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Vol. 300, Issue 2, 535-542, February 2002
Department of Pharmacology (K.Y.K., K.W.H.), College of Medicine and College of Natural Sciences (B.G.K.), Pusan National University, Pusan, Korea; Central Research Institute, Dongbu Hannong Chemical Co., Daejon, Korea (S.-O.K.); Research Institute of Chemical Technology, Daejon, Korea (S.-E.Y.); and Institute of Cardiovascular Research, Chonbuk National University, Chonju Korea (Y.-G.K., S.-W.C.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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This study describes the antiapoptotic action of
(2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31378), a novel benzopyran analog, in human umbilical vein endothelial cells (HUVECs) in comparison with its acetylated
metabolite, (2S,3S,4R)-N"-cyano-N-(6-acetylamino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31612), and with 
-tocopherol. Exposure of HUVECs to
lipopolysaccharide (LPS) (1 µg/ml) induced time- and
concentration-dependent cytotoxicity and oligonucleosomal DNA
fragmentation. KR-31378, KR-31612, and -tocopherol potently
suppressed LPS-induced cell death in association with significant
reduction in the intracellular reactive oxygen species (ROS) and tumor
necrosis factor- (TNF-) that are stimulated by LPS. KR-31378 more
effectively protected HUVECs from LPS-induced DNA fragmentation and was
more effective in peroxyl radical scavenging than -tocopherol.
Incubation with LPS markedly decreased the Bcl-2 level, which was
totally reversed by KR-31378 and to a lesser degree by KR-31612 and by
-tocopherol. In contrast, the greatly increased Bax protein and
cytochrome c release stimulated by LPS were markedly
suppressed by KR-31378 and by KR-31612, and to a lesser degree by
-tocopherol. Taken together, KR-31378 strongly inhibited cell death
in HUVECs in association with antiapoptotic effects, which were
accompanied by up-regulation of Bcl-2 protein expression and
down-regulation of Bax protein and suppression of cytochrome
c release. KR-31378 also showed the properties to scavenge the intracellular ROS and peroxyl radicals, and to reduce the
TNF- production induced by LPS.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Vascular
endothelial cell injury or activation by LPS plays a critical role in
the pathogenesis of Gram-negative bacterial inflammation and endotoxic
shock (Parrillo et al., 1990
). LPS causes systemic release of TNF-
and both may mediate their deleterious effects by direct endothelial
cell damage (Egido et al., 1993). TNF- also induces the synthesis of
ROS (Radeke et al., 1990). ROS are involved in cytokine-mediated signal
transduction by interaction with biological macromolecules (e.g.,
nucleic acid) (Shaw et al., 1995), and apoptosis (Buttke and Sandstrom,
1994). The evidence that antioxidants attenuate the apoptosis further
supported TNF--induced ROS formation (Huang et al., 1998; Lee et
al., 1999). Thus, prevention of oxidative stress-mediated cell injury
is an area of active investigation.
It is well established that the Bcl-2 family, consisting of
antiapoptotic (e.g., Bcl-2 and Bcl-XL) and
proapoptotic (e.g., Bax and Bad) members, plays an important role in
the regulation of cell death. The former is known to prevent the
release of cytochrome c to cytosol (Gross et al., 1999;
Shimizu and Tsujimoto, 2000), whereas the latter members induce the
release of cytochrome c from mitochondria and the activation
of caspase cascade and apoptosis (Jürgensmeier et al., 1998).
Thus, a number of studies have postulated that cell survival is
associated with cell's ability to maintain a homeostatic level of
Bcl-2.
Recently,
(2S,3S,4R)-N"-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31378) and
(2S,3S,4R)-N"-cyano-N-(6-acetylamino-3,4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31612), an acetyl metabolite, were synthesized by the Korea Research Institute of Chemical Technology, Daejon, Korea. In a previous
study, we observed that KR-31378 effectively inhibited the increased
cerebral infarct and swelling observed in rat cerebral cortex subjected
to 2-h occlusion of middle cerebral artery and 24-h reperfusion.
