Progress 07/01/04 to 06/30/09
Outputs
OUTPUTS: Over the life of this project several academic seminars were given at the local, state, national, and international level. Reagents developed and characterized during the life of this project (PCR primer sets, antibodies developed, novel applications of in-hand commercial reagents) were freely shared with interested parties, namely other academic institutions and PIs. PARTICIPANTS: Dr. Vince Gallichio, Clemson University. Dr. Abigail Babcock, former Ph.D. trained during the project. Dr. Alison Springs, former Ph.D. trained during the project. TARGET AUDIENCES: Academia and biomedical professionals. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The project investigated the potential immunotoxicity of plant-based aryl hydrocarbon receptor (AhR) ligands. The AhR is a ligand-activated transcription factor associated with a battery of gene products related to xenobiotic metabolism and excretion, anti-oxidant activity, cell cycle regulation, development, and several other physiological outcomes. Over-activation of the AhR leads to severe toxicity, and especially immunotoxicity and developmental abnormalities. Since many well-characterized AhR ligands are man-made pollutants in the environment, we wanted to determine if plant-based AhR ligands are toxic. While this project focused mainly on a synthetic version of Indirubin, the active ingredient in certain Chinese herbals, other synthetic derivatives of indirubin were investigated as well. Each of these indirubins was shown to be a potent AhR ligand, with strong induction of CYP1A1. In general, these indirubins were anti-inflammatory in that the cytokines IL-6, TNF-alpha, and IL-1 were reduced in LPS-stimulated mouse macrophages, and nitric oxide was inhibited. Ironically, COX-2 is induced by select indirubins (e.g., E804), which is most likely due to direct induction through AhR activation. It has been known for quite some now that AhR ligands such as TCDD and planar PCBs have therapeutic potential in the treatment of pathologies that involve over-proliferation of cells. However, the long term toxicity of these AhR-ligands prevents clinical application because these compounds are slowly metabolized and therefore the AhR remains in a state of chronic activation. Plant-based AhR ligands may hold promise in certain clinical situations because they are metabolized fairly quickly and therefore are associated with less toxicity than the more potent agents such as TCDD and planar PCBs.
Publications
- Rollins Smith, L., Rice, C.D., and Grassman, K. 2007. Immunotoxicology of Amphibians, Fish, and Wildlife; Chapter 22, In: Immunotoxicology and Immunopharmacology, Target Organ Series, 3rd edition. (Robert W. Luebke, Ian Kimber, and Robert V. House, Eds). CRC Press, Boca Raton, FL USA.
- Babcock, A.S., and Rice, C.D. 2010. Differential effects of indirubin-3-(2,3 dihydroxypropyl)-oximether (E804) and PCB-126 on AhR signaling, gene expression profiles, and cellular functions in RAW264.7 murine macrophages. in review.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in multiple aspects of cell cycle control, inflammation, steroid/drug/xenobiotic detoxication processes, and circadian rhythms. The most well characterized AhR ligands are environmental contaminants sharing a common planar aromatic structure, such as some polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs). PAHs and HAHs with the highest affinity for the AhR and with the greatest resistance to metabolism are the most potent in terms of their effects on cell cycling, inflammation, metabolism, and other consequences of AhR-binding. Natural AhR ligands, such as the plant-derived indigoids and indirubins bind very well to the AhR, but are metabolized more quickly than most environmental contaminants, and thus do not seem to exert extreme toxicity. This study examined the relative potency and toxicity of several derivatives of natural indirubin, an indigoid compound found in certain herbal remedies of far-eastern origin. Indirubin-3-monoxime is a potent anti-inflammatory agent in the mouse macrophage cell line RAW.274, but has little CYP1A activity (associated with AhR-induced metabolism of xenobiotics). However, E804 and BIO, two other synthetic derivatives of natural indirubin were potent inducers of CYP1A gene expression, and E804 in particular was very potent in inducing COX-2, a gene product associate with elevated prostaglandin synthesis and inflammation. E804 also had the most impact on cell cycling, which indicates that the affinity of E804 for the AhR is similar to that of the most toxic PAHs and HAHs, and thus may be a good candidate for anti-cancer therapies, providing this synthetic indigoid is metabolized much more quickly than PAHs and HAHs. The potential uses of plant-derived aryl hydrocarbon ligands, such as indirubins and indigoids, have been presented to several groups of interested parties. These parties include Centers for Alternative Medicine (USC) and Marine Natural Products (MUSC). PARTICIPANTS: Three graduate students were trained on the project. Several colleagues at other Institutions were used as resources for interpreting data and findings. Colleagues at MUSC, The College of William and Mary, and Woods Hole Oceanographic Institute were routinely involved in discussions and interpretations. TARGET AUDIENCES: The scientific community associated with immunopharmacology, pharmacology, environmental toxicology, immunology, and cancer biology. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
For the most part, this study focused on the basic cellular and molecular aspects of how indirubins affect cells of the immune system, including natural cell cycling of these cells. The outcome of the study is gained knowledge of how indirubins affect cells, namely that they affect cell cycling, detoxification processes, and inflammation. These findings should open the door for future studies involving Phase I/II trials in humans for various inflammatory disorders and select cancers. It has been known for some time that one of the best anti-cancer compounds is dioxin (TCDD) because of its potent effects on cell cycling. The extreme toxicity of TCDD, however, precludes its use in a clinical setting. Our findings that certain indigoid derivatives such as indirubin-3-monoxime and E804 are also potent cell cycle disruptors, but with much less potential for toxicity should provide the opportunity to re-examine AhR-ligands as potential anti-inflammatory and anti-cancer agents.
