Selective Modulation of Protein Kinase C α over Protein Kinase C by Curcumin and Its Derivatives in CHO-K1 Cells

Article abstract of DOI:10.1021/acs.biochem.6b00057

Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKC, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKC. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKC to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKC. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state.

Full text of DOI:10.1021/acs.biochem.6b00057

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Article
Selective modulation of PKC# over PKC# by
curcumin and its derivatives in CHO-K1 cells
SATYABRATA PANY, Anjoy Majhi, and Joydip Das
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00057 • Publication Date (Web): 17 Mar 2016
Downloaded from http://pubs.acs.org on March 23, 2016
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Selective modulation of PKCα over PKCε by curcumin and its derivatives in CHO-K1 cells
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Satyabrata Pany, Anjoy Majhi and Joydip Das*
Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University
of Houston, Houston, TX 77204
*
To whom correspondence should be addressed: Tel: 713ꢀ743ꢀ1708, Fax: 713ꢀ743ꢀ1884, Email:
jdas@uh.edu.
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Abstract
The protein kinase C (PKC) family of serine/threonine kinases regulate various cellular
functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of
isoformꢀselective activity of PKC by curcumin (1), the active constituent of Curcuma L., is
poorly understood and the literature data is inconsistent and obscure. Effect of curcumin (1) and
its analogs, 4ꢀ((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀoxoheptaꢀ2,6ꢀdienꢀ1ꢀyl)ꢀ2ꢀ
methoxyphenyl oleate (2), (9Z,12Z)ꢀ4ꢀ((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀ
oxoheptaꢀ2,6ꢀdienꢀ1ꢀyl)ꢀ2ꢀmethoxyphenyl octadecaꢀ9,12ꢀdienoate (3), (9Z,12Z,15Z)ꢀ4ꢀ
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((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀoxoheptaꢀ2,6ꢀdienꢀ1ꢀyl)ꢀ2ꢀ
methoxyphenyl octadecaꢀ9,12,15ꢀtrienoate (4), and (1E,6E)ꢀ1ꢀ(4ꢀ(hexadecyloxy)ꢀ3ꢀ
methoxyphenyl)ꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)heptaꢀ1,6ꢀdieneꢀ3,5ꢀdione (5) and
didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional and PKCε, a
novel PKC has been studied in CHOꢀK1 cells, in which these PKC isoforms are endogenously
expressed. Translocation of PKC from cytosol to membrane was measured using
immunoblotting and confocal microscopy. 1 and 6 inhibited the TPAꢀinduced membrane
translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with
long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this
inhibition property. Instead, 2-4 showed significant translocation of PKCα, but not of PKCε to
the membrane. No membrane translocation was observed with 1, 6 or with the analog 5 having
saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPAꢀinduced activation of
ERK1/2 and 2-4 activated it. ERK1/2 is the downstream readout of PKC. The present results
show that the hydroxyl group of curcumin is important for PKC activity and curcumin template
can be useful in developing isoform specific PKC modulators for regulating a particular disease
state.
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Keywords: Curcumin, protein kinase C, phorbol ester, diacylglycerol, isoform, membrane.
Abbreviations: PKC, protein kinase C; DAG, diacylglycerol; TPA, 12ꢀOꢀtetradecanoylphorbolꢀ
13ꢀacetate; ERK, extracellular signal regulated Kinase; CHO, Chinese hamster ovary; DOG, snꢀ
1,2ꢀdioctanoylglycerol; PC, phosphatidylcholine; PS, phosphatidylserine; NMR, nuclear
magnetic resonance; MTT, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide; ESꢀ
MS, electrospray mass spectrometry; FBS, fetal bovine serum; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; SDSꢀPAGE, sodium dodecyl sulfonateꢀ polyacrylamide gel
electrophoresis; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; clog P,
calculated value of logarithm of the partition coefficient for nꢀoctanol/water; BSA, bovine serum
albumin; DPPH, 2, 2ꢀdiphenylꢀ1ꢀpicrylhydrazyl; FITC, fluorescein isothiocyanate.
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Protein kinase C (PKC) is a family of serine/threonine protein kinases involved in the regulation
of various aspects of cell functions, including cell growth, differentiation, metabolism, and
apoptosis(1). Its role has been implicated in the pathology of several diseases such as cancer,
diabetes, stroke, heart failure, and Alzheimer’s disease (2-8). PKC has been a subject of intense
research and drug development for these disease states (9, 10).
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The PKC family has been divided into three main groups: conventional isoforms (α, βI,
2
+
βII and γ) that require Ca and diacylglycerol (DAG) for activation; novel isoforms (δ, ε, η, θ
2
+
and ꢁ) that require only DAG and atypical isoforms (ζ, ι and λ) that require neither Ca nor
DAG(11). DAG is generated by the phospholipase Cꢀcatalyzed hydrolysis of membrane
phosphatidylinositolꢀ4,5ꢀbisphosphate (PIP ). It contains two long chains and acts as a second
2
messenger(12). These two acyl chains remain embedded in the membrane during the PKC
activation process. DAG interacts with proteins containing a C1 domain and induces their
translocation to discrete subcellular compartments. In the conventional and novel PKC isoforms,
the DAGꢀsensitive C1 domain is duplicated into a tandem C1 domain consisting of C1A and
C1B subdomains. Along with the PKC family, there are about six additional families of proteins
that contain a DAGꢀresponsive C1 domain (13, 14).
