PKC activation by resveratrol derivatives with unsaturated aliphatic chain

Abstract

Resveratrol (1) is a naturally occurring phytoalexin that affects a variety of human disease models, including cardio- and neuroprotection, immune regulation, and cancer chemoprevention. One of the possible mechanisms by which resveratrol affects these disease states is by affecting the cellular signaling network involving protein kinase C (PKC). PKC is the family of serine/threonine kinases, whose activity is inhibited by resveratrol. To develop PKC isotype selective molecules on the resveratrol scaffold, several analogs (2-5) of resveratrol with a long aliphatic chain varying with number of unsaturated doubled bonds have been synthesized, their cytotoxic effects on CHO-K1 cells are measured and their effects on the membrane translocation properties of PKCα and PKCε have been determined. The analogs showed less cytotoxic effects on CHO-K1 cells. Analog 4 with three unsaturated double bonds in its aliphatic chain activated PKCα, but not PKCε. Analog 4 also activated ERK1/2, the downstream proteins in the PKC signaling pathway. Resveratrol analogs 2-5, however, did not show any inhibition of the phorbol ester-induced membrane translocation for either PKCα or PKCε. Molecular docking of 4 into the activator binding site of PKCα revealed that the resveratrol moiety formed hydrogen bonds with the activator binding residues and the aliphatic chain capped the activator binding loops making its surface hydrophobic to facilitate its interaction with the plasma membrane. The present study shows that subtle changes in the resveratrol structure can have profound impact on the translocation properties of PKCs. Therefore, resveratrol scaffold can be used to develop PKC selective modulators for regulating associated disease states.

Figure 1. Chemical structure of compounds 1–5.

Figure 2. Effect of solvent polarity on the absorption and fluorescence properties of 2 (5–10 µM).

A), Normalized absorption and B) normalized florescence emission spectra of 2 in a) water, b) ethanol, c) acetonitrile and d) hexane.

Figure 3. Effect of 1–5 on CHO-K1 cell viability.

The graph shows the percentage of viable cell after treatment with 1–5 at three different concentrations for 48 h. The cell viability was measured by MTT assay. Mean and standard deviation (SD) were obtained from three experiments done in triplicate.

Figure 4. Effect of 1–5 on the expression of PKCα and PKCε.

Upper panels, Western blot analysis of whole cell lysate of CHO-K1 cells after treatment with 1–5 (10 µM) for 24 h. Lower panel, bar graph of densitometry analysis of PKC expression (Mean ± SE, *P<0.05, n = 3). β actin was used as a reference for uniform loading. Control refers to the sample with no addition of compounds.

Figure 5. Effect of 1 on the membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (A) PKCα and (B) PKCε after the cells were treated with varying concentration 1 for 1 h. Lower panel, bar graph of densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.

Figure 6. Effect of 4 on the membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (A) PKCα and (B) PKCε after the cells were treated with varying concentration of 4 for 1 h. Lower panel, bar graph depicts the densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.

Figure 7. Effect of 1, 2, 3, and 5 on the membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (A) PKCα and (B) PKCε after the cells were treated with 100 µM of 2, 3 or 5 for 1 h. Lower panel, bar graph of densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.

Figure 8. Effect of 1 and 4 on the membrane translocation of PKCα.

Western blot analysis of the cytosolic (C) and the membrane (M) fractions of PKCα after the cells were treated with varying concentration of 1 and 4 for 24 h. Control refers to the sample with no addition of compounds.

Figure 9. Effect of 2–5 on the membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fractions of (A) PKCα and (B) PKCε after the cells were treated with 10 µM of 2, 3, 4 or 5 for 24 h. Lower panel, bar graph of densitometry analysis of the upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.

Figure 10. Effect of 3 and 4 on ERK1/2 phosphorylation in CHO-K1 cells.

Cells were either treated with 10 µM of 3 or 4 for 24 h or 100 nM of TPA for 1 h for Western blot analysis. Control refers to the sample with no addition of compounds.

Figure 11. Effect of oleic acid, linoleic acid and linolenic acid on the membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fraction of (A) PKCα and (B) PKCε after the cells were treated with 10 µM of oleic acid, linoleic acid or linolenic acid for 24 h. Lower panel, bar graph of densitometry analysis of upper panel immunoblots (Mean± SE, n = 3). Control refers to the sample with no addition of compounds.

Figure 12. Effect of 1 on TPA-induced membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and the membrane (M) fraction of (A) PKCα and (B) PKCε. Lower panel, bar graph of densitometry analysis of upper panel immunoblots (Mean ± SE, *P<0.05, n = 3). Cells were treated with 100 µM of 1 in the presence and absence of 100 nM TPA for 1 h. Control refers to the sample with no addition of compounds.

Figure 13. Effect of 2–5 on TPA-induced membrane translocation of PKCα and PKCε.

Upper panels, Western blot analysis of the cytosolic (C) and membrane (M) fraction of (A) PKCα and (B) PKCε. Lower panel, bar graph of densitometry analysis of upper panel immunoblots (Mean ± SE, n = 3). Cells were treated with 100 µM of 2–5 and 100 nM of TPA for 1 h. Control refers to the sample with no addition of compounds.

Figure 14. Molecular modeling.

A) Energy minimized structure of 4. B) Surface diagram of PKCαC1B (blue) docked with 4 (magenta). C) Overlaid structures of αC1B (blue) and εC1B (magenta). Structure of 4 was minimized using Chem3D pro 12.0.2. Molecular docking was done using sybyl 8.0. The protein structures were visualized using UCSF chimera 1.6.1.

Figure 15. Synthetic scheme for compounds 2–4.