Source: UNIV OF MASSACHUSETTS submitted to
USE OF BIOACTIVE FOOD COMPONENTS TO MODULATE VITAMIN D RECEPTOR FUNCTION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0223609
Grant No.
(N/A)
Project No.
MAS00992
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Wood, R.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Center for Agriculture
Non Technical Summary
It is especially important to pursue research on bioactive food components at this time because it has the potential of identifying a novel avenue for targeting dietary prevention strategies to help alleviate the growing medical costs and societal burden related to diet-based problems in the area of obesity and chronic disease. The current project will investigate the effects of a bioactive food component called sulforaphane, which is found in broccoli and other cruciferous vegetables, on basic cellular functions using cell culture and animal (mice) models. The aim of this work is to develop new knowledge that could help improve the dietary prevention of the bone disease osteoporosis and obesity, two important public health problems. Advancement in this area of nutrition research will help address the need to further the national goal of agriculture research to improve the health of the population. Moreover, supplying foods with enriched amounts of bioactive food components represent a potential area of future commercial argriculture opportunity for local Massachusetts farmers, as well as other stakeholders in the food distribution chain, including grocers, restaurateurs, and food manufacturers.
Animal Health Component
(N/A)
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20614401010100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
1440 - Cole crops;

Field Of Science
1010 - Nutrition and metabolism;
Goals / Objectives
Bioactive food components are minor food components that are nutritionally non-essential, in contrast to the well known essential amino acids, fatty acids, vitamins and minerals, but have potentially unique health-promoting properties. Our long-term research goal is to identify bioactive food components that could be safely used as part of the long-term consumption of a health-promoting diet designed to reduce the risk of certain chronic diseases. We envision that future developments in the understanding of the genetic determinants of chronic disease risk will lead to the need for more specifically-targeted diet prescriptions ("individualized nutrition"), which could include recommendations for increased consumption of certain bioactive food components to help reduce specific chronic disease risks and promote optimal health in the population. In the current project, our objectives are to determine to what extent sulforaphane treatment affects: (1) 1,25-dihydroxyvitamin D-mediated gene expression and calcium transport in a human intestinal cell culture system and 1,25-dihydroxyvitamin D-mediated inhibition of adipogenesis in a mouse preadipocyte cell line; and (2) 1,25-dihydroxyvitamin D-mediated intestinal calcium absorption and bone metabolism, and body fat accumulation, in mice fed a control or sulforaphane-fortified diet for 12 weeks. The measurable physiologic and body composition outcomes of the proposed research will be a rigorous test of our primary working hypothesis that sulforaphane affects important physiologic endpoints in the intestine, bone and adipose tissue; namely, an increase in intestinal calcium absorption and an increase in bone mineral density and bone functional breaking strength; as well as, a decrease in fat cell development (as demonstrated by the ability of sulforaphane treatment to increase vitamin D-mediated inhibition of differentiation of preadipocytes to adipocytes in the 3T3L1 mouse preadipocyte cell culture system) and reduce fat tissue accumulation in sulforaphane-fed mice under in vivo conditions. Identification of a bioactive food component that influences vitamin D responsiveness is important because poor intestinal response to the vitamin D hormone is believed to be a one of the factors that can contribute to the development of osteoporosis, an important age-associated chronic bone disease and public health problem. Moreover, recently published studies also indicate that vitamin D hormone can influence adipogenesis, and, therefore, may play a role in the development of obesity, another important nutritional and public health problem. Our expected output from this project is new knowledge demonstrating that a non-essential dietary component (sulforaphane) can alter the gene expression activity of a nuclear transcription factor (vitamin D receptor) to influence whole body physiology (intestinal calcium absorption and bone metabolism, and adipose metabolism). An additional output from this project will be the presentation of our findings at scientific meetings and the publication of scientific journal papers to share this knowledge with scientists and the general public.
Project Methods
In the current project, we describe a two-pronged (cell culture studies and whole animal feeding studies) research approach to test the hypothesis that a specific bioactive food component sulforaphane (found in cruciferous vegetables) affects bone metabolism and adipogenesis. Cell culture studies will be performed in human intestinal cells (Caco-2) for measures of vitamin D-induced 45-Ca transcellular transport and gene expression, and in mouse preadipocytes (3T3-L1)for measurements of adipogenesis in a 2 x 2 factorial design with 1,25-dihydroxyvitamin D and sulforaphane as main treatment factors. Mean treatment effects will be compared by analysis of variance for factorial designs with an interaction term. In addition, a 12-week study with C57BL/6J mice fed control or sulforaphane-fortified AIN93G diet will be conducted to assess sulforaphane effects on 45-Ca calcium absorption, bone mineral density (DEXA), bone breaking strength (Instron 3-point bending), body fat accumulation (DEXA)and gene (PCR) and protein (Western) expression. Success of the studies will be evaluated by scientific peer-review of manuscripts submitted for publication in scientific journals and peer evaluation of data presentations at scientific meetings.