Protective effects of veskamide, enferamide, becatamide, and oretamide on H2O2-induced apoptosis of PC-12 cells

Article abstract of DOI:10.1016/j.phymed.2011.01.025

Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on H₂O ₂-induced apoptosis in P

Full text of DOI:10.1016/j.phymed.2011.01.025

Phytomedicine 18 (2011) 843–847
Contents lists available at ScienceDirect
Phytomedicine
journal homepage: www.elsevier.de/phymed
Protective effects of veskamide, enferamide, becatamide, and oretamide on
H2O2-induced apoptosis of PC-12 cells
Jae B. Park∗
Diet, Genomics, and Immunology Laboratory, Bldg. 307C, Rm. 131, BHNRC, ARS, USDA, Beltsville, MD 20705, United States
a r t i c l e i n f o
a b s t r a c t
Keywords:
Veskamide, enferamide, becatamide, and oretamide are phenolic amides whose analogues are found in
plants. In this study, the four amides were prepared by chemical synthesis and their protective effects on
H2O2-induced apoptosis in PC-12 cells were investigated. The syntheses were relatively simple and the
yields were more than 43%. Using NMR spectroscopic methods, the chemical structures of veskamide,
enferamide, becatamide, and oretamide were confirmed. The decreasing order of the protective effects
on H2O2-induced apoptosis was becatamide > enferamide ≥ oretamide > veskamide. In fact, becatamide
suppressed H2O2-induced mitochondrial membrane depolarization in a dose-dependent manner. At the
concentration of 10 ␮M, becatamide maintained mitochondrial membrane depolarization at 16% com-
pared to 51% in H2O2-treated PC-12 cells (P < 0.05). Also, at the same concentration, becatamide inhibited
H2O2-induced caspase-9 activation and caspase-independent chromatin condensation by 68% (P < 0.05)
and 73% (P < 0.05), respectively. This is the first report about the chemical synthesis of becatamide and its
potential biological activity to inhibit H2O2-induced apoptosis of PC-12 cells via protecting mitochondrial
membrane integrity, thereby suppressing caspase-9 activation and chromatin condensation.
Published by Elsevier GmbH.
Veskamide
Enferamide
Becatamide
Oretamide
PC-12
Mitochondria
Caspase
Chromatin condensation
Apoptosis
Introduction
damage to cellular components such as lipids, proteins, and DNA
leading to apoptosis (Cutler et al. 2005; Mena et al. 2009). A num-
Certain neurodegenerative diseases progressively deteriorate
the structure and/or function of neurons (e.g., soma, axon, den-
drites) in the central nervous system, eventually leading to cell
death (Reeve et al. 2008; Martinelli and Rugarli 2010; Nakamura
and Lipton 2009; Glass et al. 2010). In actual fact, neurodegen-
erative processes are very much responsible for several neuronal
diseases such as Alzheimer’s, Parkinson’s, and Huntington’s dis-
eases (Duyckaerts et al. 2009; Winklhofer and Haass 2010; Bates
2003). Although there are several mechanisms involved in neuronal
cell death, a most common mechanism is through the well-known
intrinsic mitochondrial apoptotic pathway (Ribe et al. 2008; Rohn
and Head 2008). This pathway regulates the activation of caspase
cascade systems by releasing cytochrome c from the mitochondria
premeabilized by the pro-apoptotic Bcl-2 members such as Bax and
Bak (Jourdain and Martinou 2009). Reactive oxygen species (ROS)
can be produced as normal by-products from several sources of
human body. Among them, mitochondria are a major source of
ROS (Wei et al. 1998; Richter et al. 1995). However, along with
insufficient antioxidant defense systems and/or some types of dis-
ease conditions, the elevated levels of ROS can cause detrimental
ber of studies suggest that ROS-induced apoptosis in neuronal
cells is associated with a series of cellular events such as mito-
chondrial depolarization, cytochrome c release, and activation of
caspases and caspase-independent chromatin condensation (Mena
et al. 2009; Kirkland and Franklin 2003; Du and Yan 2010). There-
fore, the protection of mitochondria in neuronal cells from ROS is
considered as a very promising way in treating and/or preventing
neurodegenerative and other related disorders (Du and Yan 2010;
Ramassamy 2006; Sun et al. 2008).
