Curcuminoids activate p38 MAP kinases and promote UVB-dependent signalling in keratinocytes

Abstract

Curcuminoids exhibit anti-proliferative properties in many cell lines by modulating signalling pathways to inhibit cell growth. However, the specific effects of curcuminoids on human keratinocytes are not well defined, and this situation impairs mechanistic thinking regarding potential therapeutic uses. We hypothesized that curcuminoids would modulate key growth regulatory pathways in keratinocytes to inhibit cell proliferation. To test this hypothesis, the effects of curcumin and tetrahydrocurcumin (THC) on mitogen-activated protein (MAP) kinase signalling in keratinoctyes were determined. Primary human keratinocytes treated with curcumin or THC demonstrated decreased activation of p44/42 MAP kinases but increased levels of activated p38 MAP kinases. These data suggest that curcuminoids specifically activate stress-induced MAP kinases while inhibiting mitogen-induced MAP kinases. Curcuminoids also promote the phosphorylation of p53 on serine 15 in a dose-dependent and p38-dependent manner, suggesting that these compounds may activate p53. The effects of curcuminoids on keratinocytes mirrored some aspects of UVB and could be inhibited by N-acetylcysteine, suggesting that these compounds activate p38 through a mechanism that involves glutathione depletion. Both curcuminoids induced G2/M block and inhibited keratinocyte growth, and THC increased cellular levels of p21, a known p53 transcriptional target. These data demonstrate that curcuminoids can differentially regulate MAP kinases to inhibit keratinocyte growth while inducing p21. Curcuminoids also synergize with UVB to enhance p53 phosphorylation. The findings provide a rationale for testing curcuminoids in disorders associated with impaired p53 function or in which UVB-treatment is efficacious.

Figure. 1. Curcuminoids promote G2/M block, and inhibit growth in PHKs

A) PHKs were cultured for the indicated times in the presence or absence of 20μM curcumin, harvested, and stained with PI. Labeled keratinocytes were analyzed by flow cytometry to assess DNA content. Incubation times are indicated on the x-axis. The percentage of cells in the S-phase and G0/G1-phase is indicated on the y-axis B) The percentage of curcumin-treated cells from A in the G2/M-phase. N=3 C) PHKs were treated with 200μM THC for 2 or 24 hours or with DMSO (D) and analyzed as in B to determine the percentage of cells in G2/M N=3 Standard deviation indicated by error bars. In A, B, and C, asterisks indicate differences associated with p values ≤ 0.05. D) PHKs were treated with nothing, 0.1% DMSO, 20μM curcumin, or 200μM THC. After three days, total cell counts were determined in triplicate. Asterisks indicate differences with p values < 0.001 compared to DMSO condition. N=2

Figure 2. Curcuminoids inhibit p44/42 MAP kinases and activate p38 MAP kinases in PHKs

A), PHKs were treated with 20μM curcumin or DMSO for the indicated times. Lysates were subjected to SDS-PAGE followed by western blotting to detect levels of phosphorylated p44/42 MAP kinases (α-pp44/42, upper panels) and total p44/42 MAP kinases (lower panels). 0=baseline control. N=3. B) PHKs were treated with 20μM curcumin or DMSO for the indicated times, and processed as in A. N=2. C) PHKs were treated with 200μM THC or DMSO for the indicated times, and processed as in A. N=2. PHKs were treated with 20μM curcumin or DMSO for short (D) or long (E) time courses. Cells were lysed, and subjected to SDS-PAGE followed by western blotting to detect levels of activated p38 MAP kinases (pp38) and total p38 MAP kinases. N=3. F) PHKs were treated for two hours with 20μM curcumin or tetrahydrocurcumin (THC) at 50, 100, 200 μM. Control 1 lysed at the beginning of the time course, 2-at the end. Cells analyzed as in D. N=2.