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Disciplinary peers and advanced students in Nutritiobal Science Changes/Problems:We are continuing with our cell-based studies to investigate the effects of sulforaphane treatment on cell function, as specified in specific aim 1. For financial reasons, accomplishment of specific aim 2 in the stated time frame has proven to be difficult. As such, we plan to accomplish our second specific aim, focused on the in vivo effects of sulforaphane and vitamin D in an animal model, within the context of our collaborator Dr Zhenhua Liu's new NIFA study that is currently in progress. We will obtain relevant animal tissues as generated to study the effects of this bioactive food component on vitamin D metabolism and function. What opportunities for training and professional development has the project provided?During this reporting period, the work supported by this grant has provided training opportunities for 2 graduate students (B. Savage Master's thesis work and the basis of doctoral dissertation work of S. Hossain) in the Department of Nutrition. These two students were responsible for the generation of the dexperimental data during this reporting period. How have the results been disseminated to communities of interest?As yet, the project has not been completed. Generation of additional data to support publication of our findings in scientific journals is expected to accrue during the final project year. The idea of interaction between steroid hormones and bioactive food components has been put forward to NIFA for further scientific review in the form of a competitive grant application. Data generated from our sulforaphane experiments have already been used successfully in collaboration with a new Nutrition Department Assistant Professor faculty member, Dr. Zhenhua Liu, to obtain a ~$500,000 NIFA competitive grant (PI: Z. Liu, Co-Investigators R. Wood and YC Kim) to study the effects of vitamin D and sulforaphane treatment on Wnt signaling and colon carcinogenesis in a cancer-prone genetic mouse model. These studies have been initiated and will provide valuable tissue biopsies to investigate the in vivo effects of combined bioactive food component sulforaphane and vitamin D during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During this reporting period, we have investigated the effects of the bioactive food component sulforaphane, which is found in relatively high abundance in Brassica vegetables, on the expression of vitamin D-dependent genes in a human breast cancer cell line (MCF7 cells). MCF7 cells are commonly used by scientist investigating the molecular aspects of breast carcinogenesis. The MCF7 cell line has retained several characteristics particular to the mammary epithelium, including the ability of MCF7 cells to respond to estrogen, in the form of estradiol, via estrogen receptors in the cell cytoplasm. This makes the MCF7 cell line an estrogen receptor (ER) positive control cell line and a potential model system to explore the interaction of steroid hormone receptor expression and bioactive food component exposure. We studied initially the expression of genes involved in vitamin D action and metabolism in response to treatment with histone deacetylase inhibitors and 1,25(OH)2D3, the active vitamin D hormone. We found that in MCF7 human breast cancer cells, 1,25(OH)2D3 treatment alone induced the expression of VDR mRNA, CYP24 mRNA and CYP27B1. The vdr gene codes for the intracellular vitamin D receptor; the cyp24 gene codes for the 24- hydroxylase enzyme involved in the inactivation of the vitamin D hormone; and the cyp27b1 gene codes for the 1-hydroxylase gene that activates 25-hydroxyvitamin D. We then investigated to what extent histone deacetylation inhibitors altered the effects of the vitamin D hormone in MCF7 breast cancer cells. We tested the individual effects of two inhibitors, the histone deacetylase inhibitor Trichostatin A (TSA), a Streptomyces metabolite and a well-known potent histone deacetylase inhibitor, and sulforaphane, a bioactive food component from Brassica vegetables that others have shown can affect cell epigenetic status. Interestingly, we found that the inhibitor TSA alone increased expression of VDR mRNA, but SFN alone had no effect. Importantly, VDR mRNA was increased by co-treatment of 1,25(OH)2D3 and TSA compared to 1,25(OH)2D3 alone. This suggest that alterations in histone acetylation status may improve cellular responsiveness to the vitamin D hormone in breast cancer cells by altering the cellular level of the vitamin D receptor, an important controller of cellular rsponse to vitamin D. As expected, 1,25(OH)2D3 markedly increased CYP24 mRNA. Moreover, treatment with either inhibitor TSA or SFN alone increased CYP24 mRNA, suggesting that endogenous expression of the cyp24 gene may be normally suppressed in MCF7 cells by epigenetic mechanisms involving histone acetylation status. However, treatment with the inhibitor sulforaphane with 1,25(OH)2D3 did not cause a further increase in CYP24 mRNA. 1,25(OH)2D3 treatment increased CYP27B1 mRNA expression, but co-treatment with TSA had no additional effect. This may suggest that the vitamin D-activating gene, in contrast to the vitamin D-deactivating gene is not under epigenetic control in these cells. Overall, these initial findings in MCF7 cells are consistent with our working hypothesis of a potential role important role of epigenetic factors in controlling the cellular response to the vitamin D hormone. Studies are continuing to assess the possible role of the vitamin D hormone in cellular epigenetic remodelling and the role of bioactive food components on cell function.