Veskamide, enferamide, becatamide, and oretamide are N-
phenylethylbenzoylamide-type phenolic amides, whose analogues
are found in plants such as Aniba riparia, Begonia nantoensis, Haplo-
phyllum tuberculatum, and Houttuynia cordata (Thomas et al. 1994;
Wu et al. 2004; Al-Rehaily et al. 2001; Chou et al. 2009). Most likely,
these four amides are to be found in numerous plant sources. In fact,
during the course of our study, becatamide was subsequently iso-
lated from Houttuynia cordata and its structure (houttuynamide A)
was published (Chou et al. 2009). Currently, there is great inter-
est regarding potential biological activities of these amides, but
their potential effects have not been investigated much, proba-
bly due to the scarcity of the amides. Therefore, in this paper, the
four phenolic amides were prepared using a chemical synthesis
method and their protective effects on H2O2-induced apoptosis
of rat pheochromocytoma PC-12 cells were studied by investigat-
∗
Tel.: +1 301 504 8365, fax: +1 301 504 9456.
E-mail address: jae.park@ars.usda.gov
0944-7113/$ – see front matter. Published by Elsevier GmbH.
doi:10.1016/j.phymed.2011.01.025

844
J.B. Park / Phytomedicine 18 (2011) 843–847
ing mitochondrial membrane depolarization, caspase-9 activation,
and chromatin condensation, in order to search potential com-
pounds with anti-neurodegenerative activity. This study clearly
indicates that out of the four amides, becatamide is the most
potent compound able to inhibit H2O2-induced apoptosis of PC-12
cells via maintaining mitochondrial membrane polarization, sup-
pressing caspase-9 activation, and inhibiting caspase-independent
chromatin condensation.
Experimental
Materials
Benzoic acid, protocatechuic acid, vanillic acid, syringic acid, and
other chemicals were purchased from Sigma Chemical Co. (St. Louis,
MO). Dulbecco’s Modified Eagle’s Medium was purchased from
ATCC (Manassas, Virginia). The Mito Flow fluorescent dye moni-
toring mitochondria membrane potential was purchased from Cell
Technology Inc (Mountain View, CA), Caspase-Glo® 9 luminescent
assay kit was purchased from Promega (Madison, WI), and Nuclear-
IDTM Green Chromatin Condensation Detection Kit was purchased
from Enzo Life Sciences (Plymouth Meeting, PA).
Fig. 1. Chemical structures of veskamide, enferamide, becatamide, and oretamide.
Veskamide (R = OH, R1ꢀ = OCH3, R2ꢀ = OCH3), enferamide (R = OH, R1ꢀ = OCH3, R2ꢀ = H),
becatamide (R = OH, R1ꢀ = OH, R2ꢀ = H) and oretamide (R = OH, R1ꢀ = H, R2ꢀ = H).
measured using a flow cytometry with excitation at 480 and 530 nm
long-pass filter.
Caspase-9 assay
Caspase-9 activity was measured using a Caspase-Glo® 9 lumi-
nescent assay kit (Promega), according to the manufacturer’s
protocol. The PC-12 cells were seeded in 96 well-culture dish and
pre-incubated with indicated concentrations of the amides for
20 min prior to the addition of H2O2 (200 ␮M) to the medium.
Becatamide was prepared in ethanol and added at less than 0.1%
(v/v). After 3, 6, and 9 h incubation, the cells were collected and rel-
ative light units (RLU) output was measured to determine caspase-9
activity.
Syntheses of veskamide, enferamide, becatamide, and oretamide
Chemical synthesis was performed as previously described
(Park 2005a,b). Briefly, benzoic acid, protocatechuic acid, vanil-
lic acid, or syringic acid were dissolved in dichloromethane
(DCM) and converted to the symmetrical anhydride with 1,3-
diisopropylcarbodiimide (DIC). Tyramine was added to the reaction
mixture and incubated with a gentle stirring for 12 h. The syn-
thesized products were recovered and purified by HPLC (Waters,
Milford, MA) (Park 2005a).