Furthermore, KR-31378 strongly inhibited lipid peroxidation in
association with suppression of the electron paramagnetic resonance
signals of superoxide anion and hydroxyl radicals, as did
-tocopherol.
In this study, we examined how KR-31378 suppressed the DNA
fragmentation and consequent cell death in HUVECs in comparison with
KR-31612 and with -tocopherol. To identify the mechanism(s), we
further evaluated their ability to scavenge peroxyl radicals, to
suppress the LPS-stimulated hydrogen peroxide in HUVECs, and to
evaluate the effect of these compounds on the expression of Bcl-2 and
Bax protein, and on cytochrome c release from mitochondria in LPS-mediated apoptosis. -Tocopherol was used as a reference agent.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Cultures. HUVECs [American Type Culture Collection (Manassas, VA), CRL-1730, endothelial cell line derived from the vein of normal human umbilical cord] were cultured in Kaighn's F12K medium supplemented with 10% heat-inactivated fetal bovine serum, 0.1 mg/ml heparin sodium, 0.03 to 0.05 mg/ml endothelial cell growth supplement, and 1% antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were grown to confluence at 37°C in 5% CO2 on 0.1% gelatin-coated culture dishes and used for experiments at no greater than passage 8.
Cell Viability Assay. For the mitochondrial tetrazolium assay (MTT) procedure, cells were seeded 1 × 104 cells/well in 96-well gelatin-coated tissue culture plates. The confluent cells received F12K medium with 1% fetal bovine serum plus drugs for 5 h before stimulation with LPS, and then were exposed to LPS for 18 h. After incubation, 20 µl/well of MTT solution (5 mg/ml phosphate-buffered saline) was added and incubated for 2 h. The plates were shaken for 20 min and the optical density measured at 570 to 630 nm by using enzyme-linked immunosorbent assay (Bio-Tek Instruments, Winooski, VT).
DNA Fragmentation Assays.
After incubation in the absence
and presence of the drugs for 5 h, cells (1-5 × 106) were exposed to LPS (1 µg/ml) for 18 h. At harvest, trypsinized cells were pelleted by centrifugation.
Oligonucleosomal fragmentation of genomic DNA was determined as
previously described (Wyllie, 1980). Cells were lysed in 1 ml of
lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1%
sodium dodecyl sulfate, and 0.5 mg/ml proteinase K). Digestion was
continued for 1 to 3 h at 55°C, followed by addition of RNase A
to 0.1 mg/ml and running dye (10 mM EDTA, 0.25% bromophenol blue, 50%
glycerol). Equivalent amounts of DNA (15-20 µg) were loaded into
wells of 1.6% agarose gel and electrophoresed in 0.5× TAE buffer (40 mM Tris-acetate, 1 mM EDTA) for 2 h at 6 V/cm. DNA was visualized
by ethidium bromide staining. Gel pictures were taken by UV
transillumination with a Polaroid camera. Bands were quantified by
Molecular Analyst Software by using Bio-Rad's Image Analysis System
(Bio-Rad, Hercules, CA).
TNF- Assay.
Confluent cells incubated in the 48-well
plates for 5 h in the absence and presence of the drugs and
stimulated with 1 µM LPS for 18 h. TNF- levels were assessed
in supernatants by using a commercially available Quantikine M human
TNF- Immunoassay (R & D Systems, Minneapolis, MN), which is known to
be cross-reactive with human TNF-. TNF- content was assessed by
measuring absorbance at 450 nm by using enzyme-linked immunosorbent
assay (Bio-Tek Instruments) and extrapolating from a standard curve.