Naturally occurring tumorꢀpromoters, phorbol esters also bind to the C1 domain and
actually activates PKC several fold higher than the endogenous DAG (15, 16). In addition,
several other naturally occurring compounds including bryostatins, teleocidins, aplysiatoxins,
ingenols, and iridals bind to the C1 domain. However, most of the naturally occurring C1 domain
ligands are highly complex in their structure and show very poor or no isoform specificity (17,
18). Therefore, structural modification with the aim of altering the ligand specificity has been
difficult. Moreover, many of these ligands are associated with toxicity problems. To circumvent
these problems, the strategy has been to find simpler template for ligand synthesis, whose
structure could be easily modified and fineꢀtuned in order to achieve selectivity and expected to
be nonꢀtoxic.
Curcumin (1, Fig. 1) is a βꢀdiketone constituent of turmeric obtained from the powdered
root of Curcuma L. (19, 20). Not only is it used as a spice to give a specific flavor and yellow
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color to curry, which is consumed in trace quantities daily by millions of people, curcumin has
also been used as a traditional medicine for liver disease (jaundice), indigestion, urinary tract
diseases, rheumatoid arthritis, and insect bites (19-21). Curcumin is a promising therapeutic
agent for diseases such as cancer, diabetes, multiple sclerosis, Alzheimer’s, HIV and
cardiovascular disease (22-24).
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The positive interference of curcumin with the tumor promoting effects of phorbol esters
has been attributed to its effect on the phorbol ester receptor, protein kinase C (PKC)(25) (26-
2
8). There are wellꢀdocumented studies on the modulation of PKC activity by curcumin in vivo
(
29, 30) and in vitro (31, 32). However, the mechanism by which curcumin modulates PKC
activity is poorly understood. In studies involving purified proteins, curcumin (< 20 µM) was
shown to activate PKCα in the presence of membrane(32). At higher curcumin concentration (>
2
0 µM), decrease in activity was observed. In another study using purified protein it was also
shown that curcumin (6ꢀ48 ꢀM) activated calcium sensitive PKC (e.g. PKCα) in the presence of
membrane and inhibited it in the absence of membrane(31). Similarly, another study showed
that in a membraneꢀfree system, curcumin (100 ꢀM) inhibited PKC(30). All these results
+
+
indicated that membrane, Ca and curcumin concentration are important determinants for
curcumin to behave as an activator or inhibitor. Studies using cultured NIH3T3 fibroblasts have
shown that curcumin (15ꢀ20 ꢀM) alone did not affect PKC activity but it inhibited the TPAꢀ
induced PKC activity(29). Another study involving mouse skin showed that curcumin (10 ꢀmol)
inhibited TPAꢀinduced membrane translocation of both PKCα and PKCε (33).
In our previous studies (34, 35) we found that addition of long chains in curcumin
structure modified their binding to the C1 domains of PKCs. In this paper we describe the
chemical modification of curcumin with long aliphatic chain with varying number of unsaturated
double bonds on the membrane translocation properties of PKCs in CHOꢀK1 cells. Our results
indicate that curcumin inhibited the TPAꢀinduced PKC activity of PKCα but not PKCε. On the
other hand, the long chain derivatives containing unsaturated double bonds directly activated
PKCα but not PKCε.
Experimental Procedures
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General
Curcumin and TPA were from Sigma. 1,2ꢀdioctanoylꢀsnꢀglycerol (DOG) was purchased from
Avanti polar lipids. All other reagents were from Aldrich and used without further purification.
Progress of chemical reaction was monitored through thin layer chromatography (TLC) on preꢀ
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coated glass plates (silica gel 60 F254, 0.25mm thickness) purchased from EMD chemicals. H
1
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NMR and C NMR spectra were recorded on a QEꢀ300 spectrometer. Unless otherwise
specified, all NMR spectra were obtained in deuterated chloroform (CDCl ) and referenced to
3
the residual solvent peak. Chemical shifts are reported in parts per million, and coupling
constants in hertz (Hz). Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet),
m (multiplet) and br (broadened). Mass spectra were obtained on either a VG 70ꢀS Nier Johnson
or JEOL Mass Spectrometer.
Synthesis of the curcumin derivatives
For compounds 2-4, the corresponding acid chlorides (oleic, linoleic and linolenic acid) were
prepared by treatment of the acid (1 equivalent) in anhydrous dichloromethane and thionyl
chloride (1.1 equivalents) under nitrogen at 0 °C with catalytic amount DMF. Acid chloride was
distilled under vacuum and used immediately for the next step. Curcumin (1 equivalent) was
dissolved in anhydrous pyridine at 0 °C under nitrogen and treated with different acid chlorides.
The reaction mixture was allowed to stirring for 2 h at room temperature and then heated at 60
°
C for another 2 h. After cooling the mixture to room temperature, excess pyridine was
evaporated under high vacuum. The resulting residue was subjected to column chromatography.
Compounds 5 and 6 were synthesized as described earlier (34). The chemical structure of 1-6 is
shown in Fig.1.
