|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 40, 37630-37636, October 4, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Departments of Obstetrics and Gynecology and Molecular and
Medical Pharmacology and Jonsson Comprehensive Cancer Center, David
Geffen School of Medicine at UCLA, Los Angeles, California
90095-1740
Received for publication, April 15, 2002, and in revised form, July 23, 2002
We have previously reported that
N Metabolic products of arginine modulate the growth of many types
of cancer cells. Arginine is converted by arginase to ornithine, the
only source of synthesis of the polyamines putrescine, spermidine, and
spermine in mammalian cells, which are essential for cell proliferation
and regulation of the cell cycle (1, 2). On the other hand, arginine is
also catalyzed by the enzyme nitric-oxide synthase
(NOS)1 to form
N We have previously demonstrated that breast cancer cell lines that
predominately expressed arginase had a higher rate of proliferation when compared with cell lines that predominately expressed NOS (11),
thereby demonstrating how products of arginine metabolism may modulate
cell proliferation. NOHA inhibited arginase in MDA-MB-468 cells, and
this initially led to cytostasis followed by activation of caspase-3.
This was accompanied by a decrease in intracellular polyamine levels.
As this action of NOHA was abolished in the presence of exogenous
L-ornithine, we speculated that the action of NOHA in
inducing apoptosis in this cell line was most likely due to a reduction
in intracellular polyamine levels (11). However, in this cell line,
when we added difluromethyl ornithine, an inhibitor of ornithine
decarboxylase, or SAM-486A, an inhibitor of SAMDC, either alone or in
combination, there was a reduction in intracellular polyamine content,
but only cytostasis was observed, not apoptosis (17). On this basis, we
concluded that the apoptotic action of NOHA in MDA-MB-468 cells cannot
be explained solely on the basis of a reduction in intracellular
polyamine levels and that other mechanisms must also be considered. The
present work was, therefore, undertaken to elucidate the mechanism(s)
involved by which NOHA induced apoptosis in this cell line. MDA-MB-468
cells were selected for our study as this cell line expresses high
arginase activity with very little NOS (11). Thus, the effects of NOHA can be studied without any confounding influence of endogenous NOHA and
NO produced by these cells.
Chemicals--
N Cell Culture--
Human breast cancer cell line MDA-MD-468
(American Type Culture Collection) were cultured in Dulbecco's
modified Eagle's medium containing 10 mM nonessential
amino acids, 2 mM L-glutamine, 1 µg/ml
insulin, and 10% fetal bovine serum. For experimental purposes, cells
were grown in 5% fetal bovine serum, allowed to seed overnight, and
treated with drugs for various durations.
Caspase Assay--
Cells were lysed in lysis buffer as
previously described (7). The lysates were used for caspase-3 (3 µg),
caspase-9 (6 µg), and caspase-8 (15 µg) assays using respective
substrates. The released AMC (for caspase-3 and caspase-9) and
AFC (for caspase-8) after specific cleavage of respective substrates
becomes fluorescent were quantified using a fluorometer (Versa FluroTM,
Bio-Rad) with excitation at 380 nm and emission at 440 nm for AMC
substrates (7) and at excitation 410 nm and emission at 510 nm for AFC substrate (34), respectively.
Western Analysis--
Cells were lysed as described previously
(7). Lysates (30 µg) were resolved electrophoretically on 10%
SDS-polyacrylamide gel and electrotransferred to a polyvinylidine
difluoride membrane (Bio-Rad) using a tank blot procedure (Bio-Rad Mini
Protean II). The membranes were incubated with anti-caspase-8 antibody
(1:200), anti-caspase-3 antibody (1:3000), anti-BID antibody (1:1000), and anti- PARP antibody (1:1000) for 2 h at room temperature and anti-caspase-9 antibody (1:1000) overnight followed by subsequent incubation with 1:1000 dilutions of horseradish
peroxidase-linked F(ab) fragment secondary antibody (Amersham
Biosciences) for 1 h. Immunoreactive bands were visualized by the
enhanced chemiluminescence detection system (Amersham Biosciences).
Detection of Cytochrome c Release into the
Cytosol--
Cytochrome c release into the cytosol was
detected as described previously with minor modifications (7). Briefly,
6 × 106 cells were harvested and washed with
phosphate-buffered saline (PBS). The cells were suspended in Buffer A
(20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, 1× protease inhibitor mixture) and homogenized by Dounce homogenizer; unbroken cells and nuclei were removed by
centrifugation at 1,000 × g for 10 min at 4 °C. The
supernatant was further centrifuged at 10,000 × g for
20 min. The supernatant was saved as a cytosolic fraction while the
precipitate was suspended in Buffer A containing 0.5% (v/v) Nonidet
P-40 and was saved as the mitochondrial fraction. The mitochondrial and
cytosolic fractions were analyzed by Western blot with an
anti-cytochrome c monoclonal antibody or with an
anti-cytochrome c oxidase antibody.