Assay of Intracellular ROS. Measurement of intracellular ROS was based on ROS-mediated conversion of nonfluorescent 2',7'-dichlorofluororescine diacetate (DCFH-DA) into DCFH. The intensity of fluorescence reflects enhanced oxidative stress. To measure the intracellular ROS, confluent cells were preincubated for 5 h in the absence and presence of the drugs and then stimulated with LPS (1 µg/ml) for 18 h. Cells were incubated in the dark for 2 h at 37°C in 50 mM phosphate buffer, pH 7.4, containing 5 µM DCFH-DA. This agent is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the fluorescent polar derivative DCFH and thereby trapped within the cells. The quantity of DCFH fluorescence was measured at an emission wavelength of 530 nm and an excitation wavelength of 485 nm by using Fluorescence Plate Reader (Bio-Tek Instruments). All experiments were repeated at least three times. Results were expressed as percentage of control fluorescence intensity.
Peroxyl Radical Absorbing Capacity (PRAC).
The assay for
peroxyl radical scavenging is based on production of peroxyl radicals
by 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) (3 mM) with
subsequent oxidation of the reporter protein 
-phycoerythrin (-PE)
(16.7 nM), in a volume of 2 ml with 75 mM phosphate buffer, pH 7.0, in
24-well plates. After adding AAPH, loss of fluorescence was measured
every 5 min at the emission of 590 nm and excitation of 485 nm by using
Fluorescence Plate Reader (Bio-Tek Instruments). Trolox (1 µM) was
used as a reference for PRAC assay. The fluorescence just before
addition of the AAPH was estimated as the 100% value for that sample.
The PRAC values were calculated as follows: PRAC = [area of
compound 
area of blank]/[area of 1 µM trolox area
of blank], where 1 PRAC unit is the value of 1 µM trolox.
Western Blot Analyses. For determination of Bcl-2 and Bax protein levels, cells were grown in 100-mm tissue culture dishes and treated with the indicated compounds. After washing, the cells were lysed in lysis buffer containing 50 mM Tris-Cl, pH 8.0; 150 mM NaCl; 0.02% sodium azide; 100 µg/ml phenylmethylsulflonyl fluoride; 1 µg/ml aprotinin; and 1% Triton X-100. After centrifugation at 12,000 rpm, 50 µg of total protein of each sample was loaded into 12% SDS-polyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membrane (Amersham Biosciences, Inc., Piscataway, NJ). The blocked membranes were then incubated with the antibody of Bcl-2 and Bax (Santa Cruz Biotechnology, Santa Cruz, CA).
Mitochondrial cytochrome c was prepared via the following procedures. After washing cells (12 × 106) once with ice-cold phosphate-buffered saline, cell pellets were resuspended in buffer A (20 mM HEPES-KOH, pH 7.5; 10 mM KCl; 1.5 mM MgCl2; 1 mM Na-EDTA; 1 mM Na-EGTA; 1 mM dithiothreitol; 0.1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose. The cells were homogenized and then centrifuged twice at 750g for 10 min at 4°C. The harvested supernatants were centrifuged at 10,000g for 10 min at 4°C, and the resulting mitochondrial pellets were dissolved in 1× SDS sample buffer. Western blots were performed as described above with the antibody of cytochrome c (Santa Cruz Biotechnology). The immunoreactive bands were visualized using chemiluminescent reagent of the Supersignal West Dura Extended Duration Substrate kit (Pierce Chemical, Rockford, IL). The signals of the bands were quantified using the calibrated imaging densitometer (GS-710; Bio-Rad). The protein concentration of the lysate was determined using the Bio-Rad DC assay kit (Bio-Rad).Chemicals.
KR-31378 and KR-31612 (Korea Research Institute
of Chemical Technology) were dissolved in dimethyl sulfoxide as a 10 mM
stock solution. -Tocopherol (Sigma-Aldrich, Seoul, Korea) was
dissolved in ethanol (99%) as a 10 mM stock solution.