4ꢀ((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀoxoheptaꢀ2,6ꢀdienꢀ1ꢀyl)ꢀ2ꢀ
1
methoxyphenyl oleate (2): Yield: 65% H NMR (CD OD) δ 7.59 (2H, d, J = 16.0 Hz), 7.15ꢀ7.06
3
(3H, m), 7.02 (1H, d, J = 8.0 Hz), 6.91 (1H, d, J = 8.0, Hz), 6.51 (1H, d, J = 15.4 Hz), 6.47 (1H,
d, J = 15.4 Hz), 6.0 (1H, brs), 5.81 (1H, s), 5.35 (2H, m), 3.91 (3H, s), 3.85 (3H, s), 2.58 (2H, t,
13
J = 7.7 Hz), 2.01 (4H, m), 1.75 (2H, m), 1.42ꢀ1.26 (20H, m), 0.87 (3H, t, J = 6.9 Hz); C NMR
(
CD OD) δ 184.6, 181.9, 171.9, 151.4, 148.0, 146.9, 141.3, 141.2, 139.5, 134.0, 130.1, 129.8,
3
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27.5, 124.1, 123.3, 123.1, 121.7, 121.0, 114.9, 111.4, 109.7, 101.7, 56.0, 55.9, 34.1, 32.0, 29.8,
9.8, 29.6, 29.4, 29.2, 29.2, 29.1, 27.3, 27.2, 25.0, 22.8, 14.2; ESꢀMS: 633 [M+H], 655 [M+Na].
2
(9Z,12Z)ꢀ4ꢀ((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀoxoheptaꢀ2,6ꢀdienꢀ1ꢀyl)ꢀ2ꢀ
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1
methoxyphenyl octadecaꢀ9,12ꢀdienoate (3): Yield: 69% H NMR (CD OD) δ 7.59 (2H, d, J =
3
1
5.5 Hz), 7.16ꢀ7.10 (3H, m), 7.04 (2H, d, J = 8.0 Hz), 6.93 (1H, d, J = 8.0, Hz), 6.53 (1H, d, J =
16.0 Hz), 6.49 (1H, d, J = 16.0 Hz), 5.92 (1H, brs), 5.82 (1H, s), 5.35 (4H, m), 3.93 (3H, s), 3.85
(3H, s), 2.77 (2H, t, J = 6.3 Hz), 2.58 (2H, t, J = 7.4 Hz), 2.05 (4H, m), 1.76 (2H, m), 1.48ꢀ1.29
1
3
(
14H, m), 0.88 (3H, t, J = 6.8 Hz); C NMR (CD OD) δ 184.5, 181.9, 171.8, 151.5, 148.0,
3
1
46.8, 141.2, 141.0, 139.5, 134..0, 130.3, 130.1, 128.1, 127.9, 127.6, 124.2, 123.3, 123.0, 121.8,
1
21.0, 114.9, 111.4, 109.7, 101.6, 56.0, 55.9, 34.1, 31.9, 29.7, 29.4, 29.2, 29.2, 27.2, 25.7, 25.0,
+
22.3, 14.1; ESꢀMS: 630 [M ], 631 [M+H].
(
9Z,12Z,15Z)ꢀ4ꢀ((2Z,6E)ꢀ3ꢀhydroxyꢀ7ꢀ(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ5ꢀoxoheptaꢀ2,6ꢀdienꢀ1ꢀ
1
yl)ꢀ2ꢀmethoxyphenyl octadecaꢀ9,12,15ꢀtrienoate (4): Yield: 58% H NMR (CD OD) δ 7.6 (2H,
3
d, J = 15.4 Hz), 7.18ꢀ7.10 (3H, m), 7.04 (2H, d, J = 8.0 Hz), 6.93 (1H, d, J = 8.0, Hz), 6.54 (1H,
d, J = 15.4 Hz), 6.48 (1H, d, J = 15.4 Hz), 5.87 (1H, brs), 5.82 (1H, s), 5.36 (6H, m), 3.94 (3H,
s), 3.86 (3H, s), 2.80 (4H, t, J = 6.3 Hz), 2.58 (2H, t, J = 7.3 Hz), 2.07 (4H, m), 1.76 (2H, m),
13
1
.45ꢀ1.35 (8H, m), 0.96 (3H, t, J = 7.1 Hz); C NMR (CD OD) δ 184.5, 181.9, 171.8, 151.4,
3
1
48.0, 146.8, 141.3, 141.2, 139.5, 134.0, 132.0, 130.3, 128.3, 128.3, 127.8, 127.6, 127.1, 124.2,
123.3, 123.1, 121.8, 121.0, 114.9, 111.4, 109.7, 101.6, 56.0, 55.9, 34.1, 29.6, 29.2, 29.2, 29.1,
+
2
7.3, 25.7, 25.6, 25.0, 20.6, 14.3; ESꢀMS: 628 [M ], 629 [M+H].
Absorption and fluorescence measurement: Ultravioletꢀvisible absorption and fluorescence
emission spectra of curcumin (1) and its derivatives (2-6) (1ꢀ10 µM) were recorded and analyzed
as described earlier (36).
DPPH free radical scavenging activity: Antioxidant activity of the compounds was measured
using DPPH (2, 2ꢀdiphenylꢀ1ꢀpicrylhydrazyl) radical scavenging assay. For scavenging
experiments, DPPH (60 ꢁM) and varying concentration of compounds (0.25–60 ꢁM) were
incubated in a methanolic solution for 2 h at 25 °C. DPPH absorbance spectra were recorded
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from 400 to 600 nm. The percentage of inhibition of DPPH radical by each compound was
calculated using formula: (1ꢀF/F ) x100 and plotted against concentration, where F and F0
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respectively. IC50 was measured from the fitted curve using Hill equation in Sigma Plot 11
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(Systat Software Inc., San Jose, CA).
