1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) is a powerful differentiation agent, which has potential for treatment of
myeloid leukemias and other types of
cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that
carnosic acid, the major rosemary
polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D(3) in human
myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse
leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D(-) murine myelomonocytic
leukemia cells induced by 1 nM 1,25D(3) or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by
carnosic acid (10 microM) or the
carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0 + G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p.
injections of 2 microg
Ro25-4020 in Balb/c mice bearing WEHI-3B D(-)
tumors produced a significant delay in
tumor appearance and reduction in
tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than
Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 microg
Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing
hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a
vitamin D derivative can cooperate not only in inducing
leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for
chemoprevention or differentiation
therapy of
myeloid leukemia.