Publications
- Springs, A.E., and Rice, C.D. 2006. The effects of indirubin-3-monoxime, a novel AhR ligand, on stress and toxicity-related gene/protein expression in human U937 cells undergoing differentiation and activation. J. Immunotoxicology. 3:1-10.
- Babcock, A.S., and Rice, C.D. 2009. The anti-inflammatory properties of select derivatives of indirubin. Molecular Immunology. In review.
- Matsebatlela, T.M., and Rice, C.D. 2009. Didox inhibits reactive oxygen species (ROS) production, inflammatory events, phagocytosis activity and NF-B (p65) nuclear translocation in LPS-activated Raw 264.7 murine macrophages. Journal of Pharmacology and Experimental Therapeutics. In review.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: We have shown that indirubin, and indirubin-3-monoxime, and E804 have dramtic anti-inflammatory properties when tested on RAW-1 mouse macrophages. Inducible nitric oxide synthase activity, IL-6 production, and COX-2 are inhibited by very low levels. Both indirubin-3-monoxime and E804 stop cell cylcling by inducind cyclin-dependent kinase-1. Their ability to affect functions through the AhR is less than what is seen in PCB-126, a strong AhR ligand and environmental pollutant.
PARTICIPANTS: Clemson University only. Two Ph.D. students and 4 undergraduates participated.
TARGET AUDIENCES: Life Science specialists,
Impacts
Our ongoing studies confirm the potential for indirubins as anti-cancer and anti-inflammatory agents.
Publications
- None for this period (2007).
- Three publications are in preparation for submission by the summer, 2008.
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Progress 01/01/06 to 12/31/06
Outputs
Previous work with indirubins in our lab were carried out with U928 histiocytes, which are good models for gene expression analysis, but poor for evaluating macrophage functional endpoints. Current work using a more differentiated macrophage (mouse RAW) will model both gene expression and functional endpoints expected in humans. Indirubins have a biomodal impact on cells of the immune system in that that they are active through the aryl hydrocarbon receptor, thus may have potential for immunotoxicity, but also act as cyclin-dependent kinase inhibitors. Cyclin-dependent kinase inhibitors inhibit cellular proliferation, which may be good for inhibiting unwanted cellular proliferation seen in autoimmunity and cancer, and may be anti-inflammatory. In RAW cells, indirubins appear to be anti-inflammatory.
Impacts
Previous work with indirubins in our lab were carried out with U928 histiocytes, which are good models for gene expression analysis, but poor for evaluating macrophage functional endpoints. Current work using a more differentiated macrophage (mouse RAW) will model both gene expression and functional endpoints expected in humans. The impact of this work is to elucidate the effects of indirubins on inflammation.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
We treated human U937 histiocytic lymphoma cells with indirubin-3'-monoxime in four different regimens, representing various stages of PMA-induced differentiation and further activation with LPS. Gene expression profiles were monitored using commercial macroarrays for 96 select stress and toxicity genes. The four treatment regimens were (A) the monocyte model: U937 cells were treated with 1 micromolar indirubin-3-monoxime or carrier control for 24 hrs; (B) the differentiated model: PMA-differentiated U937 cells were treated with 1 micromolar indirubin-3-monoxime or carrier control for 24 hrs; (C) the differentiation process model: U937 cells were treated for 24 hrs with 1 micromolar indirubin-3-monoxime or carrier control, followed by PMA-induced differentiation over a 24 hr period; and (D) the LPS-activated model: PMA-differentiated and LPS-activated U937 cells were treated with 1 micromolar indirubin-3-monoxime or carrier control for 24 hrs. Indirubin-3-monoxime
treatment caused a 12.7-, 2.1-, 3.2-, and 4.5-fold increases in CYP1A1 in regimes A, B, C, and D, respectively. Indirubin-3-monoxime treatment also enhanced cyclooxygenase 2 expression in PMA-differentiated cells, but reduced the expression of macrophage inflammatory protein, catalase, heme oxygenase-1, glutathione peroxidase, and manganese superoxide dismutase; a scenario consistent with oxidative stress. Macroarray validity was confirmed by RT-PCR using CYP1A1, COX-2, MIP-beta, and GADPH as target genes, and at the protein level for CYP1A1, COX-2, and beta-actin. To our knowledge, this is the first study to examine the effects of indirubin-3-monoxime in a human cell line relevant to the immune system. Overall, our study suggests that indirubin-3-monoxime is either a classical AhR ligand, or that it modulates endogenous AhR-regulated activity.
