated with vehicle alone (0.1% ethanol). The average control cell number at 96 h was 624,000 ± 34,000 cells/ml. b TPA (2 μM)-stimulated nitroblue tetrazolium (NBT) reduction (myeloid differentiation marker). The percentage of cells containing black formazan deposits was evaluated by counting 200 cells. c, d Effects of deltanoids on serum calcium and animal weight gain. Healthy Balb/c mice were intraperitoneally injected with 1,25D₃, Gemini, 19-nor (all at 0.1 μg) or vehicle (0.1% ethanol), 3 times per week. c Serum calcium levels were measured weekly. d Mice from the experiment shown in c were weighed 3 times per week. Means ± SE of one experiment performed in groups of 10 animals.

Fig. 3.

Carnosic acid (CA) and rosemary extract (RE) cooperate with 1,25D₃ (D₃) and 19-nor-Gemini (19-nor) in the induction of differentiation (a, b) and inhibition of proliferation (c) in WEHI cells. Cells (5 × 10³/ml) were incubated with the indicated compounds for 96 h. Differentiation was assessed by MSE (a) and F4/80 (b) assays. c Viable cell counts. Means ± SE of four to seven independent experiments performed in duplicate. Synergistic effects of the deltanoid-carnosic acid and deltanoid-rosemary extract combinations on MSE expression were statistically significant (∗ p < 0.001).

Fig. 4.

Antioxidant effects of rosemary-derived agents and deltanoids in WEHI cells. Cells (2.5 × 10⁴/ml) were incubated with carnosic acid (CA), rosemary extract (RE), 1,25D₃ (D₃), 19-nor Gemini (19-nor) or their combinations for 48 h. a Intracellular levels of ROS determined by DCF fluorescence. b Total cellular glutathione content. Means ± SE of four independent experiments performed in duplicate.

Fig. 5.

Induction of NADP(H)-quinone oxireductase (NQO1) by rosemary-derived agents and deltanoids in WEHI cells. Cells (2.5 × 10⁴/ml) were incubated for 48 h with carnosic acid (CA), rosemary extract (RE), 1,25D₃ (D₃), 19-nor Gemini (19-nor) or their combinations. a NQO1 protein levels. Membranes were reblotted for calreticulin (CRN) to demonstrate similar protein loading. b Quantitative representation of NQO1 protein expression. Average OD ratios (NQO1 vs. CRN) of three similar experiments are shown (means ± SE). Statistically significant differences in synergistic effects of the deltanoid/carnosic acid and deltanoid/rosemary extract combinations were found (∗ p < 0.05 and ∗∗ p < 0.01).

Fig. 6.

Systemic leukemia induced by intravenous inoculation of WEHI cells in syngeneic Balb/c mice. Six animals were injected with 2 × 10⁶ WEHI cells in the lateral tail vein and monitored for 6 weeks. a WBC counts in retro-orbital blood samples. b–d Wright-Giemsa-stained smears of peripheral blood (b) and bone marrow (c) were taken from a sacrificed moribund mouse, 41 days after cell inoculation. Large blast cells are indicated by arrows. Leukemic blasts clustered in the bone marrow (d) and cultured WEHI cells (e) display similar size and morphology. ×1.000.

Fig. 7.

Combined antileukemic effect of rosemary extract (RE) and 19-nor-Gemini (19-nor) in a mouse model of systemic AML. a Balb/c mice were inoculated with 2 × 10⁶ WEHI cells i.v. and treated with 19-nor-Gemini (0.1 μg) or rosemary extract (4% w/w, mixed with food) either alone or in combination, or vehicle (0.05% ethanol, i.p.). Groups of 6 (experiments 1 and 2) and 10 (experiment 3) mice were used. Animal survival was estimated by the Kaplan-Meier analysis. WBC number (b) and differential count (c) as well as blood chemistry parameters (d) were determined on day 38 in retro-orbital blood samples of 6 mice randomly selected from each treatment group (experiment 3). Means ± SE. b ∗ p < 0.05 vs. untreated control. c^a p < 0.05 and ^b p < 0.01: statistically significant differences in neutrophil/lymphocyte ratios obtained for combination-treated mice (0.44 ± 0.05) vs. untreated controls (0.85 ± 0.11), and healthy animals (0.15 ± 0.02) vs. untreated controls (0.85 ± 0.11); ^c p < 0.05: vs. untreated control.