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. 2018 Jan 29;11(1):dmm032375.
doi: 10.1242/dmm.032375.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum

Marco Cocorocchio  1 , Amy J Baldwin  2 , Balint Stewart  3 , Lou Kim  4 , Adrian J Harwood  2 , Christopher R L Thompson  3 , Paul L R Andrews  5 , Robin S B Williams  6
Affiliations

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum

Marco Cocorocchio et al. Dis Model Mech. .

Abstract

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Keywords: Alzheimer's disease; Cancer; Curcumin; Dictyostelium discoideum; PP2A; Presenilin.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Acute cell behaviour, growth and developmental effects of curcumin on D. discoideum. (A) Curcumin, a diferuloylmethane, was used to assess multiple roles using D. discoideum as a model. (B) Time-dependent changes in D. discoideum cell behaviour (membrane protrusion) were recorded over a 15-min period (±s.e.m.) at increasing concentrations of curcumin. Data are presented normalised to control conditions, showing a significant difference between control condition (vehicle) and 3 µM (****P<0.0001) using one-way ANOVA. (C) The concentration-dependent response is illustrated as the normalised reduction of cell behaviour (protrusion formation) against the Log (concentration) of curcumin, enabling calculation of an IC50 with a 95% CI. (D) D. discoideum cells were grown with increasing concentration of curcumin, causing a complete block at 100 µM, with (E) normalised concentration-dependent response shown plotted against Log curcumin (mM) concentration, providing an IC50 with 95% CI. (F) Cells were developed on agar over 22 h in control conditions (vehicle) and in the presence of 100 µM curcumin. Scale bar: 0.25 mm for both side view images. All experiments were carried out in triplicate.
Fig. 2.
Fig. 2.
Quantification of the acute cell behaviour, growth and developmental effects of curcumin derivatives on D. discoideum. (A) Structure of natural and artificial derivatives used in a quantitative structural analysis of curcumin effects in D. discoideum. (B) Concentration-dependent responses were determined for cell behaviour (protrusion formation) and illustrated as the IC50 for each compound with errors shown as 95% CIs (Figs. S1 and S2). (C) Concentration-dependent responses were determined for cell growth and illustrated as the IC50 for each compound with errors shown as 95% CIs (Figs. 3 and 4). Data from B and C are presented as mean±s.e.m. of triplicate experiments. (D) Cells were developed on agar over 22 h in the absence of compounds (vehicle only), or the presence of curcumin derivatives at concentrations shown to block growth (100 µM FLLL31, 25 µM DMC, 20 µM BDMC, 100 µM THC, 6 µM EF24, 5 µM UBS109 and 100 µM CuPy). All images are representative of triplicate experiments. Scale bar: 0.25 mm for all side view images.
Fig. 3.
Fig. 3.
Antioxidant activity of curcumin and related structures. Reducing activity was monitored using the FRAP assay. Initial rapid (0 min) and sustained (60 min) activity was measured, using ascorbic acid as an antioxidant standard. Data are presented as mean±s.e.m. of triplicate experiments.
Fig. 4.
Fig. 4.
Loss of psrA and psenB genes provides partial growth resistance to curcumin or its derivatives. (A) Through screening a D. discoideum mutant library, a mutant showing resistance to curcumin was identified showing an insertion into the protein phosphatase 2A regulatory subunit gene (psrA), and a mutant showing resistance to EF24 was identified showing an insertion into the presenilin B gene (psenB) (blue exons and black introns). (B) Analysis of wild-type (Ax3) and recapitulated psrA− mutant growth rate confirmed that the psrA− mutant was resistant to curcumin, and additionally to EF24 and DMC, but not BDMC (Fig. S5). (C) Analysis of wild-type (Ax2) and recapitulated psenB− mutant growth rate showed that PsenB was not resistant to curcumin, but showed confirmed resistance to EF24, in addition to UBS109 (Fig. S6). Data are presented as mean±s.e.m. of triplicate experiments. *P<0.05; **P<0.01; ***P<0.001; ns, nonsignificant.
Fig. 5.
Fig. 5.
Molecular docking prediction of PsrA and curcumin analogues. Tertiary protein structures were generated with Phyre2, with docking prediction performed by SwissDock to provide the most stable binding site (deltaG; Gibbs free energy). Using this approach, curcumin, DMC and EF24 are predicted to bind to the same site on PsrA that is not shown for CuPy.
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