Publications


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience:Disciplinary peers and advanced students in Nutritiobal Science. Changes/Problems:We are continuing with our cell-based studies to investigate the effects of sulforaphane treatment on cell function, as specified in specific aim 1. For financial reasons, accomplishment of specific aim 2 in the stated time frame has proven to be difficult. As such, we plan to accomplish our second specific aim, focused on the in vivo effects of sulforaphane and vitamin D in an animal model, within the context of our collaborator Dr Zhenhua Liu's new NIFA study that is currently in progress. We will obtain relevant animal tissues as generated to study the effects of this bioactive food component on vitamin D metabolism and function. What opportunities for training and professional development has the project provided?During this reporting period, the work supported by this grant has provided training opportunities for 2 graduate students (B. Savage Master's thesis work and the basis of doctoral dissertation work of S. Hossain) in the Department of Nutrition. These two students were responsible for the generation of the dexperimental data during this reporting period. How have the results been disseminated to communities of interest?As yet, the project has not been completed. Generation of additional data to support publication of our findings in scientific journals is expected to accrue during the final project year. The idea of interaction between steroid hormones and bioactive food components has been put forward to NIFA for further scientific review in the form of a competitive grant application. Data generated from our sulforaphane experiments have already been used successfully in collaboration with a new Nutrition Department Assistant Professor faculty member, Dr. Zhenhua Liu, to obtain a ~$500,000 NIFA competitive grant (PI: Z. Liu, Co-Investigators R. Wood and YC Kim) to study the effects of vitamin D and sulforaphane treatment on Wnt signaling and colon carcinogenesis in a cancer-prone genetic mouse model. These studies have been initiated and will provide valuable tissue biopsies to investigate the in vivo effects of combined bioactive food component sulforaphane and vitamin D during the next reporting period. What do you plan to do during the next reporting period to accomplish the goals?The next reporting period will reflect the ongoing investigations in cell culture model to better understand the mechanisms underlying the effects of sulforaphane on cell function. To this end, we will perform experiments to investigate the potential role of Wnt signaling in cells treated with vitamin D and histone deacetylase inhibitors. Wnt signaling is a major cell communication systems involved in cellular proliferation and differentiation.

