Angoline and Chelerythrine, Benzophenanthridine Alkaloids That Do
Not Inhibit Protein Kinase C*
Sang Kook
Lee
,
Wei Guo
Qing
§,
Woongchon
Mar¶,
Lumonadio
Luyengi
,
Rajendra G.
Mehta§,
Kazuko
Kawanishi
,
Harry H. S.
Fong
,
Christopher W. W.
Beecher
,
A. Douglas
Kinghorn
, and
John M.
Pezzuto
§**
From the
Program for Collaborative Research in the
Pharmaceutical Sciences and Department of Medicinal Chemistry and
Pharmacognosy, College of Pharmacy and the § Department of
Surgical Oncology, College of Medicine, University of Illinois,
Chicago, Illinois 60612, ¶ Natural Products Research Institute,
Seoul National University, Seoul 110-460, Korea, and
Kobe
Pharmaceutical University, Kobe 658, Japan
 |
ABSTRACT |
Starting with an extract derived from the stem of
Macleaya cordata (Papaveraceae) that was active in the
process of inhibiting phorbol 12,13-dibutyrate binding to partially
purified protein kinase C (PKC), the benzophenanthridine alkaloid
angoline was isolated and identified. This discovery appeared in
context, as a related benzophenanthridine alkaloid, chelerythrine, has
been reported to mediate a variety of biological activities, including potent and selective inhibition of protein kinase C (PKC). However, in
our studies, angoline was not observed to function as a potent inhibitor of PKC. Moreover, we were unable to confirm the reported inhibitory activity of chelerythrine. In a comprehensive series of
studies performed with various PKC isozymes derived from a variety of
mammalian species, neither chelerythrine nor angoline inhibited
activity with high potency. To the contrary, chelerythrine stimulated
PKC activity in the cytosolic fractions of rat and mouse brain in
concentrations up to 100 µM. In addition, chelerythrine and angoline did not inhibit [3H]phorbol 12,13-dibutyrate
binding to the regulatory domain of PKC at concentrations up to 40 µg/ml, and no significant alteration of PKC-
, -
, or -
translocation was observed with human leukemia (HL-60) cells in
culture. Further, chelerythrine did not inhibit 12-O-tetradecanoylphorbol 13-acetate-induced ornithine
decarboxylase activity with cultured mouse 308 cells, but angoline was
active in this capacity with an IC50 value of 1.0 µg/ml.
A relatively large number of biological responses have been reported in
studies conducted with chelerythrine, and alteration of PKC activity
has been considered as a potential mechanism of action. In light of the
current report, mechanisms independent of PKC inhibition should be
considered as responsible for these effects.
 |
INTRODUCTION |
Protein kinase C (PKC),1
a serine/threonine kinase, has been extensively studied due to its
central role in cellular signal transduction (1). Various mitogens,
growth factors, and transmitters of secondary messengers have been
shown to mediate their effects through PKC. PKC subtypes have been
grouped as (i) conventional (
,
I,
II, and
), which are
activated by calcium, phospholipids, diacylglycerol, and
12-O-tetradecanoylphorbol 13-acetate (TPA); (ii) novel (
,
,
,
, and µ), which do not require calcium for activation;
and (iii) atypical (
and
), which are activated by phospholipids
but not by calcium, diacylglycerol, or TPA (2, 3). It is likely that
these isozymes have distinct and distinguishable functions (4-7).
Since PKC has been elaborated as a major intracellular phorbol ester
receptor (8, 9), it has become clear that this protein plays an
important role in the process of tumor promotion (10). Accordingly,
inhibition of PKC can be viewed as a rational method of blocking or
inhibiting tumor promotion, and several natural product inhibitors have
been identified. Examples include staurosporine (11), UCN-1028C (12),
and isoquinolinesulfonamide H-7 (13). In addition, the
benzophenanthridine alkaloid, chelerythrine, was described as a potent
and selective inhibitor of PKC, with an IC50 value of 0.66 µM illustrated with enzyme derived from rat brain (14).
Biological responses mediated by chelerythrine, such as cytotoxic
activity with L-1210 tumor cells (14) and antiplatelet activity (15),
have been ascribed to inhibition of PKC. Additional activities mediated
by chelerythrine include inhibition of alanine aminotransferase (16),
inhibition of Na+,K+-ATPase (17), and
antibacterial effects (18).