Cell culture: Ham’s F12 medium (Life Technologies, Grand Island, NY) supplemented with
fetal bovine serum (10 %), penicillin (100 units/ml) and streptomycin (100 ꢁg/ml) were used to
culture CHOꢀK1 cells. Cultures were maintained in humidified atmosphere containing 5% CO2
at 37°C. Cells were starved in media without FBS for 12 h before treating with the compounds.
Cytotoxicity assays: Cytotoxicity assays were performed using Vybrant® MTT cell
proliferation assay kit (Molecular Probes, Waltham, MA) as described earlier (36).
Cell lysis, sub-cellular fractionation and Western blotting: Whole cell, cytosolic and
membrane lysates were prepared for Western blot analysis as described earlier (36). Briefly, cells
were harvested and lysed in lysis buffer (Cell Signaling, Danvers, MA) for whole cell lysates
preparation. Cell lysates (25 ꢀg protein/lane) were subjected to SDSꢀPAGE and and
immunoblot to detect PKCα, PKCε and ERK1/2. For detecting PKCε and PKCα in cytosolic
and membrane lysates, cells were lysed in buffer containing 20 mM TrisꢀHCl, pH 7.4 and
protease inhibitor. Lysates were then centrifuged at 40,000 rpm for 2 h at 4 °C to separate out
cytosolic (supernatant) and membrane (pellet) fractions. Protein concentration of lysates were
measured and subjected to SDSꢀPAGE (7%). Proteins in lysates were detected using standard
Western blot methods and protein specific antibodies. Following antibody dilutions were used
for detection: antiꢀrabbit PKCα, 1:500; antiꢀrabbit PKCε, 1:500; antiꢀrabbit ERK1/2, 1:1000;
antiꢀrabbit pERK1/2, 1:500; antiꢀrabbit βꢀactin, 1:2000 and antiꢀrabbit HRPꢀconjugated, 1:5000
(Cell Signaling, Danvers, MA)
Confocal microscopy: CHOꢀK1 cells were cultured and treated on glass coverslips (VWR,
Atlanta, GA). Cells were then fixed in PBS containing 4% paraformaldehyde on ice for 10 min.
Fixed cells were rinsed in PBS at room temperature followed by incubation in PBS containing
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0% normal Goat Serum and 0.1 % Triton Xꢀ100 to block the cells. The cells were then
incubated with antiꢀPKCα antibody (dilution, 1:100) for overnight at 4 °C in blocking solution.
Cells were rinsed three times in PBS and then incubated in FITCꢀconjugated goat antiꢀrabbit
secondary antibody (Santa Cruz Biotechnology, Dallas, TX) (dilution, 1:200) for 1 h and
visualized in Leica SP8 confocal microscope (Leica Microsystems Inc, Buffalo Grove, IL). The
subꢀcellular distributions of PKCα in confocal images were quantified using ImageJ software
(http://rsb. info.nih.gov/ij/) (37) using the guidelines described earlier (38). Briefly, cytosol and
plasma membrane (~300 nm inside the cell edge) regions were defined and their respective total
fluorescence intensity was measured. The normalized intensity ratio between the cytosol and
plasmaꢀmembrane are displayed in the figures.
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Statistical Analysis: Statistical analyses were performed using Sigma Plot 11 and were based
on three independent experiments. The results were expressed as the mean ± SEM. Statistical
significance was established by oneꢀway RMꢀANOVA, followed by Bonferroni post hoc test. A
value of P < 0.05 was considered significant.
Results
Absorption and emission properties of 1-6: Table S1 lists the absorption and emission
maxima of curcumin (1) and its derivatives (2-6) in various solvents. The representative
absorption spectra of 3 in different solvents are shown in Fig S1A. Absorption maxima of
curcumin (1) in hexane and water were 404 nm and 425 nm, respectively, showing a ~20 nm
red shift in polar solvent compared to the nonꢀpolar solvent. Addition of a long aliphatic chain
containing unsaturated double bond caused significant blue shift of absorption maxima of the
derivatives 2ꢀ4 compared to curcumin in water, ethanol and acetonitrile. The extent of the blue
shift in acetonitrile was less compared to water and ethanol. In hexane, no significant changes
were observed in the absorption maxima for the derivatives 2-5. Also there were no significant
differences in the absorption maxima with the increase in the number of double bonds in the
aliphatic chain in 2-4.
Curcumin (1) and its derivatives (2-6) also showed significant solventꢀdependent change
in fluorescence emission maxima (Table S1, Fig S1B). Emission maxima for curcumin in hexane
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and water are 474 nm and 572 nm respectively, showing a ~100 nm red shift in polar solvent
compared to the nonꢀpolar solvent. Similar red shift was also observed for compounds 2-4. The
extent of red shift increased with the increasing number of double bonds in the aliphatic chain.
However, as compared to curcumin, the emission maxima of 2-4 were blueꢀshifted in ethanol
and redꢀshifted in acetonitrile. No significant differences were observed between curcumin and
the derivatives 2-4 in either hexane or in water.
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Antioxidant properties of 1-6: Antioxidant activity of curcumin (1) and derivatives (2ꢀ6) was
measured using DPPH free radical scavenging assay (Fig 2). Curcumin, with two phenolic OH
groups, exhibited DPPH radical scavenging activity with an IC value of 14.7±1 µM, a value
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which was reported earlier (39). Derivative 6, with four phenolic OH groups, showed increased
DPPH radical scavenging activity with an IC value of 2.6±1µM. In contrast, derivatives 2ꢀ4,
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with one phenolic OH group, showed reduced DPPH radical scavenging activities. The IC50
values of 2, 3 and 4 were 20.8±1, 26.2±1, and 25.8±1 ꢁM respectively. We did not observe any
DPPH radical scavenging activity by 5. This could be, in part, due to the poor solubility of
derivative 5 in methanolic solution.