Chromatin condensation
Chromatin condensation was measured using Nuclear-IDTM
Green Chromatin Condensation Detection Kit (Enzo Life Sciences),
according to the manufacturer’s protocol. The green fluorescent dye
enables monitoring of the dynamics of nucleolar changes in intra-
cellular distribution, trafficking, and localization coincident with
cell death. For the experiment, the PC-12 cells were seeded in a 6
well-culture dish, pre-incubated with becatamide for 20 min, fol-
lowed by the addition of H2O2 (200 ␮M). The cells were collected
after 24 h and chromatin condensation was measured by calculat-
ing the fluorescence intensity of the dye using a flow cytometry.
NMR analyses
For NMR experiments, the samples were prepared by dissolv-
ing veskamide, enferamide, becatamide or oretamide (20 mg) in
d6-DMSO (0.75 ml). 1H and 13C spectra were acquired at ambi-
ent temperature on the JEOL BCX-400 NMR spectrometer operating
400 MHz for 1H and 100 MHz for 13C. Chemical shifts were refer-
enced to DMSO (2.50 ppm for 1H, 39.5 ppm for 13C).
Cell culture and treatment
PC-12 cells were grown in Ham’s F12K supplemented with 15%
(v/v) heat-inactivated fetal bovine serum at 37 ◦C in a humidified
atmosphere of 5% CO2. Experiments were carried out 24 h after cells
were seeded.
Results and discussion
Syntheses and NMR analyses of veskamide, enferamide,
becatamide, and oretamide
Measurement of mitochondrial membrane depolarization
Veskamide (N-syringoyltyramine), enferamide (N-vanilloyl-
tyramine), becatamide (N-protocatechuoyltyramine) and ore-
tamide (N-benzoyltyramine) are N-phenylethylbenzoylamides,
whose analogues were found in several plants such as Aniba riparia,
Begonia nantoensis, Haplophyllum tuberculatum, Hortia regia and
Houttuynia cordata. Years ago, we synthesized and named the
four amides as veskamide, enferamide, becatamide, and oretamide,
because the amides are most likely to have important biologi-
cal activities as well as to be present in numerous plant sources.
To attest our assumption, becatamide was subsequently isolated
from Houttuynia cordata and its structure (houttuynamide A) was
published during our study (Chou et al. 2009). However, there
is currently not much information about their biological effects.
Therefore, in this study, veskamide, enferamide, becatamide, and
oretamide (Fig. 1) were prepared via a chemical synthesis method
Mitochondrial membrane depolarization was monitored by
measuring mitochondrial membrane potential (MMP) using the
Mito Flow fluorescent dye (Cell Technology Inc), a cell permeable
cationic dye to emit a strong fluorescent signal in mitochon-
dria with the highly negative membrane potential. Veskamide,
enferamide, becatamide, and oretamide were prepared in ethanol
and added to samples less than 0.1% (v/v). The PC-12 cells were pre-
incubated with indicated concentrations of the amides for 20 min,
prior to the addition of H2O2 (200 ␮M) to the cells. After 3 h, the
cells were collected, centrifuged at 1200 × g for 5 min, and resus-
pended in DMEM. Mito Flow fluorescent dye was added to the cells
at the final concentration of 1 mM. After 1 h incubation at 37 ◦C,
the intracellular fluorescence intensity associated with the dye was

J.B. Park / Phytomedicine 18 (2011) 843–847
845
t, J = 7.5 Hz, CH2-9); 13C NMR (d6-DMSO, 100 MHz) d: 169.3 (C-7),
156.3 (C-13), 149.3 (C-4), 146.1 (C-3), 131.7 (C-10), 130.5 (C-11, -
15), 127.2 (C-1), 120.5 (C-6), 116.1 (C-12, -14), 115.9 (C-5), 115.7
(C-2), 43.9 (C-8), 36.8 (C-9); Thus, the structure of the synthesized
product was determined as being 3,4-Dihydroxy-N-[2-(4-hydroxy-
phenyl)-ethyl]-benzamide (becatamide). In the following are the
NMR data for oretamide (4): 1H NMR (d6-DMSO, 400 MHz) d:
7.24 (1H, d, J = 2.0 Hz, H-2), 7.31 (1H, dd, J = 8.4 Hz, H-6), 7.01 (2H,
d, J = 8.6 Hz H-11, -15), 6.78 (1H, d, J = 1.9 Hz, H-5), 6.71 (2H, d,
J = 8.6 Hz, H-12, -14), 5.00 (1H, br s, OH), 3.51 (2H, t, J = 7.5 Hz, CH2-
8), 2.79 (2H, t, J = 7.5 Hz, CH2-9); 13C NMR (d6-DMSO, 100 MHz) d:
169.3 (C-7), 156.3 (C-13), 149.3 (C-4), 146.1 (C-10), 130.5 (C-11, -
15), 127.2 (C-1), 120.5 (C-6), 116.1 (C-12, -14), 115.9 (C-3), 115.7
(C-5), 115.7 (C-2), 43.9 (C-8), 36.8 (C-9); Thus, the structure of the
synthesized product was determined as being 4-Hydroxy-N-[2-(4-
hydroxy-phenyl)-ethyl]-benzamide (oretamide).