In searching for novel natural product cancer chemopreventive agents,
the benzophenanthridine alkaloid angoline was obtained from an extract
of Macleaya cordata (Papaveraceae); this plant extract had
previously been shown to antagonize the interaction of
[3H]phorbol 12,13-dibutyrate (PDBu) with PKC receptor.
Due to the structural similarity of angoline and chelerythrine, we
undertook a series of studies to investigate the activity of angoline,
especially in the context of affecting PKC-mediated responses. As
reported herein, neither angoline nor chelerythrine were effective
inhibitors of PKC. Alternate mechanisms should be taken into account
when considering the biological responses mediated by these
compounds.
 |
EXPERIMENTAL PROCEDURES |
Materials
Phosphatidylserine, PDBu, histone type IIIs, ATP, pyridoxal
phosphate, dithiothreitol, 1,2-diolein, Triton X-100, and staurosporine were obtained from Sigma. [
-32P]ATP (6000 Ci/mmol, 10 mCi/ml) was from Amersham Pharmacia Biotech, and
[20-3H]phorbol 12,13-dibutyrate ([3H]PDBu)
(20 Ci/mmol) was purchased from NEN Life Science Products. S-minimal
essential medium, RPMI 1640, non-essential amino acid solution (10 mM, 100×), trypsin-EDTA solution (1×), and
penicillin-streptomycin, antibiotic-antimycotic solution were purchased
from Life Technologies, Inc. Dialyzed fetal bovine serum was obtained
from HyClone (Logan, UT). Monoclonal anti-PKC (
,
, and
)
antibodies were purchased from Transduction Laboratories
(Lexington, KY).
Angoline and Chelerythrine
Angoline was isolated from the stem of M. cordata
(Willd.) R. Br. (syn. Bocconia cordata Willd.)
(Papaveraceae). Briefly, the stem bark of M. cordata (600 g)
was extracted with methanol (3 × 3 liters). The resultant extract
(50 g) was defatted with petroleum ether (300 ml), suspended in 100 ml
of H2O, and partitioned with ethyl acetate (3 × 400 ml) to afford 8 g of an ethyl acetate residue. Further
purification was performed by silica gel column chromatography using
CHCl3, and CHCl3 with an increasing amounts of
methanol (0-25%) as eluents. Through repeated column chromatography of the subfractions, angoline (Fig. 1) (also known as
9-methoxychelerythrine) was isolated with melting point 212-215 °C
(literature: 210 °C; Ref. 19), [
]D20 + 1.85 ° (literature: 0 °; Ref. 20), and exhibited UV, infrared, and electron impact mass spectroscopic data comparable with literature values (20, 21), and 1H NMR (CDCl3)
2.75 (3H, s, N-CH3), 3.45 (3H, s,
OCH3-8), 3.90 (3H, s,
OCH3-10), 3.95 (3H, s,
OCH3-9), 5.53 (1H, s, H-8), 6.02 (2H, s,
-O-CH2-O-), 7.03 (1H, dd, J = 8.70 Hz, 2.40 Hz, H-11), 7.10 (1H, s, H-4), 7.46 (1H, d,
J = 8.60 Hz, H-5), 7.60 (1H, d, J = 8.70 Hz, H-12), 7.68 (1H, s, H-1), 7.75 (1H, d, J = 8.60 Hz, H-6); 13C NMR (CDCl3)
40.59 (q,
N-CH3), 53.87 (q,
OCH3-8), 55.84 (q, OCH3-10), 61.59 (q,
OCH3-9), 86.00 (d, C-8), 100.53 (d, C-1), 100.94 (t, -O-CH2-O-), 104.52 (d, C-4), 112.85 (d,
C-11), 118.93 (d, C-12), 120.02 (d, C-6), 122.46 (s, C-13), 123.39 (d,
C-5), 125.00 (s, C-12a), 125.90 (s, C-8a), 126.40 (s, C-14a), 130.96 (s, C-4a), 138.12 (s, C-14), 146.00 (s, C-9), 147.22 (s, C-3), 148.31 (s, C-2), 152.02 (s, C-10). Chelerythrine (Fig. 1) was obtained from
various commercial sources (Sigma; LC Service Corp., Woburn, MA;
Extrasynthese, Lyon, France).
PKC Preparations and Analysis of Activity
The purification of PKC-
from calf brain and the PKC assay
were performed essentially as described by Da Silva et al.