Effect of 1-6 on cell viability: Curcumin exerts cell toxicity to cancer and nonꢀcancer cell lines
(40-42). In the present study, we examined the toxicity by measuring the viability of CHOꢀK1
cells in the presence of curcumin and its derivatives (Fig 3). Curcumin (1) and
didemethylcurcumin (6) showed a doseꢀdependent decrease in cell viability. In contrast,
derivatives (2-4) with C unsaturated chain and derivative (5) C saturated alkyl chain showed
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higher cell viability than curcumin. Additionally, no significance difference in cell viability was
observed for 2-5.
Effect of 1-6 on PKC expression: We examined the effect of curcumin (1) and its derivatives
(2-6) on the expression of PKCα and PKCε from whole cell lysate after treating CHOꢀK1 cells
with (1-6, 10 µM) for 24 h (Fig 4). Curcumin (1) has no effect on PKCα and PKCε expression.
While the expression level remains unchanged for PKCα and PKCε with C16 saturated alkyl
chain derivative (5), the derivatives 2-4, with C18 unsaturated aliphatic chain significantly
increased the expression of PKCα, but not PKCε. The increment was calculated to be 27%,
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6%, and 45% (P <0.05) for 2, 3 and 4 respectively. For didemethylcurcumin (6), the expression
level was significantly decreased for both PKCα and PKCε.
Effect of 1-6 on TPA-induced membrane translocation of PKCα and PKCε: TPA is a highly
potent PKC activator that causes translocation of PKC from cytosol to membrane. To find out if
chemical modifications of curcumin could affect PKC activity, we examined the effect of 1-6 on
TPAꢀinduced PKC membrane translocation. For this, CHOꢀK1 cells were coꢀtreated with TPA
(100 nM) and 1-6 (6.25ꢀ25 µM) for 1 h and distribution of PKC in cytosol and membrane was
analyzed. TPA alone localized both PKCα and PKCε to membrane as expected (Fig 5).
However, when curcumin was coꢀtreated with TPA, the amount of PKCα in the membrane was
reduced (P <0.05). This reduction was doseꢀdependent. At the same concentration curcumin
alone did not affect level of PKCα in the membrane. These results were further supported by
confocal analysis which showed significant increase in cytosolic to membrane ratio (P <0.05) of
PKCα on coꢀtreatment as compared to TPA alone. In contrast, the amount of PKCε in the
membrane fractions remained unchanged with the coꢀtreatment of TPA and curcumin (Fig 6).
Moreover, coꢀtreatment of 2-5 with TPA did not reduce membrane translocation of either PKCα
or PKCε (Fig S2), even after raising the concentration to as high as 100 ꢁM. Like curcumin,
didemethylcurcumin (6) inhibited TPAꢀinduced membrane translocation of PKCα in a doseꢀ
dependent manner, but, not of PKCε (Fig S3). In summary, curcumin and didemethylcurcumin
inhibited TPAꢀinduced membrane translocation of PKCα and this property was lost when the
chemical modification was done with the addition of long aliphatic chain in 2-5.
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Effect of 1-6 on the membrane translocation of PKCα and PKCε:
The results from the previous section that the curcumin derivatives 2-5 are ineffective in
inhibiting the TPAꢀinduced membrane translocation unlike curcumin and didemethylcurcumin,
we decided to check their effect on membrane translocation in the absence of TPA. Initially this
experiment was performed with 1 h incubation of 1-6 with a concentration varying in the range
0ꢀ100 ꢀM and no membrane translocation of either PKCα or PKCε was observed. Control
experiments with PKC activators TPA (100 nM) and DOG (1ꢀM) however translocated both the
PKC isoforms to the membrane as expected (Fig. S4).
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We then examined the effect of 1-6 on membrane translocation for 24 h incubation at 10
M (Fig.7). At this concentration curcumin (1) and its analogs 5 and 6 did not cause membrane
ꢀ
translocation of PKCα. In contrast, 2, 3 and 4 resulted in 46%, 27% and 36% (P< 0.05) increase
in PKCα membrane translocation, respectively. No effect on PKCε membrane translocation was
observed as cytosolic and membrane fractions remain same for any of the treated compound like
untreated cells. To summarize, curcumin with unsaturated aliphatic chains selectively
activates/translocate PKCα over PKCε.
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Effect of 1-4 and 6 on ERK1/2 phosphorylation:
To examine if inhibition/activation of PKCα by 1ꢀ4 and 6, is propagated along the signal
transduction pathway, their effect on the downstream ERK1/2 was studied. Inhibition/activation
of ERK1/2 was determined by the extent of its phosphorylation in response to 1ꢀ4 and 6. As
expected, cell treated with TPA (100 nM) significantly (>2 folds) increased the level of pERK1/2
indicating activation of ERK1/2. In contrast, no effect of curcumin was observed on basal
pERK1/2 level at 25 ꢀM (Fig. 8A). However, when cells were coꢀtreated with TPA (100 nM)
and curcumin (25 µM), TPAꢀinduced pERK1/2 level was reduced (2 fold), which is consistent
with the inhibition of TPAꢀinduced membrane translocation by curcumin. Likely, derivative 6
also inhibited TPA induced ERK1/2 phosphorylation (Fig 8B). In contrast, treatment of CHOꢀ
K1 cells with curcumin derivatives 2-4 for 24 h significantly (P <0.05) increased the pERK1/2
level, which again is consistent with our membrane translocation data for PKCα (Fig. 8C).These
results indicated that curcumin derivatives activated the ERK1/2 pathway by presumably
activating PKCα.