    Impacts
    What was accomplished under these goals? During this reporting period, we have investigated the effects of the bioactive food component sulforaphane, which is found in relatively high abundance in Brassica vegetables, on the expression of vitamin D-dependent genes in a human breast cancer cell line (MCF7 cells). MCF7 cells are commonly used by scientist investigating the molecular aspects of breast carcinogenesis. The MCF7 cell line has retained several characteristics particular to the mammary epithelium, including the ability of MCF7 cells to respond to estrogen, in the form of estradiol, via estrogen receptors in the cell cytoplasm. This makes the MCF7 cell line an estrogen receptor (ER) positive control cell line and a potential model system to explore the interaction of steroid hormone receptor expression and bioactive food component exposure. We studied initially the expression of genes involved in vitamin D action and metabolism in response to treatment with histone deacetylase inhibitors and 1,25(OH)2D3, the active vitamin D hormone. We found that in MCF7 human breast cancer cells, 1,25(OH)2D3 treatment alone induced the expression of VDR mRNA, CYP24 mRNA and CYP27B1. The vdr gene codes for the intracellular vitamin D receptor; the cyp24 gene codes for the 24-hydroxylase enzyme involved in the inactivation of the vitamin D hormone; and the cyp27b1 gene codes for the 1-hydroxylase gene that activates 25-hydroxyvitamin D. We then investigated to what extent histone deacetylation inhibitors altered the effects of the vitamin D hormone in MCF7 breast cancer cells. We tested the individual effects of two inhibitors, the histone deacetylase inhibitor Trichostatin A (TSA), a Streptomyces metabolite and a well-known potent histone deacetylase inhibitor, and sulforaphane, a bioactive food component from Brassica vegetables that others have shown can affect cell epigenetic status. Interestingly, we found that the inhibitor TSA alone increased expression of VDR mRNA, but SFN alone had no effect. Importantly, VDR mRNA was increased by co-treatment of 1,25(OH)2D3 and TSA compared to 1,25(OH)2D3 alone. This suggest that alterations in histone acetylation status may improve cellular responsiveness to the vitamin D hormone in breast cancer cells by altering the cellular level of the vitamin D receptor, an important controller of cellular rsponse to vitamin D. As expected, 1,25(OH)2D3 markedly increased CYP24 mRNA. Moreover, treatment with either inhibitor TSA or SFN alone increased CYP24 mRNA, suggesting that endogenous expression of the cyp24 gene may be normally suppressed in MCF7 cells by epigenetic mechanisms involving histone acetylation status. However, treatment with the inhibitor sulforaphane with 1,25(OH)2D3 did not cause a further increase in CYP24 mRNA. 1,25(OH)2D3 treatment increased CYP27B1 mRNA expression, but co-treatment with TSA had no additional effect. This may suggest that the vitamin D-activating gene, in contrast to the vitamin D-deactivating gene is not under epigenetic control in these cells. Overall, these initial findings in MCF7 cells are consistent with our working hypothesis of a potential role important role of epigenetic factors in controlling the cellular response to the vitamin D hormone. Studies are continuing to assess the possible role of the vitamin D hormone in cellular epigenetic remodelling and the role of bioactive food components on cell function.

    Publications


      Progress 10/01/12 to 09/30/13

      Outputs
      Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided an opportunity for training and thesis research for a Master's degree students here at the University of Massachusetts. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will complete additional studies in human colon cancer cells and investigate to what extent 1,25-D and sulforaphane interact to promote vitamin D action. In addition, we will determine the effects of 1,25-D and sulforaphane on epigenetic changes (histone acetylation status) in Caco-2 cells.

      Impacts
      What was accomplished under these goals? We found that treatment of 3T3-L1 cells, a cell culture model of adipogenesis, with the active hormone form of vitamin D (1,25-D), induced the expression of vitamin D-dependent genes. Next, we tested the hypothesis that sulforaphane, a bioactive food component from cruciferous vegetables with histone deacetylase inhibitor activity, significantly increased the expression of the cyp 24 gene. We also observed that 1,25-D increased cyp24 expression; however, in this cell line sulforaphane had no significant effect, although another histone deacetylase inhibitor TSA did increase cyp24 expression. This observation suggests that there may be different responses in certain cell types or cell lines that will need further exploration in the future.