(23). Briefly, fresh calf brain homogenates were centrifuged at
100,000 × g for 60 min. The supernatant was
concentrated using an ultrafiltration cell (Amicon Division, Beverly,
MA) equipped with a XM50 membrane and subjected to passage over a
DEAE-cellulose column equilibrated with buffer A (20 mM
Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 10% glycerol). The sample was eluted
with a linear gradient of KCl (0-0.3 M) in buffer A. Fractions were collected and enzyme activities were monitored by
[3H]PDBu binding (24). The major fractions showing
binding activity were combined and concentrated, and further
purification of PKC was conducted by sequential column chromatography
over phenyl-Sepharose, Sephacryl S-200,
poly-L-lysine-agarose, and hydroxyapatite. Additional samples of brain used for the preparation of cytosolic fractions (12,000 × g) bearing PKC activity were from mouse,
rat, rabbit, chicken, and dog (Pel-Freez® Biologicals,
Rogers, AR).
PKC catalytic activity was assayed by quantifying transfer of the
-phosphate group of [
-32P]ATP into histone IIIs.
The incubation mixture (80 µl) consisted of 20 mM
Tris-HCl buffer, pH 7.5, 200 µg/ml histone (type IIIs), 10 mM MgCl2, 0.5 mM CaCl2,
500 µg/ml phosphatidylserine in 3% Triton X-100, 10 µM
ATP containing [
-32P]ATP, and test compounds. After a
10-min preincubation at 30 °C, reactions were initiated by the
addition of 20 µl of enzyme solution (4 µg). After an additional
incubation period of 10 min, 85 µl of the reaction mixtures were
spotted onto Whatman P-81 cation exchange paper (Whatman Paper Ltd.,
Maidstone, Kent, United Kingdom). The papers were washed three times
with 0.2% aqueous phosphoric acid solution, air-dried, and subjected
to liquid scintillation counting. Test compounds were evaluated for
potential to affect basal activity (no activator added), or in the
presence of PDBu (0.4 µM) or 1,2-diolein (180 µM). Results were expressed as a percentage, relative to
solvent-treated controls, and dose-response relationships were obtained
by evaluating serial dilutions of test compounds in duplicate.
Assessment of PDBu Binding to the Regulatory Site of PKC
PDBu binding assays were performed in 96-well microtiter plates
essentially as described by De Vries et al. (24), using calf
brain homogenate or other sources of soluble PKC as receptor. Each
reaction mixture (200 µl final volume) contained homogenate (25 µg
of protein) as a source of PKC, bovine serum albumin (200 µg), 20 mM Tris-HCl buffer, pH 7.4, and [3H]PDBu (5 nM, 20 Ci/mmol). Test substances (10 µl) were added to
the reaction mixtures (in duplicate) and incubated for 1 h at
37 °C. Unbound [3H]PDBu was removed by filtration with
50 mM Tris-HCl, pH 7.4, through glass fiber filter mats
(Type B; Wallac Inc., Gaithersburg, MD) using a Tomtec Mach
III® 96-well plate harvester (Tomtec, Orange, CT).
Radioactivity was determined by scintillation counting using a Wallac
1450 MicroBeta® liquid scintillation counter (Wallac,
Inc.). The amount of [3H]PDBu bound in the presence of
nonradioactive PDBu (4 µM) was used to assess nonspecific
binding. Specific binding was defined as the difference between total
and nonspecific binding, and results were expressed as a percentage,
relative to the solvent-treated control group. IC50 values
were calculated using semi-log plots.
Translocation Studies and Western Blot Analysis of PKC with
Cultured HL-60 Cells
HL-60 Cells--
Human promyelocytic leukemia (HL-60) cells,
obtained from ATCC (Rockville, MD), were routinely cultured in RPMI
1640 medium containing 7.5% fetal bovine serum, 100 units penicillin
(100 units/ml), and streptomycin (100 µg/ml) (37 °C, 100%
humidity, 5% CO2 in air).
