Curcumin regulates transcription in Dictyostelium discoideum

Botanicals are widely used as dietary supplements and for the prevention and treatment of a myriad of diseases ranging from the common cold to depression. One in four Americans uses a botanical as part of their primary healthcare with billions of dollars spent annually. Curcumin, the active ingredient in turmeric, has gained a lot attention in recent years for its therapeutic uses and has been linked to a wide spectrum of pharmacological effects including anti-carcinogenic, anti-inflammatory, Alzheimer's prevention and antioxidant activity. Despite the large and growing interest in the use of botanicals such as curcumin in disease treatment and prevention, there is little evidence regarding their efficacy, safety and long-term effects. Importantly, the fundamental mechanisms associated with the cellular response to botanicals are generally not clearly understood, and there are often unknown off-target effects. This is supported by the bafflingly large number of effects associated with curcumin. The use of suitable model systems in pharmacogenetic analysis allows for the subsequent characterization of genes and their protein products to explain the mechanisms of drug action. Understanding the complex mechanisms associated with drug response is compounded by the use of mammalian models. In addition, the maintenance and care of such models, the large numbers of animals required for experimental study as well as ethical concerns have necessitated the use of simple and genetically tractable non-mammalian models. The social amoeba, Dictyostelium discoideum, has proven to be an excellent model for the molecular and genetic study of the mechanisms of action of drugs and their effects on the cell. D discoideum has been successfully used to find targets to improve efficacy of drugs used in psychiatry and cancer treatment. Those studies were subsequently validated in human cells. By taking advantage of this simple but powerful biological model and the molecular genetic tools available to us, we have started to investigate the complex effects of the botanical compound curcumin on cell growth, cell physiology and the underlying molecular mechanisms of action. Results from our studies revealed a rather complex pleiotropic response to curcumin including effects on proliferation, oxidative stress and a global regulation of gene expression at the transcriptional level. The relative lack of conclusive scientific evidence regarding the therapeutic benefits of curcumin, coupled with curcumin's possible deleterious effects as revealed by our research findings, underscore the importance of further research to establish curcumin's risks and benefits. Ascertaining the risk-benefits of curcumin with more conclusive scientific evidence would better equip consumers to make decisions about using curcumin, and in fact other botanicals, as part of their primary healthcare

Curcumin reduces proliferation and cell viability.

<p>A) Axenically growing AX4 cells were treated with curcumin at the indicated concentrations and cell density was monitored over four days by direct counting with a hemocytometer. B) In separate experiments, viability of curcumin treated cells was assayed by measuring ATP in metabolically active cells using CellTiter-Glo® which measures cell viability. C) Curcumin stability in HL5 growth medium was determined by adding curcumin to medium at the onset of the experiment (0 hours). Flasks were inoculated with cells at time 0, 24 and 48 hours, and each assayed for 72 hours using the CellTiter- Glo® method. Taken together, these results show that curcumin has a lasting inhibitory effect on cell proliferation. Error bars in all figures represent the standard deviation compared to the mean.</p

Curcumin affects gene expression and reactive oxygen species via a PKA dependent mechanism in <i>Dictyostelium discoideum</i>

<div><p>Botanicals are widely used as dietary supplements and for the prevention and treatment of disease. Despite a long history of use, there is generally little evidence supporting the efficacy and safety of these preparations. Curcumin has been used to treat a myriad of human diseases and is widely advertised and marketed for its ability to improve health, but there is no clear understanding how curcumin interacts with cells and affects cell physiology. <i>D</i>. <i>discoideum</i> is a simple eukaryotic lead system that allows both tractable genetic and biochemical studies. The studies reported here show novel effects of curcumin on cell proliferation and physiology, and a pleiotropic effect on gene transcription. Transcriptome analysis showed that the effect is two-phased with an early transient effect on the transcription of approximately 5% of the genome, and demonstrates that cells respond to curcumin through a variety of previously unknown molecular pathways. This is followed by later unique transcriptional changes and a protein kinase A dependent decrease in catalase A and three superoxide dismutase enzymes. Although this results in an increase in reactive oxygen species (ROS; superoxide and H₂O₂), the effects of curcumin on transcription do not appear to be the direct result of oxidation. This study opens the door to future explorations of the effect of curcumin on cell physiology.</p></div

Proposed mechanism of curcumin action.

