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. 2017 Nov 14;12(11):e0187562.
doi: 10.1371/journal.pone.0187562. eCollection 2017.

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

William S Swatson  1 , Mariko Katoh-Kurasawa  2 , Gad Shaulsky  2 , Stephen Alexander  1
Affiliations

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

William S Swatson et al. PLoS One. .

Abstract

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. D. discoideum 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Curcumin reduces proliferation and cell viability.
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.
Fig 2
Fig 2. Curcumin reduces catalase A enzyme levels in wild-type cells.
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 in vitro 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.
Fig 3
Fig 3. Curcumin reduces SOD enzyme activity in wild-type cells but not in pkaC null cells.
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 pkaC 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 Fig 2.
Fig 4
Fig 4. Curcumin negatively regulates antioxidant enzyme RNA levels.
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, catA, sodA, sodB and sod2 are reduced in cells treated with curcumin. P-values are defined in Fig 2.
Fig 5
Fig 5. Curcumin up-regulates superoxide and H2O2 in wild-type cells but not in pkaC null cells.
Logarithmically growing wild-type AX4 (A and B) or pkaC null cells (C and D) were treated with curcumin at the indicated concentrations, and superoxide (A and C) or H2O2 (B and D) levels were determined as described in Material and Methods. The results indicate that curcumin increases the level of superoxide and H2O2 in a dose-dependent manner in AX4 cells but not in the pkaC null cells. P-values are defined in Fig 2.
Fig 6
Fig 6. Oxidants cause an increase in catalase A enzyme activity.
A) In contrast to the effect of curcumin, catalase A activity is increased in cells treated with ethidium bromide for 24 hours in a dose dependent manner. B) The same effect is observed with treatment with another oxidant, menadione. These results indicate that D. discoideum cells, like mammalian cells, respond to oxidative stress by upregulating antioxidant enzymes and that the effect curcumin has on catalase A enzyme specific activity (Fig 2A and 2B) is not the direct result of oxidative stress. C) Ethidium bromide at 5 and 10 μg/ml inhibits cell proliferation of parent cells. D) Menadione at 5, 10, 20 and 50 μg/ml inhibits proliferation of parental cells. P-values are defined in Fig 2.
Fig 7
Fig 7. The antioxidant NAC affects cells differently than curcumin and does not reverse the oxidant effect of curcumin.
The antioxidant NAC, known to counter the effect of oxidative stress, does not have any effect on cell proliferation of wild-type D. discoideum 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 Fig 2.
Fig 8
Fig 8. Examination of candidate genes reveals that PKA is required for the cellular response to curcumin.
The indicated mutants, described in detail in the text, were tested for their sensitivity to curcumin over time. Each mutant was tested multiple times and in parallel with the wild-type AX4 strain. Cell proliferation was measured using the CellTiter-Glo® assay, and expressed as percent of the control untreated samples at 72 hours. Δ = null mutant, OE = over-expressing mutant.
Fig 9
Fig 9. Validation that the pkaC null and pkaR-OE mutants are more resistant to curcumin.
Wild-type, pkaC null and pkaR-OE cells were clonally plated in association with Klebsiella aerogenes on SM plates containing curcumin at the indicated concentrations. Plaques resulting from clonal growth were counted and the percent survival was calculated. The results are from multiple platings within the linear range of the assay. The data confirm that the pkaC null and pkaR-OE cells are more resistant to curcumin than the wild-type cells. P-values are defined in Fig 2.
Fig 10
Fig 10. Catalase A enzyme levels are unchanged in curcumin treated pkaC null cells.
Unlike wild-type cells, pkaC 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 Fig 2.
Fig 11
Fig 11. Transcriptome analysis of curcumin treated samples.
We averaged the values of the replicate RNA-seq samples at each time point and each curcumin concentration as indicated, and calculated the distance (Spearman’s correlation) between the samples. A) Hierarchical clustering. The hierarchical clustering dendrogram illustrates the relationships between the samples based on these distance calculations. In the dendrogram, each leaf represents one condition (time and curcumin concentration) and the vertical distances between the joints represent the dissimilarity between the samples (see scale on the left, arbitrary units). B) Multidimensional scaling. Each point on the graph represents a sample (time and curcumin concentration, as indicated) and the distances between the points represent the dissimilarity between them–the closer two point are, the more similar they are. The axes units are arbitrary.
Fig 12
Fig 12. Heat map of differentially expressed genes following curcumin treatment.
A) 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.
Fig 13
Fig 13. Proposed mechanism of curcumin action.
Curcumin inhibits growth and generates ROS in D. discoideum. 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.
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