Progress 06/01/07 to 05/31/10
Outputs
Progress Report 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. 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. The agreement was established in support of Objective 2C of the in-house project, the goal being to test the hypothesis that polyphenol-rich foods and individual plant polyphenols induce apoptotic cell death of leukemia cells in vitro and in vivo. The specific goal of this project was 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 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 was designed to place 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 was to determine if dietary resveratrol or curcumin can prevent growth of the leukemia cells. Mice that became leukemic were treated with vincristine 3x per week and continuously fed the resveratrol or curcumin diets. In this way, we could also see if dietary resveratrol or curcumin increased the efficacy of vincristine leading to an increase in leukemia cell death above vincristine alone. The dietary resveratrol and curcumin failed to prevent leukemia cell growth and did not increase the activity of vincristine. The study was monitored for appropriate progress and administration by annual progress reports sent to the NIH. This agreement expired on 5/31/2010
Impacts
(N/A)
Publications
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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 two 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 engraftment of the leukemia cells, blood was collected weekly from each mouse and the peripheral blood mononuclear cells were stained with fluorescent rat anti- mouse CD45 and mouse anti-human CD19 antibodies. 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 treatment protocol based on metabolic rate and body surface area will be used for the mice, since human doses do not translate equally to mouse doses. The new treatment will be three vincristine injections in the first week at a concentration of 0.5 mg/kg body weight each and the mice will be placed on the diets immediately after the mice go into remission. Methods for detecting bioavailability of resveratrol and curcumin in plasma and tissues of the mice are being developed based on extraction and chromatography methods. 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). Although the exact level of detection has not been determined, the sensitivity is expected to be more than adequate to measure tissue and plasma concentrations from small sample volumes.
Impacts
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Publications
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Significant Activities that Support Special Target Populations This report serves to document research conducted under the Reimbursable Agreement between ARS and the National Institutes of Health (NIH). Additional details of this research project can be found in the report for the parent CRIS 5306-51530-013-00D, Micronutrients and Immune Function. The funds for this agreement were awarded on June 1, 2007. The first experiments to understand whether the antioxidants resveratrol (from grapes) and curcumin (from the spice turmeric) can kill leukemia in an animal model began on July 31, 2007. This project is ongoing and results from the first portion of this study are expected in the next twelve months. The study is monitored for appropriate progress and administration by an annual progress report to be prepared for the National Institutes of Health, National Cancer Institute. The progress report for this study is due to the National Institutes of Health, National Cancer Institute in April 2008.
Impacts
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Publications
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