Lipopolysaccharide (Sigma-Aldrich) was dissolved in distilled water as
a 1-mg/ml stock solution. -PE (Sigma-Aldrich) and AAPH (Wako Pure
Chemicals, Osaka, Japan) were dissolved in 75 mM phosphate
buffer, pH 7.0. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide was from Sigma-Aldrich.
Statistical Analysis. The results are expressed as means ± S.E.M. Statistical differences between groups were determined by paired or unpaired Student's t test or analysis of variance. P < 0.05 was considered to be significant.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Effect on Cell Viability.
MTT assay measures the mitochondrial
activity of viable cells. LPS showed time- (6-48 h) and concentration
(1-1000 µg/ml)-dependent cytotoxic effect on HUVECs as determined by
MTT assay in the culture medium. In the present study, cytotoxicity was
examined after incubation with 1 µg/ml LPS for 18 h (Fig.
1, inset). After 18-h incubation, 1 µg/ml LPS reduced cell viability to 75.2% of control cells. Cell
death was concentration-dependently prevented by simultaneous incubation with KR-31378, KR-31612, and -tocopherol
(10
8-104 M, each).
After application of KR-31378 (104 M) in the
absence of LPS, the cells showed 94.3 ± 2.7% viability, suggestive of lack of cytotoxicity.
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Antiapoptotic Effect.
Exposure of HUVECs to LPS (1 µg/ml)
induced oligonucleosomal DNA fragmentation in time- and
concentration-dependent manner, and the maximum values were obtained
after 18 h of incubation. At 18 h after exposure to LPS,
cells showed morphological characteristics of apoptosis, including cell
shrinkage and chromatin condensation relative to control cells (data
not shown). Treatment with KR-31378 strongly suppressed the LPS (1 µg/ml)-induced DNA laddering (P < 0.001), whereas
KR-31612 and -tocopherol showed significant but less suppression
than KR-31378 (Fig. 2).
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Scavenging of Intracellular ROS and Peroxyl Radicals.
The
intracellular ROS concentration was determined by measuring the
intensity of fluorescence. Incubation of DCFH-loaded cells in the
medium containing LPS (0.01-10 µg/ml) for 18 h showed a concentration-dependent increase in fluorescence intensity. The intensity was 128.3 ± 3.8% by LPS at 1 µg/ml. KR-31378 and
KR-31612 (108, 106, and
104 M) significantly suppressed the increased
fluorescence stimulated by LPS (1 µg/ml) in a concentration-dependent
manner, as did -tocopherol (Fig. 3).
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-PE, where AAPH was used as a source of peroxyl radicals. In the
presence of 106 and 104
M of KR-31378, the extinction curve showed a right shift, suggestive of
its large scavenging effect (Fig. 4). The
relative peroxyl radical absorbing capacity (in unit) calculated for
KR-31378 (106 and 104
M) was 2.04 ± 0.45 (P < 0.01) and 3.25 ± 0.28 (P < 0.001) (Fig. 4, inset). The values of
KR-31378 were significantly higher than those for -tocopherol
(106 and 104 M),
indicating that KR-31378 possesses higher efficacy for scavenging peroxyl radical than -tocopherol.
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Effect on TNF- Levels.
The level of TNF- in the control
culture medium of HUVECs was 21.6 ± 3.4 pg/mg protein. Upon
application of LPS (0.1-100 µg/ml) for 18 h, the TNF- level
was concentration-dependently increased as shown in Fig.
5, inset. TNF- level stimulated by 1 µg/ml LPS was 383.4 ± 14.3 pg/ml, which was markedly suppressed by treatment with KR-31378 and KR-31612 (105 M,
each) to 138.9 ± 5.5 (P < 0.001) and 152.2 ± 9.0 pg/ml (P < 0.01), respectively. -Tocopherol
(105 M) demonstrated a lower suppression
(232.0 ± 14.5 pg/ml, P < 0.01) than KR-31378
(Fig. 5).
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Western Blot Analyses.