Measurement of Mitochondrial Membrane Potential by Flow
Cytometry--
1 × 106 cells were harvested, washed
twice with cold 1× PBS and incubated with 100 nM
MitoTracker Red CMX-Ros dye at 37 °C for 15 min in the dark, washed
twice with cold PBS, and analyzed immediately by flow cytometry.
Valinomycin (10 µM), a potassium ionophore, which
serves as a hyperpolarization control, and gramicidin (10 µM), a relatively non-selective ionophore, which serves
as a depolarization control (18), were also included in our experiments in order to validate our results for mitochondrial membrane potential (MMP) assay.
Time Course of Caspase-3 and PARP Induction by NOHA and
Antagonistic Effect of L-Ornithine--
We have shown
previously that NOHA when used alone induced significant caspase-3
activity in MDA-MB-468 cells after 48 h, which was blocked in the
presence of exogenous L-ornithine. To understand the
precise mechanism(s) by which NOHA induced apoptosis in this cell line,
we studied the time course of caspase-3 induction after NOHA treatment.
We observed a 4-fold induction of caspase-3 with NOHA (1 mM) at 32 h, which increased subsequently by 10-fold at 48 h (Fig. 1A).
Exogenous L-ornithine (1 mM) added along with NOHA was able to block this NOHA-induced caspase-3
induction. We also assessed caspase-3 activation by Western blot
analysis by observing its proteolytic cleavage (Fig. 1B). We
observed a 17-kDa band corresponding to cleaved caspase-3 as early as
32 h, the intensity of which increased further at 48 h of
NOHA treatment. This cleaved band (17 kDa) was undetectable in samples
that received exogenous L-ornithine along with NOHA. We
observed that PARP, a caspase-3 substrate, was cleaved as early as
32 h, generating a cleaved 85-kDa fragment (Fig. 1C).
At 48 h, there was complete fragmentation of 117-kDa PARP into
cleaved fragment (85 kDa). L-Ornithine (1 mM)
was able to efficiently block this cleavage of PARP (Fig.
1C).
Time Course of Caspase-9 Induction by NOHA and Antagonistic Effect
of Exogenous L-Ornithine--
Activation of caspase-3
could be either via death receptor signaling or due to caspase-9
activation through mitochondrial release of cytochrome c. We
observed that caspase-9 was activated during NOHA-induced apoptosis.
There was a 2-fold induction of caspase-9 at 24 h followed by a
4-fold induction at 32 h, and it increased further to ~9-fold at
48 h (Fig. 2A). Western
blot analysis further indicated that at 32 h there was a
proteolytic cleavage of caspase-9 that further increased at 48 h
(Fig. 2B). L-Ornithine was able to block this
NOHA-induced activation as well as proteolytic cleavage of
caspase-9.
Inhibitors of Caspase-3 or Caspase-9 Block the Induction of
NOHA-induced Caspase-3 Activation--
To further confirm the role of
caspase-9 and -3 in NOHA-induced apoptosis, we studied the effect of
their inhibitors. We used Z-DEVD-fmk and Z-LEHD-fmk as caspase-3 and -9 inhibitors, respectively. These inhibitors at 25 µM
concentration completely inhibited the NOHA-induced activation of
caspase-3 (Fig. 2C) and thus apoptosis suggesting a distinct
involvement of the caspase-9/-3 pathway.
Time Course of Cytochrome c Release into the Cytosol with NOHA and
Effect of Exogenous L-Ornithine--
To study the upstream
sequence of events during NOHA-induced apoptosis, we assessed the time
course of cytochrome c release into the cytosol and
correlated this with the time course of caspase-9 and -3 activation. We
isolated mitochondrial and cytosolic fractions from cells after
different treatments as described under "Materials and Methods." We
observed the release of cytochrome c into the cytosol as
early as 32 h, which further increased by 4-fold after 48 h
of NOHA treatment. However, simultaneous treatment of cells with
L-ornithine blocked this release of cytochrome c
into the cytosol (Fig. 3A). We
also confirmed that our cytosolic fractions had no mitochondrial
contamination by probing the membrane with anti-cytochrome-c
oxidase antibody (data not shown).
Measurement of MMP by Flow Cytometry--
Disruptions in MMP are
measured with a number of cationic lipophilic fluorochromes including
MitoTracker Red CMX-Ros and flow cytometry (18). Fig. 3B
(top panel) shows the validation of the method using a
negative control. Addition of dye, CMX-Ros (100 nM), caused
a shift in the fluorescence compared with the negative control.
Valinomycin, a control for hyperpolarization, further shifted the
fluorescence to the right, an indication of hyperpolarization of the
MMP. Gramicidin, a control for depolarization, shifted the intensity of
fluorescence to the left indicative of depolarized MMP. NOHA induced
hyperpolarization of the MMP as early as 3 h, and this
hyperpolarization persisted until 48 h, and
L-ornithine did not change this NOHA-induced
hyperpolarization (Fig 3B). Similar results were found when
we used other dyes like dihexyloxacarbocyanine or rhodamine 123 (data
not shown). Thus our results show that release of cytochrome
c into the cytosol was not due to the decrease in the
MMP.