      Publications


        Progress 10/01/11 to 09/30/12

        Outputs
        OUTPUTS: During the past funding period, we have conducted a series of experiments to test the hypothesis that the bioactive food compound sulphoraphane would increase vitamin D-mediated gene expression. Sulphoraphane has many biochemical effects, including histone deacetylase activity. The latter effect is relevant to vitamin D function because acetylation status is a known regulator of vitamin D function. We addressed the question of possible sulphoraphane-vitamin D interaction initially in mouse preadipocytes (3T3-L1) grown in cell culture by measuring the response of the cyp24 gene, a well-known marker of vitamin D action in cells. These activities contribute to our project goal of evaluating the effects of bioactive food components on vitamin D receptor function. We have disseminated the findings of these initial studies with the academic community by graduate student poster presentations at the University of Massachusetts. We plan in the near future to submit some of this work for presentation at a national scientific meeting in Spring 2013. PARTICIPANTS: Participation in this project during the past funding period included the three collaborators (R. Wood, YC Kim and Y. Park). Drs. Wood and Kim met on a regular basis with students to discuss the study findings, and Dr Park provided important access and technical expertise on needed laboratory equipment and student technical training. In addition to the three scientific collaborators, two graduate students from the Department of Nutrition at the University of Massachusetts (E. Ahn and B. Gray) generated important experimental data for the project, as did an undergraduate student J. Larnard from Commonwealth College at (UMass), as well as a volunteer post-baccalaureate C. Muriithi. Work conducted by the two graduate students formed the basis of their Master's thesis work, and the undergraduate student will be using this work as a basis for his honor's capstone project. TARGET AUDIENCES: The target audience for this project is academic and industry scientists where the change in knowledge made by our project activities may encourage additional investigations of the potential importance of bioactive food components in promoting health. In addition, increased awareness of the healthful components of plant-based foods will encourage the general public to consume more fruits and vegetables in their diets, which is consistent with the goals of several governmental guidelines aimed at improving the health of the population and reducing health care costs. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        We found that treatment of 3T3-L1 preadipocytes with the active form of vitamin D caused a significant and robust increase in expression of the cyp24 gene, as measured by an increase in cyp24 mRNA expression, as measured by the polymerase chain reaction. Then, using the cyp24 response as a biomarker of cellular vitamin D responsiveness, we found that treatment with sulphoraphane and vitamin D together resulted in a significant enhancement of the vitamin D response (cyp24)in these cells. These observations are consistent with the hypothesis that altering cellular acetylation status with bioactive compounds can alter cellular vitamin D responsiveness. The latter has implications for potential health benefits from the consumption of diets with an increased number of foods containing sulphoraphane, such as the Brassica vegetables. Lower vitamin D responsiveness in cells has been implicated as a possible important factor in the development of osteoporosis and the development of various cancers, including prostate, breast and colon cancer.

        Publications

        • No publications reported this period


        Progress 10/01/10 to 09/30/11

        Outputs
        OUTPUTS: Activities during the reported period include the completion of several seminal experiments in mouse 3T3L1 cells to determine to what extent the action of the active vitamin D hormone (1,25-dihydroxyvitamin D) is affected by the bioactive food component sulforaphane. PARTICIPANTS: Participants in the project during the stated period include Dr Young-Chuel Kim from the Department of Nutrition at the University of Massachusetts. Dr. Kim is a co-investigator on the project and has been consulted concerning the design of studies done in mouse 3T3L1 cells, a preadipocyte cell line. Additional participants included Ms. Brianna Gray, a Master's degree student in the Department of Nutrition who instructed Ms. Eunjee Ahn, a Master's degree student in the Department of Nutrition on the proper care and feeding of 3T3L1 cells and how to do RT-PCR for measuring gene expression. Ms. Eunjee Ahn was primarily responsible for carrying out the majority of experiments conducted with sulforaphane during the time period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The findings to date has been consistent with our original working hypothesis. Thus, we will continue to pursue our original specific aims, which will be conducted in both cell culture and in vivo animal models, during the upcoming time periods.

        Impacts
        Our activities in the laboratory during the stated period has resulted in a change in knowledge such that we now have shown that sulforaphane can increase the responsiveness of gene expression when the active vitamin D hormone is presented to 3T3L1 cells. We will continue our studies of this bioactive food component to evaluates its effect on physiological processes in fat cells and in intestinal cells grown in cell culture.

        Publications

        • No publications reported this period


        Progress 10/01/09 to 09/30/10

        Outputs
        OUTPUTS: New PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        New

        Publications

        • No publications reported this period