Figure. 3. Curcuminoids enhance UVB-induced activation of p38 MAP kinases

A) PHKs were not stimulated or stimulated with 20μM curcumin, 200μM tetrahydrocurcumin (THC), or DMSO for two hours and lysed. Where indicated, cells were treated with curcumin, THC, or DMSO then subsequently irradiated with UVB (18 mJ/cm²). Two hours post-irradiation, cells were lysed and subjected to SDS-PAGE followed by western blotting to detect phosphorylated p38 and total p38. N=2 B) Densitometric analysis of p38 activation in A. Curcumin and THC pre-treatment enhance the ability of UVB to activate p38 MAP kinases. Standard deviation indicated by error bars. Asterisks indicate differences with UVB condition associated with a p < 0.05. N=2 C). Left panel: PHKs were either treated with DMSO, 5, 10, or 20 μM curcumin for two hours, lysed, and analyzed as in A. N=2. Right panel: PHKs were treated with DMSO, UVB (18 mJ/cm²), UVB and DMSO, or Curcumin 20 μM followed immediately by UVB exposure and lysed two hours later. Cur then UVB: PHKs were treated with 20μM curcumin for 1 hour then exposed to UVB and incubated for an additional 1 hour, and then lysed. UVB then Curcumin: PHKs were exposed to UVB then treated with 20μM curcumin for two hours before lysis. Lysates were analyzed as above. N=2 D) PHKs were treated with 5 μM or 20 μM curcumin, no stimulus, DMSO, or 10 mM N-acetylcysteine (NAC) for two hours. Some cells were pre-incubated with 10 mM NAC for 45 minutes before curcumin treatment. Cells were lysed, and analyzed as in A. N=2

Figure. 4. Curcuminoids promote p53 phosphorylation, enhance UVB-induced p53 phosphorylation, and induce p21 levels

A) PHKs were treated for two hours with 20μM curcumin or tetrahydrocurcumin (THC) at 50, 100, 200 μM. Control 1 was lysed at the beginning of the time course, 2-at the end. Cells subjected to western blot analysis to detect phosphorylated p53 (serine 15) and total p53. N=3 B) PHKs were treated with DMSO or 200μM tetrahydrocurcumin (THC) for the indicated times (hours). Cells were analyzed as in A. N=2. C) PHKs were not stimulated or stimulated with 20μM curcumin, 200μM tetrahydrocurcumin (THC), or DMSO. Where indicated, cells were irradiated with UVB (18 mJ/cm²). Two hours post-irradiation, cells were lysed and subjected to analysis as in A. N=2 D) Densitometric analysis of p53 activation in experiment C. Curcuminoids promote UVB-induced phosphorylation of p53. Error bars indicate standard deviation. Asterisk indicates a p=0.09 compared to UV alone. E) PHKs were treated with 200μM tetrahydrocurcumin (THC) or DMSO for the indicated times (hours). Cell lysates were analyzed by western blotting to detect levels of p21 and β-actin. N=2

Figure. 5. p38 MAP kinase inhibition decreases curcuminoid-induced p53 phosphorylation

PHKs were not stimulated or stimulated with DMSO or 200μM tetrahydrocurcumin (THC). Where indicated, cells were irradiated subsequently with UVB (18 mJ/cm²). Duplicate cell sets were exposed to the p38 MAP kinase inhibitor SB202190 or its control compound SB202474 for 30 minutes prior to lysis. Two hours post-irradiation, cells were lysed and subjected to SDS-PAGE followed by western blotting to detect phosphorylated p38, total p38, phosphorylated p53 (serine 15), and total p53. N=3.

Figure 6. Schematic diagram of the curcuminoid-induced effects on PHKs

Curcuminoids differentially regulate p44/42 and p38 MAP kinases. Activation of p38 leads to p53 phosphorylation, and increased p21 levels. The result of these cellular events is a block in G2/M and decreased S-phase. Curcuminoids enhance p53 function in PHKs.