Time Course of Caspase-8 Induction by NOHA and Effect of
L-Ornithine--
We studied the time course of activation
of caspase-8, an upstream caspase, usually known to act upstream of
mitochondrial events. Recent reports have suggested that activation of
caspase-8 followed by truncation of BID leads to release of cytochrome
c that is independent of decrease in MMP. Fig.
4A shows that caspase-8 activity was induced by 2-fold at 8 h of NOHA treatment and
reached a maximum of 7-fold after 32 h. Exogenous
L-ornithine had no direct effect on the NOHA-induced
activation of caspase-8 (data not shown). We observed a similar pattern
of caspase-8 proteolytic cleavage by NOHA in Western blot analysis as
shown in Fig. 4B.
Time Course of BID Cleavage after NOHA Treatment and Effect of
Exogenous L-Ornithine--
We studied the time course of
BID cleavage in order to correlate the sequence of events during
NOHA-induced apoptosis. Fig. 4C shows a slight cleavage of
BID at 4 h, and maximum cleavage was observed at 32 h. A
cleaved band could be observed at 15 kDa (p15 BID) corresponding to the
truncated BID (tBID). Again, exogenous L-ornithine did not
inhibit the NOHA-induced BID cleavage, and the time course of caspase-8
activation was correlated with the time course of BID cleavage.
Effect of Different Caspase Inhibitors on the NOHA-induced
Cytochrome c Release into the Cytosol--
To confirm that caspase-8
activation works upstream of the mitochondrial events leading to
cytochrome c release, we performed experiments where
inhibitors of different caspases were incubated along with NOHA at
concentrations reported in different cell lines to block their
activation after 48 h. Fig.
5A shows that NOHA-induced cytochrome c release into the cytosol could be prevented by
the caspase-8 inhibitor or a pan-caspase inhibitor but not by
caspase-3 or caspase-9 inhibitors.
TNF- The primary objective of our study was to elucidate the
mechanism(s) by which NOHA led to apoptosis of MDA-MB-468 cells and to
assess the specific site of action and mechanism(s) by which L-ornithine prevented NOHA-induced apoptosis (Fig.
6). Mitochondria play a central role in
the commitment of cells to apoptosis (19, 20). Most studies indicate
that the initial event observed following application of the apoptotic
stimulus is a decrease in the inner transmembrane potential followed by
an increase in the permeability of the outer mitochondrial membrane by
forming the permeability transition pores and subsequent release of
cytochrome c from the intermembranous space into the cytosol
(21). This released cytochrome c then forms a complex with
the apoptosis activating factor-1 (Apaf-1) and procaspase-9 called
"apoptosome" to activate caspase-9, which in turn activates
caspase-3, a downstream caspase (22, 23). Once activated, caspase-3
cleaves its substrate PARP and, subsequently, leads to fragmentation of
DNA (24). In our flow cytometry analysis using the
mitochondria-sensitive dye CMX-Ros during NOHA-induced apoptosis, we
did not observe any decrease in MMP. Rather, we observed release of
cytochrome c from the mitochondria during NOHA-induced
hyperpolarization of the MMP. Our method for assessing changes in
transmembrane potential was validated using valinomycin, which causes
hyperpolarization of MMP and which increased the fluorescence, whereas
gramicidin, which causes depolarization of MMP, decreased the
fluorescence. The hyperpolarization of MMP caused by NOHA was further
confirmed using other dyes like dihexyloxacarbocyanine and rhodamine
123 that showed similar results (data not shown). Our results indicate
that the collapse of the inner MMP is not a universal early event that
triggers cytochrome c release leading to apoptosis and
moreover is not always an essential part of the central apoptotic
machinery. In this regard, our results are similar to those reported by
other investigators who observed that HL-60 cells underwent apoptosis
in response to the cytotoxic insults of actinomycin D, etoposide, and
staurosporine without showing significant loss of mitochondrial inner
transmembrane potential (25). We therefore considered other potential
mechanisms by which NOHA may affect the permeability of the outer
mitochondrial membrane and thereby lead to cytochrome c
release into the cytosol independent of a depolarization of the MMP. In
this regard two major mechanisms have been proposed (26, 27).
One mechanism proposed is that BID, a cytosolic BH3
interacting domain only member of the Bcl-2 family of proteins, is a
substrate for caspase-8, which is activated by the FAS/TNF-R1 pathway
and cleaves BID at the C-terminal to generate tBID. This tBID then
translocates to the mitochondria and induces cytochrome c
release either in a Bax-dependent or independent manner
(26, 28). More recently, caspase-8-mediated cleavage of BID was also
shown to induce N-myristoylation of tBID, and this
modification enhanced its targeting to lipid membranes and mitochondria
(29). One possible reason for the targeting of tBID to mitochondria has
been attributed to its propensity for binding to cardiolipin (30), a
membrane lipid unique to mitochondria (31). Another mechanism suggested
is that after apoptotic insult, Bax, another multidomain pro-apoptotic
cytosolic protein, integrates to the outer mitochondrial membrane and
causes cytochrome c release (27) in a BID-independent
manner.