Discussion
Current literature data on the modulation of isoformꢀselective PKC activity by curcumin is
obscure and inconsistent. This is, in part, due to the variation in the assay system and the purity
of PKC used by different groups. In the present study we selected a simple system of CHOꢀK1
cells in which PKCα, a conventional PKC and PKCε, a novel PKC are endogenously expressed.
Expression of these two PKC isoforms was higher than the other isoforms such as, PKCβ and
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PKCθ (data not shown). We chose to measure the effect of curcumin and its derivatives on PKC
using membrane translocation assay, because in its mechanism of function, PKC first
translocates to the membrane from cytosol, binds to the endogenous DAG and then
phosphorylates target proteins (43). Our results show that curcumin by itself does not affect the
membrane translocation of either PKCα or PKCε, but inhibits the TPAꢀinduced membrane
translocation of PKCα, and not PKCε. Further, to elucidate the role of the hydroxyl group of
curcumin, we made analogs 2-5 by modifying one of its hydroxyl groups with long aliphatic
chain having varying degree of unsaturation and analog 6 in which number of hydroxyl group
was increased to four from two in curcumin, and measured their effects on membrane
translocation. The modification with the long aliphatic chain was done because both endogenous
DAG and most potent phorbol ester, TPA contain long aliphatic chain(43). It was observed
earlier that addition of aliphatic chains in the structure of PKC activators such as benzolactams at
different positions could modify their binding to PKCs (44, 45). Our results show that
modification of the hydroxyl group of curcumin in 2-5 completely abolished inhibition of TPAꢀ
induced membrane translocation shown by curcumin for PKCα. Instead, the derivatives with
unsaturated long aliphatic chain (2-4) showed activation of PKCα, but not PKCε at a
concentration of 5ꢀ10 ꢀM, indicating selectivity of curcumin and its derivatives for PKCα over
PKCε.
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Our earlier studies with another polyphenol, resveratrol showed similar selectivity
towards PKCα over PKCε in cellular environment (36, 46). The only difference was the extent
of activation observed for the long chain derivatives with varying number of unsaturated double
bonds. Whereas in the case of resveratrol, the derivative with three double bonds in the long
chain (linolenic acid) caused highest membrane translocation of PKCα, the curcumin derivatives
with one double bond (oleic acid) in the long chain caused highest translocation of PKCα.
Derivatives with two and three double bonds in the aliphatic chain also showed substantial extent
of membrane translocation. The relative extent of PKCα translocation caused by the curcumin
derivatives 2-4 is similar to the extent of membrane translocation caused by the respective fatty
acid chains in these derivatives, which follows the order: oleic acid ≈ linolenic acid >> linoleic
acid (36). This also indicates that in derivatives 2-4, the fatty acid component of the molecule
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dictates its activation property rather than the curcumin moiety. In terms of selectivity, our
results are also consistent with an early report that oleic acid, linoleic acid and linolenic acid
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Several studies suggested that phenolic group substituted curcumin analogs often showed
aberrant or loss in cytotoxicity, which was dependent on the size and nature of the substituted
group. For example, modifications of curcumin with electron donating in the ring showed less
toxicity towards cancer cells (48, 49). Consistent to this, compounds 2ꢀ5 in which phenolic group
was modified, displayed reduced cytotoxicity. The logP value is also an important factor for
polyphenol cytotoxicity(50). For example, curcumin is more cytotoxic with log P value of 2.5
compared to bisꢀeugenol (a curcumin related compound) with log P value 4.8(51). A recent study
also found a correlation between logP values of several curcumin analogs and the cellular uptake
efficiency in several cancer cell lines (39). Our observation that the chemical modifications on
the phenolic OH group of curcumin increased the logP values of 2-5 significantly (Table S1) and
reduced cytotoxicity, suggests that lipophilicity of curcumin derivatives has an effect on the
viability of CHOꢀK1 cells.
Our result that derivatization of the hydroxyl group completely reversed the modulation
properties highlights the role of the hydroxyl group of curcumin in PKC modulation.
Interestingly, these phenolic OH groups are also important for curcumin’s antioxidant (39, 52)
and cytotoxic properties(48).Consistent with this, when one of the phenolic hydroxyl groups was
modified in 2-5, antioxidant activity was reduced and these compounds did not inhibit TPAꢀ
induced membrane translocation at all. Compound 6 with four hydroxyl groups, on the other
hand, showed similar membrane translocation and ERK1/2 phosphorylation property like
curcumin although it shows five times more antioxidant activity as compared to curcumin. This
indicates that antioxidant property of the compound does not have direct correlation with its PKC
activity.
Where is the site of action of curcumin in PKC? That curcumin inhibits the TPAꢀinduced
membrane translocation of PKCα, it is tempting to suggest that curcumin competes with phorbol
ester for binding to this isoform. In fact, our previous modeling studies with curcumin predicted
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this binding site (34). However, the inertness of curcumin towards PKCε suggests that either the
residues present in the phorbol ester binding cleft in PKCα that are different from PKCε, exert
optimum binding to curcumin or the calciumꢀbinding C2 domain of PKCα is responsible for
curcumin binding. While studies using a mixture of conventional PKCs, Mahmmoud predicted
that curcumin competes with the calcium binding site in C2 domain (31), using purified PKCα,
PerezꢀLara et al discarded this suggestion by showing that PKC modulation, in fact, was
dependent on the presence and absence of membrane rather than the calcium concentration (32).