using tyramine and the acids (syringic acid, vanillic acid, protocat-
echuic acid, and benzoic acid), according to the method described
in “Experimental” section (Park 2005a, 2009). The syntheses were
relatively simple, and their yields were greater than 43%. Each
synthesized product was purified by HPLC, and analyzed using
NMR spectroscopic methods described in “Experimental” sec-
tion. In the following are the NMR data for veskamide (1): 1H
NMR (d6-DMSO, 400 MHz) d: 7.27 (1H, d, J = 8.4 Hz, H-2), 7.30
(1H, dd, J = 8.4 Hz, H-6), 7.01 (1H, d, J = 8.6 Hz H-11, -15), 6.71
(1H, d, J = 8.6 Hz, H-12, -14), 5.00 (1H, br s, OH), 3.51 (2H, t,
J = 7.5 Hz, CH2-8), 2.79 (2H, t, J = 7.5 Hz, CH2-9); 13C NMR (d6-DMSO,
100 MHz) d: 169.3 (C-7), 156.3 (C-13), 146.5 (C-4), 150.1 (C-3),
131.7 (C-10), 130.5 (C-11, -15), 128.1 (C-1), 108.8 (C-6), 116.1
(C-12, -14), 150.2 (C-5), 108.7 (C-2), 56.2 (C-1ꢀ), 56.1 (C-2ꢀ), 43.9
(C-8), 36 (C-9); Thus, the structure of the synthesized product was
determined as being 4-hydroxy-N-[2-(4-hydroxy-phenyl)-ethyl]-
3,5-dimethoxy-benzamide (veskamide). In the following are the
NMR data for enferamide (2): 1H NMR (d6-DMSO, 400 MHz) d:
7.27 (1H, d, J = 8.4 Hz, H-2), 7.30 (1H, dd, J = 8.4 Hz, H-6), 7.01
(2H, d, J = 8.6 Hz H-11, -15), 6.78 (1H, d, J = 1.7 Hz, H-5), 6.71
(2H, d, J = 8.6 Hz, H-12, -14), 5.00 (1H, br s, OH), 3.51 (2H, t,
J = 7.5 Hz, CH2-8), 2.79 (2H, t, J = 7.5 Hz, CH2-9); 13C NMR (d6-DMSO,
100 MHz) d: 169.3 (C-7), 156.3 (C-13), 146.5 (C-4), 148.7 (C-3),
131.7 (C-10), 130.5 (C-11, -15), 127.1 (C-1), 120.1 (C-6), 116.1 (C-
12, -14), 115.7 (C-5), 114.7 (C-2), 56.1 (C-1ꢀ), 43.9 (C-8), 36.8 (C-9);
Thus, the structure of the synthesized product was determined
as being 4-Hydroxy-N-[2-(4-hydroxy-phenyl)-ethyl]-3-methoxy-
benzamide (enferamide). In the following are the NMR data for
becatamide (3): 1H NMR (d6-DMSO, 400 MHz) d: 7.24 (1H, d,
J = 2.0 Hz, H-2), 7.31 (1H, dd, J = 8.4 Hz, H-6), 7.01 (2H, d, J = 8.6 Hz
H-11, -15), 6.78 (1H, d, J = 1.9 Hz, H-5), 6.71 (2H, d, J = 8.6 Hz, H-12,
-14), 5.00 (1H, br s, OH), 3.51 (2H, t, J = 7.5 Hz, CH2-8), 2.79 (2H,
Effect of veskamide, enferamide, becatamide, and oretamide on
H2O2-induced mitochondrial membrane depolarization
Mitochondrial membrane depolarization (MMP) was deter-
mined using a cell permeable cationic fluorescent dye (Mito Flow,
Cell Technology Inc). Depolarization of mitochondria membrane
potential induced by the H2O2-induced damage of the outer mem-
brane resulted in the loss of the dye from the mitochondria and
a decrease in intracellular fluorescence (Lemasters et al. 1998).