We, therefore, assessed the possibility as to whether NOHA-induced
activation of caspase-3 and apoptosis occurred by a
BID-dependent or BID-independent mechanism. As caspase-8
activation is required for truncation of BID, we initially assessed the
time course of activation of caspase-8, its proteolytic cleavage, and
the appearance of tBID and how this correlated with the release of
cytochrome c from the mitochondria. In this regard, we
observed that activation of caspase-8 was significantly increased after
8 h following NOHA treatment, and peak activation occurred at
32 h (Fig. 4A). Slight truncation of BID was seen as
early as 4 h that peaked between 24 and 32 h. Following
exposure of the cells to NOHA, the peak release of cytochrome
c release coincided with the peak caspase-8 activation and
significant truncation of BID. We then considered the possibility as to
whether the early and late release of cytochrome c may have
been due to BID-mediated integration of Bax at the outer mitochondrial
membrane. However, this does not appear to be the case as we were not
able to demonstrate Bax integration into the mitochondrial membrane at
any time point, and therefore the release of cytochrome c by
tBID must have occurred by a Bax-independent mechanism. Activation of
caspase-3 started around 32 h and peaked at 48 h following
treatment of cells with NOHA. This activation of caspase-3, therefore,
was initiated only following caspase-8 activation and truncation of
BID, which then induced a massive release of cytochrome c
and ultimately was responsible for the activation of caspase-3, thereby
committing the cells to the apoptotic pathway. We next assessed the
mechanism by which NOHA induced activation of caspase-8. We observed
that NOHA along with TNF- We have demonstrated previously that L-ornithine inhibited
NOHA-induced apoptosis. We, therefore, elected to use
L-ornithine as a tool to further identify the sequence of
events during NOHA-induced apoptosis (Fig. 6). L-Ornithine
did not inhibit the NOHA-induced activation of caspase-8 and cleavage
of BID, suggesting that the action of L-ornithine must have
occurred further downstream. NOHA-induced hyperpolarization of MMP was
not changed by L-ornithine indicating that
hyperpolarization was not the triggering event that led to cytochrome
c release. On the other hand, L-ornithine
antagonized the effect of NOHA in releasing cytochrome c
from the mitochondria and all subsequent steps downstream leading to
apoptosis. It, therefore, appears that the antagonistic effect of
L-ornithine during NOHA-induced apoptosis was most likely
at the level of the mitochondria where it prevented the release of
cytochrome c. In this regard, it is interesting that other
investigators have demonstrated (32) that extracellularly applied
arginase inhibited neuronal apoptosis induced by multiple stimuli.
Furthermore, arginase was identified by mass spectrometry as one of the
proteins released from the mitochondria during apoptosis (33). On this basis, it was speculated that the protective effect of arginine in
inhibiting neuronal apoptosis was dependent on depletion of arginine
metabolized by arginase. As arginase converts arginine to ornithine, it
would be interesting to speculate whether L-ornithine was
the anti-apoptotic factor in these studies similar to our observation in studies related to NOHA-induced apoptosis. Macrophages as well as myoepithelial cells at the site of breast cancer express NOS
(35) and therefore have the capacity to generate both NOHA and NO. Thus
NOHA and NO by independent mechanisms may induce apoptosis and
therefore complement each other.
In conclusion, our studies indicate that NOHA-induced apoptosis
occurs upstream of the mitochondria, most likely by activation of
caspase-8 followed by cleavage of BID to tBID. Our studies also
indicate that L-ornithine has anti-apoptotic action and
acts at the level of the mitochondria to inhibit NOHA-induced release of cytochrome c and thereby apoptosis. Further studies are
in progress to assess the precise mechanism(s) by which
L-ornithine acts at the level of the mitochondria as an
anti-apoptotic factor and whether this is specific to NOHA or whether
it may be applicable to some other apoptotic agents that act by
activating caspase-8.
We thank Janis Cuevas and
Svetlana Arutyunova for excellent technical assistance. We
thank Dr. Fuyuhiko Tamanoi, Dr. Catherine F. Clarke, and Dr.
Jon M. Fukuto for constructive criticisms during the preparation of the
manuscript. Flow cytometry was performed in the core facility of UCLA
Jonsson Comprehensive Cancer Center and Center for AIDS Research Flow
Cytometry Core Facility that is supported by National Institute of
Health Awards CA-16042 and AI-28697, by the Jonsson Cancer Center, the
UCLA AIDS Institute, and the UCLA School of Medicine.
*
This work was supported in part by Palomba Weingarten, the
Allegra Charach Cancer Research Fund, and United States Public Health
Service Grant CA-78357 (to G. C).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.
