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, 1α,25-dihydroxyvitamin D₃ (1,25D₃), is a promising approach to treatment of acute myeloid leukemia (AML); however, 1,25D₃ 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 1,25D₃ 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 1,25D₃, in the induction of differentiation (EC₅₀ = 0.059 ± 0.011, 0.275 ± 0.093 and 0.652 ± 0.085 nM, respectively) and growth arrest (IC₅₀ = 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 1,25D₃ 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 1,25D₃ analogues and rosemary agents against mouse AML warrants further exploration of this therapeutic approach in translational models of human leukemia.

A non‐functioning vitamin D receptor predisposes to leukaemoid reactions in mice

“…The latter scenario could explain the myelodysplastic changes present in affected VDR mutant mice, and is also supported by in vitro studies [5] showing that 1,25(OH) 2 D 3 suppresses the proliferation of murine granulocyte-macrophage progenitor cells, and is able to induce differentiation in myeloid leukaemia cells [6,7,31]. In addition, vitamin D analogues decrease clonal growth and induce differentiation in human acute myeloid leukaemia (AML) cells [32,33], and prolong survival in murine models of AML [34,35]. Therefore, vitamin D signalling through the VDR may also be an important inhibitory factor in the onset and progression of myeloproliferative and myelodysplastic diseases.…”

Cytotoxic Effect of Rosmarinus officinalis L. Leaf Extracts on Tumor Cell Line

“…The cytotoxic activity in the present study may due to presence of compounds that were detected such as alkaloids, flavonoids, phenols, saponins, glycosides, terpene and tannins especially the most active antioxidative constituents of rosemary are phenolic diterpenes (carnosic, carnosol, rosmanol, rosmadial, 12-methoxycarnosic acid, epi-, and iso-rosmanol) and phenolic acids (rosmarinic and caffeic) [16]. Compound in rosemary have shown a variety of pharmacological activities for cancer chemoprevention and therapy in in vitro and, in vivo models [17]. Study indicated that ethanoilc extract of rosemary given to rats showed hepatoprotective and antimutagenic effects due to high content of phenolic compounds with high antioxidant activity [18].…”

Developmental Ultrastructure of Glandular Trichomes of Rosmarinus officinalis: Secretory Cavity and Secretory Vesicle Formation

“…These include Artemisia (Polichuk et al 2010), Cannabis (Marks et al 2009), Humulus (Wang et al 2008), Mentha (Turner et al 2000), Montanoa (Robles- Zepeda et al 2009), Ocimum (Iijima et al 2004), Salvia (Baran et al 2010), Stevia (Bondarev et al 2010), Tetradenia (Gairola et al 2009), Zeyheria (Machado et al 2006), and Rosmarinus (Shabtay et al 2008).…”

“…In particular, the essential oil secreted by glandular trichomes of rosemary leaves has been used as an antiseptic, astringent, and food preservative for a long time (Pokorny 2008). These glandular trichomes of the genus Rosmarinus are major accumulating sites for various secondary metabolites that have the helpful properties of inhibiting skin tumorigenesis (Huang et al 1994), exhibiting antioxidant activity (Richheimer et al 1999), protecting red blood cells (Haraguchi et al 1995), and exhibiting antileukemic activity (Shabtay et al 2008).…”