Preparation and Fractionation of HL-60 Cells--
HL-60 cells in
logarithmic phase (1 × 107 cells) were treated with
test compounds for 3 or 24 h. Cells were harvested by
centrifugation (500 × g, 10 min), washed twice with 10 ml of ice-cold phosphate-buffered saline, pH 7.4, and homogenized
(4 °C) in 1.0 ml of buffer (20 mM Tris-HCl, pH 7.4, 5 mM NaCl, 1 mM EDTA, 5 mM
MgCl2, 2 mM dithiothreitol, 200 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin,
10 µg/ml leupeptin) for 30 s using a Brinkmann
Polytron® PT-3000 homogenizer (Littau, Switzerland). The
homogenates were centrifuged at 100,000 × g for 1 h (4 °C), and the resulting supernatant fractions (used as a source
of cytosolic PKC) were stored at
80 °C. The pellets were
solubilized in buffer containing 1% Triton X-100 by vortexing and
incubating on ice for 2 h, and the suspensions were centrifuged at
100,000 × g for 1 h (4 °C) to obtain
membrane/microsomal PKC. The resulting supernatant fractions were used
as a source of membrane-associated particulate PKC, and stored at
80 °C. All protein contents were quantified using the
bicinchoninic acid (BCA) method and bovine serum albumin as a
standard.
Western Blot Analysis of PKC--
Each protein fraction (50 µg) was subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Proteins were transferred to polyvinylidene fluoride
membranes by electroblotting, and membranes were treated for 1 h
with blocking buffer (1% dry milk in 10 mM Tris-HCl
buffer, pH 7.5, 100 mM NaCl, 0.1% Tween 20). Membranes
were then incubated overnight at 4 °C with primary antibodies (mouse
anti-PKC-
, -
, and -
monoclonal antibodies) diluted in blocking
buffer (
, 1:1,000;
and
, 1:500), washed with buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% Tween
20) for 1 h, incubated for 1 h at 37 °C with
horseradish-peroxidase conjugated anti-mouse IgG (Amersham Pharmacia
Biotech) diluted 1:1,000 in blocking buffer, washed for 1 h, and
developed using enhanced chemiluminescence reagent (Amersham Pharmacia
Biotech).
Assessment of TPA-induced Ornithine Decarboxylase (ODC) Activity
with ME 308 Cells
Mouse epidermal 308 cells were cultured in S-minimal essential
medium containing non-essential amino acids (1×), dialyzed fetal
bovine serum (5%), Ca2+ (0.05 mM), and
antibiotic-antimycotics (1×) at 37 °C in a 5% CO2
atmosphere. For determination of TPA-induced ODC activity, cells were
distributed to 24-well plates at an initial density of 2 × 105 cells/ml/well. After an 18-h preincubation, test
materials dissolved in Me2SO were added in duplicate (5 µl, 0.5% final Me2SO concentration) before the induction
of ODC activity with TPA (200 nM). After an additional
incubation period of 6 h, plates were washed twice with
phosphate-buffered saline and stored at
80 °C until tested. ODC
activity was assayed directly in the 24-well plates as described previously (25). In brief, frozen cells were lysed by quickly thawing
the bottom of the culture plates in a warm water bath (37 °C, 2 min). A substrate and cofactor mixture (200 µl containing 2 µl of
L-[1-14C]ornithine (200 nCi, 56 mCi/mmol, 100 µCi/ml), 50 µl of sodium phosphate buffer (0.2 M, pH
7.2), 16 µl of EDTA (12.5 mM), 10 µl of dithiothreitol
(50 mM), 4 µl of pyridoxal phosphate (5 mM in
10 mM NaOH) and 118 µl of unlabeled
L-ornithine (78 µg/ml)) were added to each well. Released
[14C]CO2 was captured by paper discs, which
were moistened with 30 µl of 1 N NaOH during incubation
of plates at 37 °C for 1 h while shaking. The amount of
radioactivity captured on NaOH-impregnated filter disks was determined
by scintillation counting using a Wallac 1450 MicroBeta®
liquid scintillation counter. Protein was determined by the Bio-Rad method using bovine serum albumin as a standard. Results were calculated as nanomoles of [14C]CO2/h/mg of
protein and expressed as a percentage in comparison with a control
treated with Me2SO and TPA.
 |
RESULTS |
Effects of Chelerythrine and Angoline on PKC Activity--
Based
on the structural similarity of angoline and chelerythrine (Fig.
1), initial studies were performed to
assess the potential of angoline, isolated from M. cordata,
to inhibit the catalytic activity of PKC. Surprisingly, angoline
demonstrated little inhibitory activity with calf brain PKC;
IC50 values for basal and PDBu-stimulated activity were
approximately 100 µg/ml (Fig. 2). We
then examined chelerythrine in this assay system. In contrast to the
results of Herbert et al. (14), chelerythrine did not
mediate potent inhibitory activity with calf brain PKC.