Also, the activation shown by compounds 2-5, having long aliphatic chain, suggests the role of
lipid membrane in PKC modulation since long aliphatic chain can interact with the membrane
lipids. The presence of unsaturated fatty acyl chain in the lipid bilayer greatly influences PKC
activity (53-55) and in some cases shows isoform preference (56, 57). For example, the presence
of unsaturation in PC and PS elicits different effects on the activity of PKCα and PKCε (57). In
terms of phospholipid selectivity, whereas PKCα shows high selectivity for PS (58), PKCε
shows little selectivity for it (59). Now, that curcumin is also known to alter the structure and
properties of lipid bilayer (60-62), it is highly likely that the affinity of PKCα, PKCε or their
complex with curcumin derivatives for plasma membrane would be different. It is also possible
that binding of curcumin to PKC affects its interaction with the anchoring protein, RACK in an
isoformꢀdependent manner resulting in translocation variation of PKC isoforms. Taken together,
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the different membrane translocation of different PKC isoforms. In this regard, structural studies,
specifically a crystal structure of a fullꢀlength PKCα complexed with curcumin is expected to
shed light on pinpointing the exact site of action of curcumin in PKC.
Our results that the effects of curcumin and its derivatives on PKC is transduced in its
downstream readout ERK1/2, either by the Ras →Raf →MEK1/2 →ERK1/2 or the Raf
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MEK1/2 →ERK1/2 pathways, these compounds may have therapeutic potential for treating
disease states involving PKCα, such as cardiac contractility, atherogenesis, cancer and arterial
thrombosis (63-65). Since curcumin is a low affinity PKC ligand as compared to phorbol ester or
DAG, further structureꢀactivity relationship (SAR) studies on curcumin moiety are required for
developing highꢀaffinity curcuminꢀbased PKC ligand.
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In conclusion, curcumin and its derivatives show selectivity towards PKCα over PKCε in
CHOꢀK1 cells. The long chain derivatives are less cytotoxic than the parent curcumin and
activate PKCα and its downstream target ERK1/2. The hydroxyl group of curcumin is important
for modulating its PKC activity. This study shows that simple chemical scaffold like curcumin
can be suitably modified to develop isoform selective PKC modulator.
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Supporting Information Available.
Absorption and emission maxima of curcumin derivatives; Effect of curcumin derivatives on the
membrane translocation of PKCα and PKCε at various concentrations.
Funding Information
This research was supported by the startꢀup funds from the University of Houston.
Figure Legends
Figure 1: Chemical structure of curcumin (1) and its derivatives (2-6)
Figure 2: Effect of 1ꢀ6 on DPPH radical scavenging activity. Percent of free DPPH (60 ꢁM) in
presence of 0.25ꢀ60 µM of 1(●), 2(■), 3(▲), 4(▼), 5(♦), and 6( ).Values are the average of
triplicate experiments and represented as mean ± SEM. Solid lines indicate the Hill fit of dose
response curves. The corresponding IC for 1, 2, 3, 4, and 6 are 14.7±1, 20.8±1, 26.2±1, 25.8±1,
5
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and 2.6±1 ꢁM, respectively and the corresponding Hill slopes are 0.96, 0.98, 0.98, 0.99, and 0.97
respectively. Absorbance was measured at 517 nm. For compound 5, IC50 could not be
measured.
Figure 3: Effect of 1ꢀ6 on CHOꢀK1 cell viability. The bar graph shows the percentage of viable
cells after treatment with a) 0 ꢁM, b) 25 ꢁM, c) 50 ꢁM, d) 100 ꢁM of 1, 2, 3, 4, 5 and 6 for 48 h.
Data are expressed as mean ± SD of three independent experiments. Statistical analysis was
performed using oneꢀway ANOVA, followed by Bonferroni post hoc test. *P< 0.05 compared to
control (0 ꢁM). The cell viability was measured by MTT assay.
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Figure 4: Effect of 1-6 on the expression of PKCα and PKCε in CHOꢀK1 cells. Upper panels,
Western blot analysis of the whole cell lysate of CHOꢀK1 after treatment with 1ꢀ6 (10 µM) for
24 h. βꢀactin was used as a reference for equal loading. Control (ctrl) refers to the sample with
vehicle (0.1% DMSO) treated cells. Lower panel, bar graph of densitometry analysis of PKC
expression, Mean ± SEM, n = 3. Statistical analysis was performed using oneꢀway ANOVA,
followed by Bonferroni post hoc test.*P <0.05 compared to control.
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Figure 5: Effect of curcumin (1) on TPAꢀinduced membrane translocation of PKCα and PKCε in
CHOꢀK1 cells. Upper panels, Western blot analysis of the cytosolic (C) and membrane (M)
fraction of (A) PKCα and (B) PKCε after cells either treated with a) 25 ꢁM, b) 12.5 ꢁM, c) 6.25
ꢁ
M of 1 or coꢀtreated with d) 100 nM of TPA for 1h. Control (ctrl) refers to the sample with
vehicle (0.1% DMSO) treated cells. Lower panel, bar graph of densitometry analysis of upper
panel immunoblots, Mean ± SEM, n = 3. Statistical analysis was performed using oneꢀway
ANOVA, followed by Bonferroni post hoc test.*P <0.05 compared to TPA.
