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1α,25-dihydroxyvitamin D3 (1,25D3) and all-trans retinoic acid (ATRA) are powerful differentiation agents, which have potential for the treatment of myeloid leukemias. However, these compounds have marked toxicity at therapeutic doses. Recently, we have shown that different plant polyphenols (e.g., carnosic acid from rosemary, curcumin from turmeric, and silibinin from milk thistle) markedly enhance the differentiating and antiproliferative effects of low, non-toxic concentrations of 1,25D3 and ATRA in various human leukemia cell lines (HL-60, U937, NB4). In this study, we determined the effects of several polyphenols (carnosic acid and silibinin), vitamin D compounds (1,25D3 and its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol [Ro25-4020]) and their combinations on differentiation and proliferation of the vitamin D3 responsive murine myelomonocytic leukemia cell line (WEHI-3B D-) in vitro. Furthermore, the anticancer effects of Ro25-4020, dry ethanolic extract of rosemary (RosEx) containing 35% carnosic acid, and silibinin alone and in combinations were investigated in syngeneic Balb/c mice bearing WEHI-3B D- cell-derived tumors in vivo. When added alone to the cell culture, carnosic acid and silibinin dose-dependently inhibited the growth of WEHI-3B D- cells, but did not significantly induce cell differentiation, as determined by nitroblue tetrazolium reduction and the expression of F4/80 murine monocyte/macrophage surface marker. However, the combinations of either polyphenol with low, almost ineffective concentrations of 1,25D3 or Ro25-4020 (0.1-1.0 nM) synergistically induced strong differentiating and antiproliferative effects, comparable with those induced by much higher concentrations of the vitamin D compounds (10-100 nM) added alone. Balb/c mice inoculated i.p. with WEHI-3B D- cells developed both a mild leukemia and well-palpable tumors located on the anterior abdominal wall. Intraperitoneal injections of Ro25-4020 (2 μg/mouse, 3 times a week) resulted in an increase in the animal survival rate as compared to the untreated mice. This was associated with a decrease in the total number of white blood cells, a delay in tumor formation and a reduction in tumor size. Similar effects were observed in animals which were fed diet supplemented with RosEx (1% w/w) or silibinin (0.05% and 0.1%, w/w). Importantly, the combined treatment with Ro25-4020 and RosEx or silibinin produced a strong synergistic antitumor effect. These treatments were not accompanied by significant toxicity, as determined by the animal appearance and weight, as well as by blood chemistry analysis. Our results may suggest novel protocols for differentiation therapy of myeloid leukemia that include plant polyphenols and their combinations with differentiation inducers. (Supported by the USA-Israel Binational Science Foundation grant 2001-041)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]