Figure 6
shows the concentration-dependent levels of Bcl-2 and Bax protein, and
release of cytochrome c in the absence and presence of LPS
(0, 0.5, 1, and 5 µg/ml). In the absence of LPS, Bcl-2 protein was
present in high levels (control samples, relative density = 1),
and Bax protein was at very low levels (control, relative density = 1). In contrast, cytochrome c release was not manifested
in the control cells. Both Bax level and cytochrome c
release were significantly elevated with increasing concentrations of
LPS from 0.5 to 5 µg/ml, whereas Bcl-2 level was
concentration-dependently decreased.
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6, 105, and
104 M) to 0.56 ± 0.07, 0.76 ± 0.1, and 0.98 ± 0.07 relative density, respectively. The last level
indicates total restoration of Bcl-2 protein (Fig.
7A). The suppressed Bcl-2 level was also
reversed by KR-31612 and -tocopherol (105 M,
each), but to a lower degree than KR-31378 (105
M) (Fig. 7B).
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6-104 M)
in a concentration-dependent manner (Fig.
8A). KR-31612 and -tocopherol
(105 M, each) also significantly decreased the
Bax protein level (Fig. 8B).
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6-104 M) as shown
in Fig. 9A. KR-31612 showed a similar
inhibitory activity, as did KR-31378, but -tocopherol at
105 M concentration showed weak inhibition
(Fig. 9B).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present study, the major findings were that 1) KR-31378
effectively protected HUVECs from LPS-induced cell death accompanied by
oligonucleosomal DNA fragmentation; 2) KR-31378 and KR-31612 significantly reduced the increased production of intracellular ROS and
TNF- that are stimulated by LPS, and KR-31378 more effectively scavenged peroxyl radical than -tocopherol; and 3) incubation with
LPS markedly decreased the Bcl-2 protein and increased the Bax protein
in association with increase in cytochrome c release, which
was significantly reversed by KR-31378 and to a lesser degree by
KR-31612 and by -tocopherol.
LPS, a bacterial endotoxin, is a proinflammatory mediator that induces
the production of significant amounts of endogenous TNF-, and both
may mediate endothelial cell damage (Egido et al., 1993). The
importance of TNF- and ROS endogenously released after exposure to
LPS has been associated with the genesis of apoptosis and cell death
(Böhler et al., 2000). TNF- also may activate a cell survival
pathway that protects against its apoptotic effects (Karsan et al.,
1996) and HUVECs are demonstrated to hardly undergo programmed cell
death in response to TNF- alone (Polunovsky et al., 1994).
Therefore, in the present study, LPS was used as an inducer of
apoptosis instead of TNF-.
In our results, LPS-induced cytotoxicity as determined by MTT assay was
concentration-dependently suppressed by incubation of HUVECs with
108 to 104 M of
KR-31378, KR-31612 as well as was by -tocopherol. The cell death was
accompanied by DNA fragmentation in a time- and concentration-dependent manner. Treatment with KR-31378 strongly suppressed the LPS (1 µg/ml)-induced DNA laddering. Under incubation of HUVECs with LPS,
both intracellular ROS and TNF- significantly and
concentration-dependently increased in the HUVECs. The current results
of suppression of ROS and peroxyl radicals by KR-31378 and the chemical
properties of KR-31378 to scavenge not only the hydroxyl radical but
also superoxide anion highlighted the ability of KR-31378 to react with
a wide spectrum of radicals.
In the pilot study, KR-31378 did not exert any effect on the action
potential duration in the ventricular myocytes as contrasted to
pinacidil, an ATP-sensitive K+ channels.