Published, JBC Papers in Press, July 26, 2002, DOI 10.1074/jbc.M203648200
2
R. Singh, S. Pervin, and G. Chaudhuri,
unpublished observation.
The abbreviations used are:
NOS, nitric-oxide
synthase;
AMC, 7- amino-4-methylcoumarin;
AFC, 7-amino-4-trifluoromethylcoumarin;
BID, BH3 interacting
domain;
tBID, truncated BID;
CMX-Ros, chloromethyl X-Rosamine;
fmk, fluoromethyl ketone;
MMP, mitochondrial membrane potential;
NOHA, N
Caspase-8-mediated BID Cleavage and Release of Mitochondrial
Cytochrome c during
N
-Hydroxy-L-arginine-induced
Apoptosis in MDA-MB-468 Cells
ANTAGONISTIC EFFECTS OF L-ORNITHINE*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxy-L-arginine (NOHA), a
stable intermediate product formed during the conversion of L-arginine to nitric oxide, induced apoptosis in MDA-MB-468
cells, and this action was antagonized in the presence of
L-ornithine. We also reported that apoptosis induced
by NOHA in this cell line could not be explained on the basis of a
reduction of intracellular polyamines. In the current study, we
investigated other potential mechanism(s) by which NOHA may have
induced apoptosis in this cell line. We observed that NOHA initially
activated caspase-8 and induced cleavage of BH3 interacting
domain. This was followed by release of cytochrome c and
subsequently, activation of downstream caspases-9 and -3 to cleave
poly(ADP-ribose) polymerase. We also observed that NOHA induced a rapid
and persistent hyperpolarization of the mitochondrial membrane
potential rather than depolarization indicating that the release of
cytochrome c by NOHA was by a mechanism independent of the
mitochondrial transition pore. Exogenous L-ornithine did
not inhibit NOHA-induced caspase-8 activation and cleavage of
BH3 interacting domain but acted at the mitochondrial level and inhibited the NOHA-induced cytochrome c release and apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxy-L-arginine (NOHA) as an
intermediate that subsequently forms nitric oxide (NO) (3). NO causes
cytostasis (4-6) and apoptosis of cancer cells (7-10) and also
affects the cell cycle (4). Thus, depending on the predominant
metabolic pathway for arginine present in cells, products of arginine
metabolism can cause either cell proliferation or cytostasis followed
by apoptosis. We (11) and others (12) have previously demonstrated that polyamines are essential for proliferation of some breast tumor cells
and that inhibition of polyamine biosynthesis led to inhibition of cell
proliferation followed by apoptosis (11). The polyamine biosynthesis
occurs from arginine, which initially is converted by arginase to
ornithine. Ornithine is then converted to putrescine by ornithine
decarboxylase (13). S-Adenosylmethionine
decarboxylase (SAMDC), which converts S-adenosylmethionine
(SAM) to decarboxylated SAM, is required for the conversion of
putrescine to spermidine (13). Inhibitors of ornithine decarboxylase
and SAMDC have been shown to reduce proliferation of various types of
malignant cells (14, 15) including breast cancer cells (16), and this
was accompanied by a reduction in intracellular polyamine levels.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxy-L-arginine
(NOHA) was purchased from Cayman Biochemicals (Ann Arbor, MI).
L-Ornithine hydrochloride was purchased from Aldrich.
Ac-DEVD-AMC and Ac-IETD-AFC were obtained from BD PharMingen.
Ac-LEHD-AMC was purchased from Alexis Biochemicals (San Diego, CA).
Z-DEVD-fmk, Z-IETD-fmk, Z-LEHD-fmk, and Z-VAD-fmk were purchased from
Calbiochem. Antibodies were obtained from the following suppliers:
rabbit polyclonal anti-caspase-3 and mouse monoclonal anti-cytochrome
c antibodies from BD PharMingen; rabbit polyclonal
anti-caspase-8, rabbit polyclonal anti-caspase-9, and mouse monoclonal
anti-PARP antibodies were from Santa Cruz Biotechnology (San Diego,
CA); and anti-cytochrome c oxidase antibody was from
CLONTECH (Palo Alto, CA). BID antibody was a kind
gift from Dr. S. Korsmeyor (Harvard Medical School). MitoTracker Red chloromethyl X-Rosamine (CMX-Ros) was purchased from Molecular Probes
(Eugene, OR). Valinomycin and gramicidin were purchased from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Time course of caspase-3 and PARP activation
by NOHA in MDA-MB-468 cells. a, cells were treated with
either NOHA (1 mM) alone or in combination with
L-ornithine (1 mM) for various time points and
lysed, and caspase-3 activities were analyzed as described under
"Materials and Methods" using 3 µg of total cell lysates. Results
are expressed as mean of three different experiments ± S. E. (*
indicates that the values are significantly different from the control,
p < 0.01.) b, 30 µg of total cell lysates
obtained after various treatments as in a were analyzed by a
Western blot with anti-caspase-3 antibody and anti-PARP antibody
(c).