PC-12 cells were prepared in 6 groups: control (A), H2O2 (B),
veskamide (C), enferamide (D), becatamide (E), and oretamide
(F) groups. The cells in C-F groups were treated with veskamide,
enferamide, becatamide or oretamide (10 ␮M) for 20 min, then
treated with H2O2 (200 ␮M) for 3 h. The cells in the B group were
only treated with H2O2 (200 ␮M) for 3 h and the control A group
Fig. 2. Effects of veskamide, enferamide, becatamide, and oretamide on mitochondrial membrane depolarization. Mitochondria depolarization was determined using Mito
Flow dye, a cell permeable cationic dye that has a strong fluorescent signal and exhibits strong mitochondria membrane potential (MMP) dependent binding. PC-12 cells
were treated with veskamide (C), enferamide (D), becatamide (E), and oretamide (F) (all at 10 ␮M), followed by the treatment of H2O2 (200 ␮M) except control (A). After 3 h,
the cells were collected, centrifuged, re-suspended, and analyzed using flowcytometer as described in “Experimental” section.

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J.B. Park / Phytomedicine 18 (2011) 843–847
Fig. 4. The inhibition of hydrogen-peroxide induced caspase
9 activation by
Fig. 3. Effects of becatamide on mitochondrial membrane depolarization. Mito-
chondria integrity was determined as described in Fig. 2. PC-12 cells were
pre-treated with becatamide 5, 10 and 20 ␮M for 20 min and then treated with
200 ␮M H2O2 for 3 h. The cells were collected and analyzed using flowcytometer.
becatamide: PC-12 cells were pre-treated with becatamide at the concentration
of 10 ␮M for 20 min, then treated with 200 ␮M H2O2. The capase-9 activation was
measured by the Caspase-Glo® 9 Assay kit (Promega) at 0, 3, 6, and 9 h. Data points
represent the mean SD of more than 3 samples. Becatamide pre-treated cells pro-
duced significant inhibition of H2O2-induced caspase-9 activation at all time periods
measured compared to cells treated only with H2O2 (P < 0.05, ANOVA, Holm-Sidak
method).
received no treatment. After the treatment, PC12 cells were col-
lected and analyzed using a flowcytometer. As shown in Fig. 2,
the pre-exposure of H2O2-treated cells with becatamide (Fig. 2E)
significantly maintained mitochondrial membrane polarization
(83%), based on the quantization of intracellular fluorescence com-
pared to non-treated cells (P < 0.001). In contrast, H2O2-treated
cells maintained the polarization at only 49% of the control cells
(Fig. 2B). Since becatamide (10 ␮M) exhibited significant inhibi-
tion of H2O2-induced mitochondrial membrane depolarization, we
investigated the dose-dependent protective effects of becatamide
(5 and 20 ␮M). As shown in Fig. 3, at the lower concentration
(5 ␮M), the mitochondrial membrane polarization was main-
tained at 69%, but the effect was not increased further at the
higher concentration (20 ␮M), suggesting that the highest pro-
tective effect can be achieved at a range of less than 20 ␮M.