IC50 values were approximately 60 and 80 µg/ml with basal
and PDBu-stimulated activity, respectively (Fig. 2). The lack of
efficacious inhibition by chelerythrine was not due to the nature of
the stimulator, since similar dose-response patterns were observed in
the presence of either PDBu or diolein (Fig.
3). Using the same experimental
conditions, staurosporine (133 ng/ml) inhibited activity over 90%,
irrespective of which stimulator was employed (data not shown).
Chelerythrine was also ineffective as an inhibitor using mouse and rat
brain cytosol fractions as sources of PKC. In fact, with these
preparations, chelerythrine enhanced PKC activity in concentrations
ranging up to 100 µM (Fig.
4).

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Fig. 2.
Effect of angoline and chelerythrine on
purified calf brain protein kinase C activity. PKC activity was
determined in the presence of the indicated concentrations of angoline
( ) or chelerythrine ( ) in the absence of stimulator (PKC basal
activity), or in the presence of PDBu (0.4 µM)
(angoline ( ) or chelerythrine ( )). Basal and PDBu-induced
specific activities of 32P incorporation were 9.7 ± 0.3 and 45.9 ± 1.3 pmol of 32P/mg of protein/min,
respectively. Values represent the mean percent ± S.D. of the
respective solvent-treated controls of two independent
experiments.
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Fig. 3.
Effect of chelerythrine on calf brain PKC
activity treated with PDBu or diolein. PKC activity was measured
in the presence of the indicated concentrations of chelerythrine. Tests
were performed in the absence of any activator (basal level) ( ), or
in the presence of PDBu ( , 200 ng/ml) or diolein ( , 180 µM). Results were expressed as a percentage, relative to
the corresponding solvent-treated control incubations. Diolein (180 µM), TPA (200 ng/ml), and PDBu (200 ng/ml) enhanced basal
activity by 450 ± 31, 490 ± 23, and 480 ± 65%,
respectively. Basal specific activity was 10.5 ± 1.0 pmol of
32P/mg of protein/min.
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Fig. 4.
Effect of chelerythrine on the catalytic
activity of PKC derived from various sources. PKC activity was
determined in the presence of the indicated concentrations of
chelerythrine using diolein (180 µM) as an activator.
Cytosolic fractions (12,000 × g, supernatant) from rat
brain ( ), calf brain ( ) or mouse brain ( ), were used as
sources for PKC. Diolein-induced incorporation of 32P with
each respective enzyme source was 63.2 ± 9.2, 42.9 ± 1.9, and 39.9 ± 1.4 pmol of 32P/mg of protein/min. Data
represent the mean percent ± S.D. of control (diolein-induced
level). Tests were performed in duplicate.
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|
Effects of Chelerythrine and Angoline on PDBu Binding--
In
order to investigate the effect of chelerythrine and angoline on the
regulatory domain of PKC, PDBu binding activity was assessed with crude
calf brain particulate as a source of PKC. At concentrations of 2 µg/ml, chelerythrine and angoline enhanced [3H]PDBu
binding. This was followed by diminution of binding to the levels of
control. When tested at a concentration 200 µg/ml, chelerythrine and
angoline reduced PDBu binding to levels corresponding to 60 and 45% of
control, respectively (Fig. 5). Using
other sources of soluble PKC, including mouse, rat, dog, chicken, and
rabbit brain cytosol, chelerythrine and angoline did not show
inhibitory effects at concentrations ranging up to 20 µg/ml (data not
shown). In each experiment, unlabeled PDBu (0.4 µM)
inhibited [3H]PDBu binding to PKC by over 95% (data not
shown). Therefore, chelerythrine and angoline did not affect binding to
the regulatory domain of PKC with strong potency.

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Fig. 5.
Effect of angoline and chelerythrine on
[3H]PDBu binding. The binding of
[3H]PDBu to partially purified PKC from calf brain was
measured in the presence of the indicated concentrations of angoline
( ) or chelerythrine ( ). Specific binding activity of the control
group was 3.10 ± 0.14 pmol of [3H]PDBu/mg of
protein, and this was reduced to 0.15 ± 0.01 pmol of
[3H]PDBu/mg of protein by the addition of unlabeled PDBu
(0.4 µM). Data represent the average percent of control.