View larger version (17K):
[in a new window]
Fig. 2.
Time course of caspase-9 activation by NOHA
(1 mM) and inhibition of NOHA-induced caspase-3 activity by
caspase-3 and -9 inhibitors. a, cells were treated with
either NOHA (1 mM) alone or in combination with
L-ornithine (1 mM) for various time points and
lysed, and caspase-9 activities were assayed using 6 µg of total cell
lysates. Results are expressed as mean of three different
experiments ± S.E. (* indicates that the values are significantly
different from the control, p < 0.01.) b,
30 µg of total cell lysate obtained after various treatments as in
a were analyzed by Western blot with anti-caspase -9 antibody. c, cells were treated with either NOHA (1 mM) alone or in combination with caspase-3 and caspase-9
inhibitors for 48 h and lysed, and 3 µg of total cell lysates
were analyzed for caspase-3 activities. Results are expressed as mean
of three different experiments ± S. E. (* indicates that the
values are significantly different from the control (p < 0.01), ** indicates that the values are significantly different from
* values (p < 0.01).)
View larger version (23K):
[in a new window]
Fig. 3.
Time course of cytochrome c
release in the cytosol with NOHA and effect of NOHA on the MMP.
A, cells were treated with either NOHA (1 mM) or L-ornithine (1 mM) for
various time points, and cytosolic fractions were collected as
described under "Materials and Methods." 15 µg of cytosolic
fractions were analyzed by Western blot with anti-cytochrome
c antibody. B, MMP was analyzed with cells after
various treatments using flow cytometry. Within B,
A shows the validation of MMP analysis using gramicidin and
valinomycin as depolarization and hyperpolarization controls,
respectively. B, C, and D show NOHA (1 mM)-induced hyperpolarization after 3, 24, and 48 h.
View larger version (38K):
[in a new window]
Fig. 4.
Time course of NOHA-induced caspase-8
activation and cleavage of BID. a, cells were treated with
NOHA (1 mM) for various points and lysed, and caspase-8
activities were analyzed using Ac-IETD-AFC as substrate. Results are
expressed as mean of three different experiments ± S. E. (*
indicates that the values are significantly different from the control,
p < 0.01.) b, cells were treated as in
a, and 30 µg of total cell lysates were analyzed by
Western blot analysis using anti-caspase-8 antibody. c,
cells were treated with either NOHA (1 mM) alone or in
combination with L-ornithine (1 mM) for
various time points. Lysis and Western blot analysis were done using
anti-BID antibody.
View larger version (23K):
[in a new window]
Fig. 5.
Inhibition of NOHA-induced cytochrome
c by caspase-8 inhibitor and potentiation of
NOHA-induced caspase-8 by TNF- 
. a, only inhibitor of
caspase-8 blocked the NOHA-induced release of cytochrome c
into the cytosol. Cells were treated with either NOHA (1 mM) alone or in combination with different caspase
inhibitors for 48 h, and cytosolic fractions obtained were
analyzed by Western blot using anti-cytochrome c antibody.
b, TNF- acts with NOHA to induce caspase-8 at earlier
time points. Cells were treated with NOHA (1 mM) and
TNF- (100 ng/ml) either alone or in combination for different time
points and lysed, and 15 µg of total cell lysates were assayed for
caspase-8 activity. Results are expressed as mean of three different
experiments ± S.E. (* indicates that the values are significantly
different from the control, p < 0.01.)
Potentiates the Action of NOHA in Inducing Caspase-8
Activity--
Caspase-8 is activated in many cell lines after
activation of the TNF-R1/Fas pathway. We therefore assessed the role of
TNF- during NOHA-mediated caspase-8 activation. TNF- was not able to activate the caspase-8 or -3 in MDA-MB-468 cells. However, when
combined with NOHA (1 mM), we observed that TNF-
potentiated the action of NOHA in inducing caspase-8 (and caspase-3)
activity at a much earlier time point. Fig. 5B shows that
after 4 h there was a 2-fold induction in the caspase-8 activation
with a combination of TNF- and NOHA compared with the caspase-8
activation with NOHA alone, which further increased to ~3.5-fold
after 8 h of NOHA treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 6.
Proposed model for NOHA-induced apoptosis in
MDA-MB-468 cells.
led to a significant activation of
caspase-8 activity when compared with cells treated with NOHA or
TNF- alone (Fig. 5B). This action of NOHA therefore may
have contributed to the apoptosis as TNF- is released by this cell
line.2
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Obstetrics
and Gynecology and Molecular and Medical Pharmacology and Jonsson
Comprehensive Cancer Center, David Geffen School of Medicine at UCLA,
10833 Le Conte Ave., Los Angeles, CA 90095-1740. Tel.: 310-206-6575;
Fax: 310-206-3670; E-mail: gchaudhuri@mednet.ucla.edu.