Figure 6: Effect of curcumin (1) on TPAꢀinduced membrane translocation of endogenous PKCα
in CHOꢀK1 cells. Confocal analysis of PKCα after cells were either treated with 1 (6.25ꢀ25 ꢁM)
or coꢀtreated with 1 (6.25ꢀ25 ꢁM) and TPA (100 nM) for 1 h. 100 nM TPA was used as a
positive control. Control (ctrl) refers to the sample with vehicle (0.1% DMSO) treated cells.
Images are representative of three independent experiments for each condition. Lower panel, bar
graph shows quantification of cytosol to plasma membrane fluorescence intensity ratio of upper
panel confocal images, Mean ± SEM, n = 5. Statistical analysis was performed using oneꢀway
ANOVA, followed by Bonferroni post hoc test.*P <0.05 compared to TPA.
Figure 7: Effect of 1ꢀ6 on the membrane translocation of PKCα and PKCε in CHOꢀK1 cells.
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 1-6 for 24 h. Control (ctrl) refer
to the sample with vehicle (0.1% DMSO) treated cells. Lower panel, bar graph of densitometry
analysis of upper panel immunoblots, Mean± SEM, n=3. Statistical analysis was performed
using oneꢀway ANOVA, followed by Bonferroni post hoc test. * P< 0.05 compared to control.
Figure 8: Effect of curcumin 1ꢀ4 and 6 on ERK activation in the presence and absence of TPA in
CHOꢀK1 cells. Cells were either treated with 25 µM of 1(A) and 6(B) in presence or absence of
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panel, bar graph of densitometry analysis of upper panel immunoblots, Mean ± SEM, n = 3.
Statistical analysis was performed using twoꢀway RMꢀANOVA with a post hoc Bonferroni
test.*P <0.01 compared to control and **P<0.05 compared to control
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Figure 1: Chemical structure of curcumin (1) and its derivatives (2-6)
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of 0.25ꢀ60 µM of 1(●), 2(■), 3(▲), 4(▼), 5(♦), and 6( ).Values are the average of triplicate experiments
and represented as mean ± SEM. Solid lines indicate the Hill fit of dose response curves. The corresponding
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corresponding Hill slopes are 0.96, 0.98, 0.98, 0.99, and 0.97 respectively. Absorbance was measured at
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treatment with a) 0 µM, b) 25 µM, c) 50 µM, d) 100 µM of 1, 2, 3, 4, 5 and 6 for 48 h. Data are expressed
as mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA,
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Figure 4: Effect of 1ꢀ6 on the expression of PKCα and PKCε in CHOꢀK1 cells. Upper panels, Western blot
analysis of the whole cell lysate of CHOꢀK1 after treatment with 1ꢀ6 (10 µM) for 24 h. βꢀactin was used as a
reference for equal loading. Control (ctrl) refers to the sample with vehicle (0.1% DMSO) treated cells.
Lower panel, bar graph of densitometry analysis of PKC expression, Mean ± SEM, n = 3. Statistical analysis
was performed using oneꢀway ANOVA, followed by Bonferroni post hoc test.*P <0.05 compared to control.
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Biochemistry
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Figure 5: Effect of curcumin (1) on TPAꢀinduced membrane translocation of PKCα and PKCε in CHOꢀK1 cells.
Upper panels, Western blot analysis of the cytosolic (C) and membrane (M) fraction of (A) PKCα and (B)
PKCε after cells either treated with a) 25 ꢁM, b) 12.5 ꢁM, c) 6.25 ꢁM of 1 or coꢀtreated with d) 100 nM of
TPA for 1h. Control (ctrl) refers to the sample with vehicle (0.1% DMSO) treated cells. Lower panel, bar
graph of densitometry analysis of upper panel immunoblots, Mean ± SEM, n = 3. Statistical analysis was
performed using oneꢀway ANOVA, followed by Bonferroni post hoc test.*P <0.05 compared to TPA.
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Biochemistry
Page 28 of 31
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Figure 6: Effect of curcumin (1) on TPAꢀinduced membrane translocation of endogenous PKCα in CHOꢀK1
cells. Confocal analysis of PKCα after cells were either treated with 1 (6.25ꢀ25 ꢁM) or coꢀtreated with 1
(6.25ꢀ25 ꢁM) and TPA (100 nM) for 1 h. 100 nM TPA was used as a positive control. Control (ctrl) refers to
the sample with vehicle (0.1% DMSO) treated cells. Images are representative of three independent
experiments for each condition. Lower panel, bar graph shows quantification of cytosol to plasma membrane
fluorescence intensity ratio of upper panel confocal images, Mean ± SEM, n = 5. Statistical analysis was
performed using oneꢀway ANOVA, followed by Bonferroni post hoc test.*P <0.05 compared to TPA.
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Biochemistry
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Figure 7: Effect of 1ꢀ6 on the membrane translocation of PKCα and PKCε in CHOꢀK1 cells. 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 1ꢀ6 for 24 h. Control (ctrl) refer to the sample with vehicle (0.1%
DMSO) treated cells. Lower panel, bar graph of densitometry analysis of upper panel immunoblots, Mean±
SEM, n=3. Statistical analysis was performed using oneꢀway ANOVA, followed by Bonferroni post hoc test. *
P< 0.05 compared to control.
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