Tests were conducted in duplicate.
|
|
Effects of Test Compounds on PKC Translocation--
In order to
evaluate the potential of chelerythrine and angoline to effect PKC
translocation, Western blot analyses were performed with preparations
derived from cultured HL-60 cells treated with test compounds. Mouse
anti-PKC-
, -
, or -
antibodies were used as probes (Fig.
6). As expected, treatment with TPA (200 nM) for 3 h induced translocation of PKCs from the
cytosol to particulate fractions (Fig. 6, A and
B), and increasing exposure time to 24 h resulted in
down-regulation (Fig. 6, C and D). When tested at concentrations as high as 1.0 µg/ml, however, alterations in PKC levels or distribution patterns were not facilitated by chelerythrine or angoline (Fig. 6). When co-incubated with TPA for 24 h,
down-regulation of PKCs was slightly potentiated by the test
compounds.

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Fig. 6.
Effect of chelerythrine and angoline on
translocation of PKC. Human leukemia HL-60 cells were treated with
solvent (Me2SO, 0.2% final concentration), chelerythrine
(1.0 µg/ml), or angoline (1.0 µg/ml) in the presence or absence of
TPA (200 nM) for 3 or 24 h. As described under
"Experimenal Procedures," cytosol and membrane-associated
particulate fractions were obtained by differential centrifugation.
Protein samples (50 µg per lane) were analyzed by Western blotting
with monoclonal antibodies specific for PKC- , - , or - .
A, cells were incubated with solvent (Me2SO) or
chelerythrine in the presence or absence of TPA for 3 h.
Lane 1, control cytosol; lane 2, control
particulate; lane 3, TPA cytosol; lane 4, TPA
particulate; lane 5, chelerythrine cytosol; lane
6, chelerythrine particulate; lane 7, chelerythrine in
the presence of TPA cytosol; lane 8, chelerythrine in the
presence of TPA particulate. B, cells were incubated with
solvent (Me2SO) or angoline in the presence or absence of
TPA for 3 h. Lane 1, control cytosol; lane
2, control particulate; lane 3, TPA cytosol; lane
4, TPA particulate; lane 5, angoline cytosol;
lane 6, angoline particulate; lane 7, angoline in
the presence of TPA cytosol; lane 8, angoline in the
presence of TPA particulate. C, treatment conditions were
the same as in A, except the incubation period was 24 h. D, treatment conditions were the same as in B,
except the incubation period was 24 h.
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|
Effects of Chelerythrine and Angoline on TPA-induced ODC
Activity--
ODC is the key enzyme in the biosynthesis of polyamines
and is inducible by stimuli such as growth factors, hormones, and tumor
promoters, including phorbol esters (26). Activity is controlled by
various factors, including expression, stability and transcription rate
of ODC mRNA, stability and translation rate of the ODC enzyme, and
post-translational modifications (27, 28). Using mouse 308 cells as a
model system, chelerythrine did not inhibit TPA-induced ODC activity
within the non-cytotoxic range (IC50 >1.0 µg/ml), but
promoted a slight enhancement of activity at lower concentrations.
Angoline did not alter the TPA-induced effect at lower concentrations,
but reduced activity by approximately 50% at a test concentration of
1.0 µg/ml (Fig. 7).

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Fig. 7.
Effect of chelerythrine or angoline on
TPA-induced ODC activity. Mouse 308 cells were simultaneously
treated with TPA (200 nM) and the indicated concentrations
of angoline ( ) or chelerythrine ( ) for a period of 6 h. ODC
activity was then determined as described under "Experimental
Procedures." ODC activity of TPA-treated solvent control was
1.80 ± 0.08 nmol of [14C]CO2/mg of
protein/h; without TPA treatment, ODC activity was 0.070 ± 0.005 nmol of [14C]CO2/mg of protein/h. Data
represent the average percent ± S.D. of the TPA-treated control
group. Tests were performed in duplicate.
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|
 |
DISCUSSION |
Since PKC plays an important role in signal transduction and
phorbol ester-promoted tumorigenesis, various inhibitors have been
studied in detail. One such substance, chelerythrine, has been reported
as a potent and specific inhibitor. The phosphorylation reaction
mediated by PKC was reduced with an IC50 value of 0.66 µM. Inhibition was competitive with respect to phosphate
acceptor (histone IIIs), and noncompetitive with respect to ATP. No
substantive competition was observed with respective to
[3H]PDBu binding, thus suggesting interaction does not
occur at the regulatory site of PKC. Other kinases, such as protein
kinase A, tyrosine protein kinase, and
Ca2+/calmodulin-protein kinase, were not inhibited with
great efficacy, indicating specificity for PKC (14).