KR-31378, however, showed opening of large conductance Ca2+-activated K+ channels
(maxi-K channels) in isolated vascular myocytes of rat basilar artery,
which was not blocked by glibenclamide (ATP-sensitive K+ channel blocker) but by iberiotoxin (large
conductance Ca2+-activated
K+ channel blocker). Because maxi-K channels are
present in many brain regions, including the cortex and hippocampus
(Knaus et al., 1996) and maxi-K channels have been identified in
endothelial cells (Rusko et al., 1992), it is thus predictable that,
when opened, maxi-K channels may reduce voltage-dependent
Ca2+ entry into the cells after restoration of
membrane potential (Latorre et al., 1989). At present time, it is
undetermined whether suppression of LPS-induced TNF- production and
DNA fragmentation by KR-31378 are attributable to the opening of maxi-K
channels and to the reduction in intracellular
Ca2+ accumulation. Although the ability of
KR-31378 to open maxi-K channels provided a satisfactory explanation
for its efficacy in protecting cells against LPS insults, it was
unclear whether this mechanism was also involved in suppression of
TNF- formation or secretion. Future study will be required to
address this issue.
KR-31378 suppressed LPS-induced increased TNF- levels. It is
considered that KR-31378 might inhibit the LPS-induced cell death with
DNA fragmentation by a dual pathway: 1) inhibition of synthesis and/or
action of TNF-, and 2) inhibition of oxidative stress. LPS has been
demonstrated to induce apoptosis in bovine endothelial cells via a
soluble CD14-dependent pathway (Frey and Findlay, 1998). However, it is
undefined whether LPS-induced apoptosis in HUVECs is mediated through
its interaction with soluble CD14. K+ channel
openers, including diazoxide and levcromakalim, protected cultured rat
hippocampal neurons against oxidative injury induced by exposure to
FeSO4 and amyloid -peptide by suppressing the generation of peroxides, even in the presence of the
K+ channel blockers glibenclamide and
4-aminopyridine (Goodman and Mattson, 1996). They suggested that the
protective mechanism of K+ channel openers
involves antioxidant cell protective action other than
K+ channel opening effect. Most recently,
Sanlioglu et al. (2001) further emphasized the importance of ROS in the
TNF- secretion after LPS challenge in macrophages. They demonstrated
that Rac1 (a GTP-binding protein) activation-linked ROS formation
constituted a major pathway involved in nuclear factor-
B-mediated
TNF- secretion independent of CD14. Based on these reports, it is
likely that the reduction in TNF- formation under incubation of
HUVECs with KR-31378 is attributable to the high antioxidant potency of
KR-31378.
-Tocopherol is known as biologically and chemically the most active
form of vitamin E and as a lipid peroxyl radical trapping and
chain-breaking antioxidant. Although data are not shown, KR-31378 showed no pro-oxidant effect in contrast to -tocopherol
(Neu
il et al., 1997).
On the other hand, apoptosis is introduced as a regulated series of
energy-dependent molecular and biochemical processes orchestrated by a
genetic program (Hale et al., 1996). ROS, including hydrogen peroxide
and hydroxyl radical, and lipid hydroperoxides are all importantly
implicated in the processes of apoptosis as second messengers in the
cytokine (i.e., TNF- and IL-1)-induced apoptosis (Kroemer et al.,
1995; Li et al., 1997). Accumulating evidence points to a significant
role for Bcl-2 and its family of cell death-regulating proteins in
promoting cell survival and cell death (Bredesen, 1995). Martinou
(1999) suggested that overexpression of Bcl-2 in transgenic mice
appears to protect neurons from ischemia-induced cell death.
