2009 Annual Report
1a.Objectives (from AD-416)
The goal of these studies is to determine whether the antioxidant phytochemicals resveratrol and curcumin can increase survival of individuals with high-risk leukemia. The specific aims are to determine if these plant-derived compounds can kill this leukemia and prevent relapse in vivo by using a mouse model for this disease, and to also analyze the mechanisms of actions, as well as bioavailability of these antioxidants in serum and tissues.
1b.Approach (from AD-416)
Non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice will be engrafted with leukemic cells by tail vein injection. Engraftment will be positively established when the proportion of human leukemia cells in the peripheral blood reaches 1%, as determined by flow cytometric analysis using anti-human CD45 antibody. Both the SEM cell line and underived leukemic cells from patients with t(4;11) leukemia will be used. Cancer preventive efficacy will be determined by two approaches. Leukemic mice will be treated with the chemotherapeutic agent vincristine to induce remission and then fed diets containing either 0.2% w/w resveratrol or 0.5% w/w curcumin to determine if dietary intervention can prevent relapse. Leukemic mice will also be given the phytochemicals intraperitoneally to determine if these phytochemicals can prevent leukemia growth. Minimal residual disease and re-engraftment will be monitored by PCR and flow cytometry, respectively. Sixteen mice will be used in each group. Efficacy of resveratrol and curcumin in the prevention of leukemia will be measured in all experiments by length of survival of the mice compared to control animals and results from necropsy of bone marrow, blood, spleen, and liver. Survival data will be analyzed by Kaplan-Meier survival curves and differences between curves evaluated with the log-rank test. Cell cycle, caspase activation, Bcl-2 levels, and mitochondrial membrane depolarization will be analyzed in the leukemic cells by flow cytometry to determine in vivo mechanisms of cell death induced by resveratrol and curcumin. Bioavailability of parent compounds and metabolites will be measured in serum and tissues for all animals by UPLC-mass spectrometry. Documents Reimbursble with NIH. Log 29934.
This project is part of the goal to study mechanisms of antioxidant activity at a cellular level in leukemic and normal lymphocytes as stated under milestone one in the in-house project. The goal of this project is to determine whether resveratrol, from grapes, and curcumin, from turmeric can kill leukemia cells in an animal model. Forty-eight female NOD/SCID mice were randomly assigned to 3 groups (n = 16 per group). The SEM leukemia cell line carrying the t(4;11) (q21;q23) chromosomal translocation was used for this experiment. Mice were injected with 5 x 106 SEM leukemia cells through the tail vein. To monitor growth of the leukemia cells (engraftment), blood was collected weekly from each mouse and the peripheral blood mononuclear cells were stained with fluorescent antibodies against human cells. Analysis was performed on a flow cytometer to detect the percent of human leukemia cells in the blood. One hundred percent engraftment was achieved in these mice by three weeks post-injection. All mice were treated with treated with 0.5 mg vincristine/kg body wt. to induce remission. The vincristine treatment followed the treatment protocol for humans, which is once per week for four weeks. However, the vincristine was unable to completely inhibit the leukemia growth with this protocol and by the fourth treatment week, the mice began to succumb to leukemia. Therefore, we were unable to place the mice on the diets containing resveratrol or curcumin to determine if these agents could prevent relapse of the leukemia cells. A new experiment has been designed to put 5 week old mice on the resveratrol and curcumin diets for a total of 3 weeks before injecting the leukemia cells. In this experiment, the goal is to determine if dietary resveratrol or curcumin can prevent or delay growth of the leukemia cells. Any mice that become leukemic will be treated with vincristine 3x per week and continuously fed the resveratrol or curcumin diets. In this way, we can also see if dietary resveratrol or curcumin can act together with vincristine to increase killing of the leukemia cells.
Methods for the detecting bioavailability of resveratrol and curcumin in plasma and tissues of the mice have been developed. Separation and quantitation of resveratrol and curcumin was achieved by collaborators using a mass spectrometer. Injections of as little as 1.7 fmole of curcumin and 110 fmole of resveratrol are easily detected over background noise. Recoveries of resveratrol and curcumin from spiked mouse serum was 60% and 73%, respectively (n=6). Plasma and tissue samples will be collected from the mice fed the resveratrol and curcumin diets and analyzed for bioavailability.