ABBREVIATIONS
-hydroxy-L-arginine;
PARP, poly(ADP-ribose) polymerase;
PBS, phosphate-buffered saline;
SAMDC, S-adenosylmethionine decarboxylase;
TNF-, tumor necrosis
factor-;
TNF-R1, tumor necrosis factor-receptor 1.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Jenkinson, C. P.,
Grody, W. W.,
and Cederbaum, S. D.
(1996)
Comp. Biochem. Physiol. B. Biochem. Mol. Biol.
114,
107-132[CrossRef][Medline]
[Order article via Infotrieve]
2.
Pegg, A. E.,
and McCann, P. P.
(1982)
Am. J. Physiol.
243,
C212-C221 3.
Fukuto, J. M.
(1996)
Methods Enzymol.
268,
365-375[CrossRef][Medline]
[Order article via Infotrieve]
4.
Pervin, S.,
Singh, R.,
and Chaudhuri, G.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3583-3588 5.
Buga, G. M.,
Wei, L. H.,
Bauer, P. M.,
Fukuto, J. M.,
and Ignarro, L. J.
(1998)
Am. J. Physiol.
275,
R1256-R1264 6.
Kwon, N. S.,
Stuehr, D. J.,
and Nathan, C. F.
(1991)
J. Exp. Med.
174,
761-767 7.
Pervin, S.,
Singh, R.,
Gau, C. L.,
Edamatsu, H.,
Tamanoi, F.,
and Chaudhuri, G.
(2001)
Cancer Res.
61,
4701-4706 8.
Lee, Y. J.,
Lee, K. H.,
Kim, H. R.,
Jessup, J. M.,
Seol, D. W.,
Kim, T. H.,
Billiar, T. R.,
and Song, Y. I.
(2001)
Oncogene
20,
1476-1485[CrossRef][Medline]
[Order article via Infotrieve]
9.
Secchiero, P.,
Gonelli, A.,
Celeghini, C.,
Mirandola, P.,
Guidotti, L.,
Visani, G.,
Capitani, S.,
and Zauli, G.
(2001)
Blood
98,
2220-2228 10.
Saio, M.,
Radoja, S.,
Marino, M.,
and Frey, A. B.
(2001)
J. Immunol.
167,
5583-5593 11.
Singh, R.,
Pervin, S.,
Karimi, A.,
Cederbaum, S.,
and Chaudhuri, G.
(2000)
Cancer Res.
60,
3305-3312 12.
Thomas, T.,
Balabhadrapathruni, S.,
Gardner, C. R.,
Hong, J.,
Faaland, C. A.,
and Thomas, T. J.
(1999)
J. Cell. Physiol.
179,
257-266[CrossRef][Medline]
[Order article via Infotrieve]
13.
Marton, L. J.,
and Pegg, A. E.
(1995)
Annu. Rev. Pharmacol. Toxicol.
35,
55-91[CrossRef][Medline]
[Order article via Infotrieve]
14.
Regenass, U.,
Mett, H.,
Stanek, J.,
Mueller, M.,
Kramer, D.,
and Porter, C. W.
(1994)
Cancer Res.
54,
3210-3217 15.
Dorhout, B.,
te Velde, R. J.,
Ferwerda, H.,
Kingma, A. W.,
de Hoog, E.,
and Muskiet, F. A.
(1995)
Int. J. Cancer
62,
738-742[Medline]
[Order article via Infotrieve]
16.
Kaneko, H.,
Hibasami, H.,
Mori, K.,
Kawarada, Y.,
and Nakashima, K.
(1998)
Anticancer Res.
18,
891-896[Medline]
[Order article via Infotrieve]
17.
Singh, R.,
Pervin, S., Wu, G.,
and Chaudhuri, G.
(2001)
Carcinogenesis
22,
1863-1869 18.
Shapiro, H. M.
(1997)
Current Protocols in Cytometry
, pp. 9.6.1-9.6.10, Wiley and Sons, New York
19.
Green, D.,
and Kroemer, G.
(1998)
Trends Cell Biol.
8,
267-271[CrossRef][Medline]
[Order article via Infotrieve]
20.
Green, D. R.,
and Reed, J. C.
(1998)
Science
281,
1309-1312 21.
Goldstein, J. C.,
Waterhouse, N. J.,
Juin, P.,
Evan, G. I.,
and Green, D. R.
(2000)
Nat. Cell Biol.
2,
156-162[CrossRef][Medline]
[Order article via Infotrieve]
22.
Li, P.,
Nijhawan, D.,
Budihardjo, I.,
Srinivasula, S. M.,
Ahmad, M.,
Alnemri, E. S.,
and Wang, X.
(1997)
Cell
91,
479-489[CrossRef][Medline]
[Order article via Infotrieve]
23.