Conversely, a decrease in immunoreactivity of Bcl-2 and increase in Bax
protein contributing to neuronal apoptosis were observed in neurons
within ischemic cortex and thalamus (Gillardon et al., 1996). Haendeler
et al. (1996) have discussed the regulation of the Bcl-2 protein family
by demonstrating that the antioxidant N-acetylcysteine and
the combination of vitamin C and E (10 µM) inhibited LPS-induced
apoptosis, and the reduction of LPS-induced apoptosis by vitamin C and
E was paralleled by an increase in Bcl-2 and a decrease in Bax protein
levels. These results indicate that cell survival is associated with
the ability of cells to maintain high levels of Bcl-2. Consistent with
these reports, our results showed that KR-31378 totally restored the
suppressed Bcl-2 levels induced by LPS. These are well correlated with
inhibition of DNA fragmentation by KR-31378 as assessed by DNA ladder
pattern on agarose electrophoresis. On the other hand, Bax is one of
the Bcl-2 family homologous to Bcl-2, and Bcl-2 heterodimerizes with Bax, which accelerates programmed cell death (Oltvai et al., 1993). Bax
was envisioned as a cell death effector whose activity is neutralized
by binding with Bcl-2 (Sato et al., 1994).
Recent studies have implicated mitochondria as an important regulatory
site of the apoptotic process (Kroemer, 1998) and the rise of
cytochrome c release from mitochondria to cytosol as one of
the main pathways governing apoptosis (Zhang et al., 2000). It was
suggested that Bcl-2 and Bcl-XL prevented the
loss of the mitochondrial membrane potential and the release of
cytochrome c to cytosol (Gross et al., 1999), whereas Bax
promoted apoptosis by triggering the release of cytochrome c
from mitochondria, activating caspase cascade (Jürgensmeier et
al., 1998). ROS produced endogenously are known to enhance the
permeability of the mitochondrial membrane and the release of
cytochrome c to the cytosol (Marzo et al., 1998; Shimizu et
al., 1999). As shown in the profile of Bax, LPS-induced up-regulation
of cytochrome c release was also significantly reduced by
KR-31378 and to a less degree by KR-31612. It remains, however, undetermined in the present study whether Bax stimulates cytochrome c release and whether Bcl-2 acts primarily at the
mitochondria to prevent Bax-mediated cytochrome c.
Taken together, KR-31378 and its acetyl metabolite KR-31612 exert a
strong antiapoptotic effect in HUVECs with scavenging of intracellular
ROS and peroxyl radicals, and with reduction of TNF- production,
thereby eliciting up-regulation of Bcl-2 levels and down-regulation of
Bax levels and cytochrome c release.
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Acknowledgments |
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We are grateful to Jonathan Kaskin for reading and commenting on the manuscript.
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Footnotes |
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Accepted for publication November 2, 2001.
Received for publication August 9, 2001.
This study was supported with funding from the Center for Bioactive Substances, Korea Research Institute of Chemical Technology, Daejon, and from the Korea Science and Engineering Foundation, Korea.
Address correspondence to: Dr. Ki Whan Hong, Department of Pharmacology, College of Medicine, Pusan National University, 10 Ami-Dong, 1-Ga, Seo-Gu, Pusan 602-739, Korea. E-mail: kwhong{at}hyowon.pusan.ac.kr
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Abbreviations |
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LPS, lipopolysaccharide;
TNF-, tumor
necrosis factor-;
ROS, reactive oxygen species;
HUVEC, human
umbilical vein endothelial cell;
MTT, mitochondrial tetrazolium;
DCFH-DA, 2',7'-dichlorofluororescine diacetate;
DCFH, 2',7'-dichlorofluororescine;
PRAC, peroxyl radical absorbing capacity;
AAPH, 2,2'-azobis(2-amidino-propane) dihydrochloride;
-PE, -phycoerythrin.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 K. W. Hong, K. Y. Kim, J. H. Lee, H. K. Shin, Y. G. Kwak, S.-O. Kim, H. Lim, and S.-E. Yoo Neuroprotective Effect of (2S,3S,4R)-N""-cyano-N-(6-amino-3, 4-dihydro-3-hydroxy-2-methyl-2-dimethoxymethyl-2H-benzopyran-4-yl)-N'-benzylguanidine (KR-31378), a Benzopyran Analog, against Focal Ischemic Brain Damage in Rats J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 210 - 216. [Abstract] [Full Text] [PDF]
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