The Center supports four primary
Research Project 1: Botanicals Targeting 5 Signaling Pathways to Prevent Prostate Cancer
Botanical dietary supplements are used by consumers to prevent and to treat prostate cancer. Recently, an animal prostate cancer model has allowed the safety and efficacy of these supplements to be tested. We have established colonies of TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, estrogen receptor-deficient (ERαKO and ERβKO) mice, and ER-deficient mice expressing the TRAMP transgene.
Exciting data from our model clearly show that ERαKO mice are highly protected against PDC (poorly differentiated carcinoma) of the prostate and ERβKO mice are highly susceptible to PDC. In addition, we recently have found that phytoestrogens, phytosterols, and oxysterols can inhibit hedgehog signaling, a pathway important in prostate cancer. And surprisingly we find that oxysterols can bind ERb 100X better than ERa with a Kd of ~20nM.
Our overall hypothesis is that differential regulation by 5 botanicals and their antioxidant/phytoestrogen/phytosterol components, whose actions are mediated by 5 key pathways, will be effective in prostate cancer prevention and treatment. The 5 key pathways to be examined are the (anti)oxidant(Nrf2/Keap1 and NADPH oxidase) pathways and their interactions with the NF-kappaB-, estrogen-, and hedgehog-signaling pathways. Our goals are to characterize responses of key prostate tumor biomarkers to these botanicals and to provide novel molecular mechanisms for these responses useful in conducting later clinical trials.
Aim 1: Determine in vitro in prostate cancer cell lines the effects of the 5 botanicals and selected pure compounds on the activities, protein concentrations, and mRNAs expressed in 5 key pathways;
Aim 2: Perform in vivo cancer prevention trials in single and double transgenic TRAMP mice to test if the cancer protective activity of some pure compounds and botanicals will be dependent upon Keap1/Nrf2 and hedgehog pathway components;
Aim 3: Test potential active ingredients and complex botanical extracts from Aims #1 or #2 with the same ingredients used in much faster in vivo human xenograft models of prostate cancer; and
Aim 4: Identify responses associated with cell proliferation, differentiation, apoptosis, and the five key pathways in vivo in prostate tissues in response to botanicals, correlate these responses with tumorigenesis, and continue the correlations with the components of the oxidant, inflammatory, hedgehog, and/or estrogen pathways. These studies will provide fundamental insights into prostate tumor biology, and help provide biomarkers to assess the role of these botanicals in modifying the incidence and progression of prostate tumors in humans.
Research Project 2: Botanical Phenolics on Oxidative/Nitrosative Signaling Pathways: Implication for Cerebral Ischemia
Excessive oxidative and nitrosative (O/N) activities in the brain have been recognized as the basis for the neuronal excitotoxicity and glial cell inflammatory responses that underlie many neurodegenerative diseases, including stroke. Recent studies have discovered the role of NADPH oxidase as an important source of reactive oxygen species (ROS) in brain cells. Activation of NADPH oxidase is associated with a number of downstream signaling pathways, including the NF-kB pathway that induces iNOS and produces nitric oxide (NO). Among other reactions, NO can regulate protein structure and function by interacting with protein reactive cysteine thiol residues to form S-nitrosothiol. Many proteins associated with NADPH oxidase signaling pathways appear to be modified by S-nitrosylation. While the mechanisms are unknown, there is considerable evidence to suggest that a number of botanical compounds may protect the brain against O/N insults, including those resulting from cerebral ischemia.
In keeping with the central goal of this Center, Project 2 will identify new botanicals and test the hypothesis that stroke-mediated neuronal excitotoxicity and glial inflammatory responses are due in part to ROS from NADPH oxidase and subsequent S-nitrosylation of key proteins in the NADPH oxidase signaling pathways.
In Aim 1, neurons and astrocytes/microglia will be used to screen botanical extracts and pure compounds isolated from the extracts for their ability to suppress NMDA-mediated neuronal excitotoxicity and cytokine-induced inflammatory responses in glial cells. The effects of botanicals on S-nitrosylation of proteins and differentially-expressed mRNA and proteins in neurons and glial cells will be investigated using the newly developed NitroDIGE protocol and mRNA mega-sequencing facilities in the Interactions Core.
In Aim 2, the mouse focal cerebral ischemia model induced by occlusion of the middle cerebral artery will be used to investigate whether dietary supplementation with specific botanicals offers protective effects against cerebral ischemia/reperfusion damage. These studies will include using mice deficient in NOX2 and iNOS to test the effects of botanicals on behavioral outcome, neuronal survival, glial activation, and inflammatory responses.
Aim 3 will test the hypothesis that identified botanicals offers neuroprotective effects by reducing cerebral ischemia/reperfusion damage through activation of the Nrf2/Keap1 antioxidant signaling pathway. In these studies, astrocyte- or neuron-targeted conditional Keap1-null mice will be used.
In Aims 2 and 3, we will also identify changes in S-nitrosylated and inflammatory proteins and mRNA associated with oxidative and anti-oxidative signaling pathways in distinct brain regions. The in vitro and in vivo approaches of Project 2, together with support from the unique and state-of-the-art core facilities of this Center, will combine to identify specific cellular and molecular mechanisms through which novel botanicals and/or their active components may exert beneficial effects and protect against stroke damage.
Research Project 3: Antioxidant Botanicals and Antimicrobial Defenses
The primary aim of this project is to conduct pre-clinical screening of selected antioxidant botanicals for their potential to modulate in vivo anti-microbial and anti-viral activity. Crude extracts as well as putative bioactive components of each botanical will be tested both in vivo and in vitro. Two murine bacterial infection models (i.e., E. coli-mediated sepsis and systemic listeriosis) and a novel murine HIV model will be used. Clinically relevant outcomes, such as fever, food intake, lethargy, and bacterial/viral clearance will be monitored along with a common set of inflammation/infection biomarkers (e.g., TNF-a, IL-1b, IL-6, MCP-1, IL-12, IL-10).
The working hypothesis is that botanicals with potent antioxidant activity will affect the redox status of host phagocytes such that inflammatory, anti-bacterial and anti-viral responses will be altered. As a consequence of this altered cellular redox the host will experience discernable benefits (e.g., lower fever, enhanced pathogen clearance, reduced systemic inflammation).
Exciting and novel preliminary findings show that Sutherlandia and elderberry extracts affect bacterial clearance and survival in the E. coli sepsis model. A murine macrophage cell line, RAW 264.7 cells, will be used to profile alterations in cellular events of importance to the inflammatory, anti-bacterial, and anti-viral pathways in response to the botanical compounds including: NADPH oxidase (NOX) and inducible nitric oxide synthase (iNOS) activity, NF-kB activation and Nrf2-Keap1 signaling.
Additionally, mice with a conditional deletion of Keap1 in cells from the myeloid lineage (i.e., phagocytes) will be used to evaluate the interaction between Nrf2-dependent gene expression and antioxidant botanicals in shaping host response to infection. Mice with a myeloid-specific deletion of Keap1 should have greatly enhanced Nrf2 signaling in phagocytes such that redox status within these innate immune cells is altered resulting in diminished inflammation. These unique mice will be used to explore interactions between Nrf2 signaling in phagocytes and effects of various botanicals on the innate immune system and the host response to infection.