That seemed very reasonable because mitochondrial membrane
polarization was generally around 89% in the control cells with-
out the H2O2 treatment (data not shown here). We also tested
the protective effects of the precursors (e.g., protocatechuic acid
and tyramine) and other well-known phenolic and anti-oxidant
compounds (e.g., caffeic acid, rosmarinic acid, catechin, resvera-
trol, quercetin, vitamin-C). But, the same level protective effect
of becatamide could not be achieved in the tested concentrations
(data not shown here). Together, these data indicated that among
the four amides, becatamide was the most potent compound able
to maintain mitochondrial membrane polarization, probably via
protecting mitochondrial membrane integrity from H2O2.
procaspase-7, which are responsible for several cellular apoptosis
processes including poly ADP ribose polymerase (Garrido et al.
2006). Since becatamide was a most potent inhibitor of mitochon-
drial membrane depolarization, the effect of this amide on caspase
9 activity in H2O2-treated PC12 cells was investigated. As shown in
Fig. 4, the cells pre-treated with becatamide exhibited significant
suppression of H2O2-induced caspase-9 activation, compared to
H2O2-treated cells (P < 0.05). The data suggest that becatamide may
inhibit activation of caspase-9 via protecting mitochondrial mem-
brane integrity from H2O2 and suppressing cytochrome c release
from the mitochondrial inter-membrane space.
Effect of becatamide on H2O2-induced chromatin condensation
Since there are also caspase-independent apoptotic events, we
investigated the effects of becatamide on caspase-independent
chromatin condensation. The treatment of PC-12 cells with H2O2
makes the outer mitochondrial membrane more permissible,
thereby triggering the release of apoptosis-inducing factor (AIF)
that is normally located in the mitochondrial inter-membrane
space. Upon the release, AIF translocates to the nucleus, binds
to DNA, and invokes caspase-independent chromatin condensa-
tion. During this process, chromatin undergoes a phase change
from a heterogeneous, genetically active network to an inert highly
condensed form that is subsequently fragmented and packaged
into apoptotic bodies. Recent studies suggested that AIF was a
major factor determining caspase-independent neuronal death,
emphasizing the central role of mitochondria in the control of
physiological and pathological cell death (Candé et al. 2002). There-
fore, caspase-independent chromatin condensation was measured
in H2O2-treated PC-12 cells with and without the pre-treatment
with becatamide (Fig. 5). Chromatin condensation was evident
in cells treated with H2O2 for 24 h (Fig. 5B), but the chromatin
condensation was significantly attenuated by 73% (P < 0.05) in
becatamide-pre-treated PC-12 cells (Fig. 5C). This level of the
protective effect could not be demonstrated in the PC-12 cells pre-
treated with veskamide, enferamide, oretamide or their precursors
(data not shown here). In summary, becatamide may be able to
suppress caspase-independent chromatin condensation as well as
Effect of becatamide on caspase 9 activity in H2O2-treated PC12
cells
Caspases (cysteine-aspartic proteases or cysteine-dependent
aspartate-directed proteases) are a family of cysteine proteases that
play essential roles in apoptosis (programmed cell death) (Vaux
and Strasser 1996; Alnemri 1997). Caspase-9 is an initiator cas-
pase linked to the mitochondrial death pathway and activated
during programmed cell death (apoptosis) (Caroppi et al. 2009).
After treatment of cells with apoptotic agents including H2O2,
cytochrome c is released from mitochondrial intermembrane space
and binds to the apoptosis protease activation factor (APAf-1)
to forms an apoptosome complex. This complex activates cas-
pase 9. Once initiated, caspase-9 cleaves procaspase-3 as well as

J.B. Park / Phytomedicine 18 (2011) 843–847
847
Fig. 5. The protection of hydrogen-peroxide induced DNA condensation by becatamide. Chromatin condensation was measured using Nuclear-IDTM Green Chromatin
Condensation Detection Kit (normal chromatin in the left and chromatin condensation in the right). Chromatin condensation was evident in cells treated with H2O2 (200 ␮M)
for 24 h (B) compared to control untreated PC-12 cells (A). In PC-12 cells pre-treated with becatamide (C), chromatin condensation was significantly attenuated (P < 0.05)
compared to that in H2O2 only-treated cells (B).
caspase-9 activation, probably via protecting mitochondrial mem-
brane integrity from H2O2-induced damage.
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