<p>Curcumin inhibits growth and generates ROS in <i>D</i>. <i>discoideum</i>. Curcumin induced major changes in transcription which included the reduction of catalase A and superoxide dismutase enzyme levels through a PKA mediated pathway. The results of this study suggest that the increase in ROS is not the cause of the decrease in antioxidant enzyme levels, but rather that the decrease in the enzymes results in the increase in ROS levels.</p

Heat map of differentially expressed genes following curcumin treatment.

<p><b>A)</b> A differential expression analysis (using baySeq) of the 4 hour samples treated with 0 and 10 μg/ml curcumin (see two red asterisks). The yellow-blue heat map shows the differentially expressed genes at 4 hours. In the heat maps, each row represents the abundance levels of one transcript (scale indicated in the box) and each column represents one condition (time and curcumin concentration). Transcripts that exhibited increased abundance with increased curcumin concentration are clustered above the line (up-regulated), and transcripts that exhibited reduced abundance are clustered below the line (down-regulated). The number of genes in each cluster is indicated. B) Heat map showing differentially expressed genes with at least 3-fold change between 0 and 10 μg/ml curcumin at 4 hours. The above analyses in A) and B) were repeated for 0 and 10 μg/ml samples at 12 hours, C) and D), respectively. E) Heat map showing the expression patterns of the genes that are differentially expressed between untreated and treated (10 μg/ml curcumin) at all time points. Genes that were differentially expressed in the absence of curcumin were subtracted.</p

Curcumin reduces SOD enzyme activity in wild-type cells but not in <i>pkaC</i> null cells.

<p>A) SOD enzyme activity in curcumin treated parental AX4 cells (5 μg/ml and 10 μg/ml) is reduced by nearly half relative to untreated cells. In contrast, there is much less effect on SOD enzyme activity in <i>pkaC</i> null cells treated with curcumin for 24 hours. B) Curcumin has no effect on the rate of generation of superoxide in the assay. P-values are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187562#pone.0187562.g002" target="_blank">Fig 2</a>.</p

Catalase A enzyme levels are unchanged in curcumin treated <i>pkaC</i> null cells.

<p>Unlike wild-type cells, <i>pkaC</i> null cells treated with curcumin for 24 hours did not show a change in catalase A activity/mg protein enzyme activity. P-values are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187562#pone.0187562.g002" target="_blank">Fig 2</a>.</p

Curcumin reduces catalase A enzyme levels in wild-type cells.

<p>A) Catalase A enzyme specific activity is reduced in curcumin treated cells in a dose dependent manner; B) Reduction in catalase A specific activity is not immediate and takes up to 24 hours to manifest itself, indicating that it is not due to enzyme inhibition; C) Curcumin by itself does not have an effect on the stability of H₂0₂; D) Curcumin by itself does not have a direct effect on the rate of catalase A activity in cell extracts (5 μl of an extract of 2 x 10⁷ cells in 1 ml lysis buffer); and E) Curcumin by itself does not have an effect on the extent of the <i>in vitro</i> catalase A activity (5 μl of an extract of 2 x 10⁷ cells in 1 ml lysis buffer). Error bars represent the standard deviation of the mean. Statistical analyses were carried out using a two-tailed t-test. *p<0.05, **p<0.01, ***p<0.001.</p

The antioxidant NAC affects cells differently than curcumin and does not reverse the oxidant effect of curcumin.

<p>The antioxidant NAC, known to counter the effect of oxidative stress, does not have any effect on cell proliferation of wild-type <i>D</i>. <i>discoideum</i> cells: A) Cell Titer Glo assay and B) direct cell counting. C) NAC did not counter the effect of curcumin on cells treated for 24 hours, indicating that the effect of curcumin on catalase A specific enzyme activity was not directly due to oxidative stress. D) Increased NAC concentrations inhibit cell proliferation at very high concentrations. E) However, these increasing concentrations of NAC still do not counter the effect of cells treated with curcumin for 24 hours. P-values are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187562#pone.0187562.g002" target="_blank">Fig 2</a>.</p

Curcumin negatively regulates antioxidant enzyme RNA levels.

<p>Total RNA was prepared from 5×10⁶ axenically growing cells treated with 10 μg/ml curcumin for 24 hours. Transcript levels of the antioxidant enzymes, <i>catA</i>, <i>sodA</i>, <i>sodB</i> and <i>sod2</i> are reduced in cells treated with curcumin. P-values are defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187562#pone.0187562.g002" target="_blank">Fig 2</a>.</p