Accordingly, when we obtained angoline from a plant extract
demonstrating antagonism of PDBu binding with PKC, it was suspected that this substance would function in a mode similar to chelerythrine. However, angoline was found ineffective as an inhibitor of PKC activity. Thus, as a positive control, the inhibitory potential of
chelerythrine was investigated, obtaining this substance from a
commercial source (Sigma). However, in repeated attempts, chelerythrine was found to be practically inactive as an inhibitor of partially purified calf brain PKC. Although the compound appeared pure and stable
(HPLC analysis), additional samples were procured (LC Service Corp.,
Extrasynthese) and evaluated, and also found to be inactive. This lack
of inhibitory activity was observed with PKC obtained from a variety of
species, either in the presence or absence of activators (PDBu or
diolein). In fact, to the contrary, when evaluated with cytosol
fractions derived from rat or mouse brain, chelerythrine enhanced PKC
activity, and, with calf brain preparations, [3H]PDBu
binding was elevated. These types of activities are consistent with
recent reports by Lombardini (29, 30) who demonstrated chelerythrine-stimulated phosphorylation of a ~20-kDa protein present
in the mitochondrial fraction of rat retina. Further, using intact
cells, Gopalakrishna et al. (31) noted that chelerythrine and sanguinarine could induce an initial activation of membrane translocation from the cytosol, and subsequent down-regulation of PKC.
These responses parallel those of tumor promoters, such as phorbol
esters, and would not be anticipated by an inhibitor of PKC. Along
these lines, we evaluated angoline and chelerythrine for potential to
facilitate translocation of PKC-
, -
, and -
with cultured HL-60
cells, but no significant alterations were observed. Interestingly,
however, with cultured ME 308 cell system, chelerythrine stimulated
TPA-induced ODC activity at lower concentrations, whereas angoline was
inactive at lower concentrations but reduced activity approximately
50% at a test concentration of 1.0 µg/ml. Polyamines play essential
roles in normal cell proliferation and differentiation, but are
overexpressed in various cancer cells. Since ODC is a key component
that regulates intracellular polyamines, inhibitors of ODC activity are
of interest. Accordingly, it should be of value to explore the
mechanism by which angoline functions in this capacity.
Chelerythrine is known to mediate a variety of biological responses.
For example, rat liver alanine aminotransferase is inhibited by adduct
formation between thiol groups of the enzyme and the iminium bond of
chelerythrine (16). Chelerythrine also inhibits taxol-mediated
polymerization of rat brain tubulin (32), histone kinase activity using
partially purified PKC from the rat lacrimal gland (33),
pyrogen-induced expression of tissue factor in endothelial cells,
histamine release induced by aggregation of IgE-receptor on human
basophils (34, 35), Na+,K+,2Cl
cotransporter in Ehrlich mouse ascites tumor cells (36), and invasion
of metastatic human follicular thyroid cancer (37). We have recently
found that angoline and chelerythrine inhibit 7,12-dimethylbenz(a)anthracene-induced preneoplastic lesion
formation in mouse mammary gland organ culture by approximately 60% at
a test concentration of 10 µM (data not shown). Based on
the report of Herbert et al. (14), some of these effects
have been attributed to PKC inhibition. As indicated by the current
report, however, chelerythrine is not a general and specific inhibitor
of PKC. Accordingly, PKC-independent pathways should be considered when exploring the biological potential of chelerythrine and related compounds. For example, as noted above, interaction of the iminium bond
of chelerythrine and active sites in various target molecules can play
an important role in biological responses that are mediated by the
compound. An interaction of this type, of course, would not be specific
for or limited to PKC, and could be more broadly related to the
mechanism by which chelerythrine functions.
 |
FOOTNOTES |
*
This work was supported by National Cancer Institute Grant
P01 CA48112.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Program for
Collaborative Research in the Pharmaceutical Sciences (M/C 877),
College of Pharmacy, University of Illinois, 833 S. Wood St., Chicago, IL 60612. Tel.: 312-996-5967; Fax: 312-996-2815; E-mail:
jpezzuto{at}uic.edu.
1
The abbreviations used are: PKC, protein kinase
C; PDBu, phorbol 12,13-dibutyrate; TPA,
12-O-tetradecanoylphorbol 13-acetate; ODC, ornithine
decarboxylase.
 |
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