Impacts
Understanding the molecular mechanisms assosicated with plant-based neutraceutical toxicity allows us to predict the relative toxicity of other compounds with similar chemical structures. Our research clearly shows how this compound (indirubin) can be both an anti-cancer agent and and a immunomodulator.
Publications
- Springs, E.B.A., Rice, C.D. The effects of indirubin-3-monoxime, a novel AHR ligand, on stress and toxicity-related gene/protein expression in human U937 cells ungergoing differentiation and activation. Journal of Immunotoxicology. 3:1-10, 2006
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Progress 01/01/04 to 12/31/04
Outputs
Nutraceuticals are a multibillion dollar industry in the USA. For reasons of both marketing strategy and antidotal evidence, oriental herbals are touted as having great promise for ameliorating a variety of diseases. Oriental herbal supplements are perhaps the best example of products readily consumed by the American public without adequate knowledge of efficacy or safety. As a case in point, an active ingredient in Chinese herbals prepared from Polygonum tinctorium has been isolated that inhibits cyclin-dependent kinases and glycogen synthase kinase, thereby inhibiting proliferation of leukemia cells. Extracts from this plant enhance detoxication processes and they have potent anti-inflammatory properties. The biological activity of the active ingredient has been traced to an indole-metabolite of tryptophan that is further metabolized to two end products, indigo and indirubin. Indirubin is now known to be the active anti-neoplastic and anti-inflammatory ingredient in
Polygonum tinctorium extracts. Indirubin binds to the cytosolic aryl hydrocarbon receptor (AhR), a transcription factor, with high affinity and activates the translocation of the ligand-receptor complex to the nucleus, leading to the expression of a suite of genes. Surprisingly, indirubin binds to the AhR with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like affinity. High affinity binding of indirubin to the AhR is an alarming observation because most agents that bind with high affinity to the AhR are immunotoxic. Our preliminary data show that indirubin is a potent inducer of CYP1A1 in differentiated human macrophages. Furthermore, indirubin potentiates LPS-stimulated macrophage activation. In addition, indirubin alters the expression of indoleamine 2,3,-dioxygenase (IDO), an enzyme not yet described as being linked to Ahr activation. IDO modulation by indirubin is intriguing because several recent studies confirm that tryptophan metabolism and reduction by IDO alters T-cell
responses. Taken together with other preliminary data, including gene array analysis, RT-PCR, and protein expression profiles, it appears that indirubin has the potential to act as a TCDD-like compound. To date, information regarding potentially adverse effects of novel plant-derived AhR ligands like indirubin on immune function is lacking. Of particular importance to immune function, cellular and molecular events involved in macrophage differentiation and activation leading to altered proinflammatory events may be affected by plant-based AhR- ligands. This project tests the hypothesis that indirubin is a potent immunotoxic compound. Thus far, the expression profile of 96 stress and toxicity genes in phagocytes have been quantified following expsoure to indirubin. Results show clearly that this compound has sever immunotoxic potential.
Impacts
For reasons of both marketing strategy and antidotal evidence, oriental herbals are touted as having great promise for ameliorating a variety of diseases. Oriental herbal supplements are perhaps the best example of products readily consumed by the American public without adequate knowledge of efficacy or safety. As a case in point, an active ingredient in Chinese herbals enhances detoxication processes and has potent anti-inflammatory properties. The biological activity of the active ingredient has been traced to an indole-metabolite of tryptophan that is further metabolized to two end products, indigo and indirubin. Indirubin is now known to be the active anti-neoplastic and anti-inflammatory ingredient in Polygonum tinctorium extracts. Indirubin binds to the cytosolic aryl hydrocarbon receptor (AhR), a transcription factor, with high affinity and activates the translocation of the ligand-receptor complex to the nucleus, leading to the expression of a suite of genes.
Surprisingly, indirubin binds to the AhR with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like affinity. High affinity binding of indirubin to the AhR is an alarming observation because most agents that bind with high affinity to the AhR are immunotoxic. To date, information regarding potentially adverse effects of novel plant-derived AhR ligands like indirubin on immune function is lacking. Of particular importance to immune function, cellular and molecular events involved in macrophage differentiation and activation leading to altered proinflammatory events may be affected by plant-based AhR- ligands.
Publications
- The effects of indirubin-3"-monoxime, a novel AhR ligand, on stress and toxicity-related gene/protein expression in human U937 cells. 2005. Journal of Immunotoxicology, submitted
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