Susin, S. A.,
Lorenzo, H. K.,
Zamzami, N.,
Marzo, I.,
Snow, B. E.,
Brothers, G. M.,
Mangion, J.,
Jacotot, E.,
Costantini, P.,
Loeffler, M.,
Larochette, N.,
Goodlett, D. R.,
Aebersold, R.,
Siderovski, D. P.,
Penninger, J. M.,
and Kroemer, G.
(1999)
Nature
397,
441-446[CrossRef][Medline]
[Order article via Infotrieve]
24.
Decker, P.,
Isenberg, D.,
and Muller, S.
(2000)
J. Biol. Chem.
275,
9043-9046 25.
Finucane, D. M.,
Waterhouse, N. J.,
Amarante-Mendes, G. P.,
Cotter, T. G.,
and Green, D. G.
(1999)
Exp. Cell Res.
251,
166-174[CrossRef][Medline]
[Order article via Infotrieve]
26.
Wei, M. C.,
Lindsten, T.,
Mootha, V. K.,
Weiler, S.,
Gross, A.,
Ashiya, M.,
Thompson, C. B.,
and Korsmeyer, S. J.
(2000)
Genes Dev.
14,
2060-2071 27.
Wolter, K. G.,
Hsu, Y. T.,
Smith, C. L.,
Nechushtan, A., Xi, X. G.,
and Youle, R. J.
(1997)
J. Cell Biol.
139,
1281-1292 28.
Eskes, R.,
Desagher, S.,
Antonsson, B.,
and Martinou, J. C.
(2000)
Mol. Cell. Biol.
20,
929-935 29.
Zha, J.,
Weiler, S., Oh, K. J.,
Wei, M. C.,
and Korsmeyer, S. J.
(2000)
Science
290,
1761-1765 30.
Lutter, M.,
Fang, M.,
Luo, X.,
Nishijima, M.,
Xie, X.,
and Wang, X.
(2000)
Nat. Cell Biol.
2,
754-761[CrossRef][Medline]
[Order article via Infotrieve]
31.
Hatch, G. M.
(1998)
Int. J. Mol. Med.
1,
33-41[Medline]
[Order article via Infotrieve]
32.
Esch, F.,
Lin, K. I.,
Hills, A.,
Zaman, K.,
Baraban, J. M.,
Chatterjee, S.,
Rubin, L.,
Ash, D. E.,
and Ratan, R. R.
(1998)
J. Neurosci.
18,
4083-4095 33.
Patterson, S. D.,
Spahr, C. S.,
Daugas, E.,
Susin, S. A.,
Irinopoulou, T.,
Koehler, C.,
and Kroemer, G.
(2000)
Cell Death Differ.
7,
137-144[CrossRef][Medline]
[Order article via Infotrieve]
34.
De Nadai, C.,
Sestili, P.,
Cantoni, O.,
Lievremont, J-P.,
Sciorati, C.,
Barsacchi, R.,
Moncada, S.,
Meldolesi, J.,
and Clementi, E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5480-5485 35.
Thomsen, L. L.,
Lawton, F. G.,
Knowles, R. G.,
Beesley, J. E.,
Riveros-Moreno, V.,
and Moncada, S.
(1995)
Br. J. Cancer.
72,
41-44[Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Pericolini, E. Gabrielli, E. Cenci, M. De Jesus, F. Bistoni, A. Casadevall, and A. Vecchiarelli Involvement of Glycoreceptors in Galactoxylomannan-Induced T Cell Death J. Immunol., May 15, 2009; 182(10): 6003 - 6010. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Redlak, J. J. Power, and T. A. Miller Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells Am J Physiol Gastrointest Liver Physiol, October 1, 2005; 289(4): G731 - G738. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Pervin, R. Singh, W. A. Freije, and G. Chaudhuri MKP-1-Induced Dephosphorylation of Extracellular Signal-Regulated Kinase Is Essential for Triggering Nitric Oxide-Induced Apoptosis in Human Breast Cancer Cell Lines: Implications in Breast Cancer Cancer Res., December 15, 2003; 63(24): 8853 - 8860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Pervin, R. Singh, and G. Chaudhuri Nitric-Oxide-induced Bax Integration into the Mitochondrial Membrane Commits MDA-MB-468 Cells to Apoptosis: Essential Role of Akt Cancer Res., September 1, 2003; 63(17): 5470 - 5479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. M. Storey, M. Gomez-Angelats, C. D. Bortner, D. L. Armstrong, and J. A. Cidlowski Stimulation of Kv1.3 Potassium Channels by Death Receptors during Apoptosis in Jurkat T Lymphocytes J. Biol. Chem., August 29, 2003; 278(35): 33319 - 33326. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, P. Shan, J. Alam, R. J. Davis, R. A. Flavell, and P. J. Lee Carbon Monoxide Modulates Fas/Fas Ligand, Caspases, and Bcl-2 Family Proteins via the p38{alpha} Mitogen-activated Protein Kinase Pathway during Ischemia-Reperfusion Lung Injury J. Biol. Chem., June 6, 2003; 278(24): 22061 - 22070. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |