Morphine treatment induced release of cytochrome c and activation of caspase-9 and caspase-3 Previous studies have shown that a mitochondria-dependent pathway is implicated in morphine-i
Trang 1cell apoptosis via c-Jun N-terminal kinase-mediated
activation of mitochondria-dependent pathway
Xin Lin1,*, Yu-Jun Wang2,*, Qing Li2, Yuan-Yuan Hou1, Min-Hua Hong1, Ying-Lin Cao2, Zhi-Qiang Chi1and Jing-Gen Liu1
1 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China
2 School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, China
Opioids, in addition to their well-recognized analgesic
effects, may act as modulators of cell proliferation and
cell death It has been shown that opioids can protect
astrocytes from apoptosis triggered by
apoptosis-promoting agents [1], delay neuronal death in the
avian ciliary ganglion [2], and promote the growth of
tumor cells [3–5] On the other hand, opioids have also
been demonstrated to induce apoptosis of immuno-cytes [6,7], cancer cells [8,9], neuroblastoma cells such
as SK-N-SH, NG108-15 and PC12 cells [10–12], and neuronal cells [13,14], as well as human microglia [15] The effects of opioid-mediated cell proliferation and death appear to be dependent on the concentrations and durations employed for treatment
Growth-Keywords
apoptosis; JNK signaling; mitochondria;
morphine; ROS
Correspondence
J.-G Liu, State Key Laboratory of Drug
Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Fax: +86 21 50807088
Tel: +86 21 50807588
E-mail: jgliu@mail.shcnc.ac.cn
*These authors contributed equally to this
work
(Received 4 December 2008, revised 22
January 2009, accepted 28 January 2009)
doi:10.1111/j.1742-4658.2009.06938.x
Chronic high doses of morphine inhibit the growth of various human cancer cell lines However, the mechanisms by which such high-dose morphine inhibits cell proliferation and induces cell death are not fully understood Here we show that c-Jun N-terminal kinase (JNK) plays a pivotal role in high-dose morphine-induced apoptosis of SH-SY5Y cells in
a mitochondria-dependent manner Activation of JNK by morphine led to reactive oxygen species (ROS) generation via the mitochondrial permeabil-ity transition pore, because the mPTP inhibitor cyclosporin A significantly inhibited ROS generation ROS in turn exerted feedback regulation on JNK activation, as shown by the observations that cyclosporin A and the antioxidant N-acetylcysteine significantly inhibited the phosphorylation of JNK induced by morphine ROS-amplified JNK induced cytochrome c release and caspase-9⁄ 3 activation through enhancement of expression of the proapoptotic protein Bim and reduction of expression of the antiapop-totic protein Bcl-2 All of these effects of morphine could be suppressed by the JNK inhibitor SP600125 and N-acetylcysteine The key role of the JNK pathway in morphine-induced apoptosis was further confirmed by the observation that decreased levels of JNK in cells transfected with specific small interfering RNA resulted in resistance to the proapoptotic effect of morphine Thus, the present study clearly shows that morphine-induced apoptosis in SH-SY5Y cells involves JNK-dependent activation of the mitochondrial death pathway, and that ROS signaling exerts positive feed-back regulation of JNK activity
Abbreviations
ALP, allopurionol; CsA, cyclosporin A; DCFH2-DA, 2,7-dichlorodihydrofluorescein diacetate; DPI, diphenylene iodonium; FITC, fluorescein isothiocyanate; HE, hydroethidine; JNK, c-Jun N-terminal kinase; mPFC, medial prefrontal cortex; mPTP, mitochondrial permeability transition pore; NAC, N-acetylcysteine; PI, propidium iodide; PTX, pertussis toxin; ROS, reactive oxygen species; SEM, standard error of the mean; siRNA, small interfering RNA; SRB, sulforhodamine B; VTA, ventral tegmental area.
Trang 2promoting effects occur at low concentrations or single
doses of opioids, whereas growth-inhibitory effects
occur with chronic opioid treatment or relatively high
in vitro concentrations [16] For example, it was
reported by previous studies that morphine and its
derivatives inhibited the growth of various human
cancer cells, including neuroblastoma cells, with IC50
values over the millimolar level [17,18]
For humans, high plasma concentration of opioids
occur under two circumstances One is the application
of high doses of opioids for pain treatment in cancer
patients Chronic high-dose morphine therapy has been
widely used for severe cancer pain in palliative care
[19] The other is the abuse of opioids In support of
this, it was reported that, in animal models of
addic-tion, the plasma concentration of morphine is as high
as 2.5 mm [20] It is possible that such high plasma
concentrations of opiate could result in neuronal
toxic-ity and death Indeed, accumulating evidence
demon-strates that chronic exposure to morphine or other
opiates leads to alterations in the morphology,
struc-ture and function of neurons in certain brain regions
associated with the development of opioid dependence,
such as the ventral tegmental area (VTA), nucleus
accumbens, and medial prefrontal cortex (mPFC) For
example, chronic morphine exposure has been found
to reduce the size of neurons and spine density in the
VTA [21], and decrease the number of dendritic spines
and alter the complexity of dendritic branches in the
nucleus accumbens and mPFC [22] Also, reduction of
the immunodensity of neurofilament proteins, the
major intermediate filaments of the neuronal
cytoskele-ton, has been observed in the VTA of brains from
chronic morphine-treated rats [23], and in the mPFC
from chronic opioid abusers [24] In addition, a
signifi-cant loss of ventricular and cortical volume was found
in the brains of human opioid addicts in a clinical
study [25] All of these findings suggest that the abuse
of opioids may induce neuronal toxicity and affect
neuronal survival
Although opioids have been clearly shown to
modu-late cell proliferation and cell death, the molecular
mechanisms have not been fully elucidated We
recently found that morphine at relatively low
concen-trations was able to inhibit doxorubicin-induced
apop-tosis through inhibition of reactive oxygen species
(ROS) accumulation and mitochondrial cytochrome c
release, and blockade of nuclear factor-jB
transcrip-tional activation, in SH-SY5Y cells [26] The present
study was undertaken to study the mechanisms by
which chronic high doses of morphine inhibit cell
pro-liferation and induce cell death in SH-SY5Y cells,
which constitute a subclone derived from the parent
SK-N-SH cell line, a human neuroblastoma cell line that possesses the growth, biochemical and cytogenetic properties of neurons [27] and expresses both d-opioid and l-opioid receptors [28] SH-SY5Y cells have been used extensively in the study of neuronal death [29–31]
Results
Morphine inhibited SH-SY5Y cell proliferation and induced cell apoptosis
Previous studies reported that morphine inhibited the growth of various human cancer cells, including neuro-blastoma cells, with IC50values of 2.7–8.8 mm [17,18]
To assess the effect of morphine on SH-SY5Y cell pro-liferation, equal numbers of cells were treated with various concentrations of morphine (0.5–4 mm) for
48 h Cell viability was detected by the sulforhod-amine B (SRB) assay As shown in Fig 1A, morphine (0.5–4 mm) caused dose-dependent inhibition of cell proliferation, with a significant reduction at 0.5 mm and an almost 80% reduction at 4 mm
The cell death caused by morphine could be medi-ated by several different mechanisms To determine whether cell death was caused by apoptosis, an extreme consequence of neurotoxicity, we examined the apoptotic percentage of morphine-treated cells by flow cytometric analysis of permeabilized cells double stained with annexin V–fluorescein isothiocyanate (FITC)⁄ propidium iodide (PI) SH-SY5Y cells were treated with morphine (1–4 mm) or left untreated (con-trol) for 48 h As shown in Fig 1B, morphine-treated cells exhibited significantly greater apoptosis than control cells In the presence of 4 mm morphine, the percentage of apoptotic cells reached 60%, which confirmed the results obtained using the SRB assay
To determine whether morphine mediates its effect
on inhibition of cell proliferation and induction of apoptosis via an opioid receptor-related mechanism, SH-SY5Y cells were treated for 48 h with morphine in the presence of the opioid receptor antagonist nalox-one (0.1 mm) or the Gi⁄ o protein inhibitor pertussis toxin (PTX, 0.1 lgÆmL)1), and cell viability and the apoptotic percentage of morphine-treated cells were then assessed The inhibitory effects of morphine on cell growth were not antagonized by either 0.1 mm nal-oxone or 0.1 lgÆmL)1 PTX (Fig 1C) Even naloxone
up to 1 mm and PTX up to 0.4 lgÆmL)1had no effect
on morphine-induced inhibition of cell proliferation (data not shown) Furthermore, the results from flow cytometric analysis and SRB assay agreed well with each other, showing that naloxone and PTX
Trang 3pretreat-ment were not able to block morphine-induced
apop-tosis in SH-SY5Y cells (Fig 1D), suggesting that a
typical opioid receptor-related mechanism was not
involved, consistent with previous findings that an
opioid receptor-related mechanism is not involved in
opioid-induced apoptosis of tumor cells [9,32]
Morphine treatment induced release of
cytochrome c and activation of caspase-9
and caspase-3
Previous studies have shown that a
mitochondria-dependent pathway is implicated in morphine-induced
apoptosis in neuroblastoma cells and neurons [12,14]
To determine the molecular pathway for
morphine-induced apoptosis, we examined the effects of
morphine treatment on the release of cytochrome c and
the activation of caspase-9 and caspase-3 in SH-SY5Y
cells by western blot analysis Because activation of
caspase-9 and caspase-3 is required for
mitochondria-dependent apoptosis [33], we first detected activation
of caspase-9 and caspase-3 upon morphine treatment
Activation of caspases can be manifested either by a
reduction in the amount of procaspase (inactive form)
or by an increase in the amount of cleaved caspase (active form) As shown in Fig 2A,B, a concentration-dependent decrease in procaspase-3 or procaspase-9 expression and an increase in cleaved caspase-3 expres-sion were observed in SH-SY5Y cells treated with morphine (1–4 mm) for 48 h, indicative of caspase-9 and caspase-3 activation Release of cytochrome c from mitochondria to the cytosol is essential for cas-pase-9 activation [34] Next, we examined the effect of morphine treatment on cytochrome c release The cytosolic fractions from cells were isolated, and the presence of cytochrome c was detected by antibody against cytochrome c Treatment of cells with morphine (1–4 mm) for 48 h led to large amounts of cytochrome c release into the cytosol, as compared with control cells (Fig 2C)
Cytochrome c release is a common event in the cell death pathway, initiated by diverse apoptosis-inducing agents Cytochrome c can be either a trigger or a con-sequence of caspase activation Although cytochrome c release occurs mostly upstream of caspase activation,
in some models of apoptosis, such as death
receptor-80
60
40
Apoptosis (%) 20
0
80
100
120
B D
60
40
Cell viability (%) 20
0
80
100
140
120
60
40
Cell viability (%) 20
0
80
60
Mor 0.1 µg·mL –1 PTX + Mor 0.1 m M Nx + Mor
Mor 0.1 µg·mL –1
PTX + Mor 0.1 m M Nx + Mor
40
Apoptosis (%) 20
0 Con
Concentration (m M ) Concentration of Mor (m M )
Con Concentration (m M )
1
4
0
Concentration of Mor (m M )
2
0
Fig 1 Morphine-induced cell death in a naloxone-irreversible and PTX-irreversible manner in SH-SY5Y cells (A) Cells were treated with vari-ous concentrations of morphine (Mor) for 48 h, and cell viability was determined using the SRB assay as described in Experimental proce-dures (B) Cells were treated with various concentrations of morphine for 48 h, and then permeabilized, double stained with annexin V–FITC ⁄ PI, and detected by flow cytometry as described in Experimental procedures (C) Cells were treated with 2 m M morphine in the absence or presence of 0.1 m M naloxone or 0.1 lgÆmL)1PTX, as indicated, for 48 h (D) Cells were treated with 4 m M morphine in the absence or presence of 0.1 m M naloxone or 0.1 lgÆmL)1PTX, as indicated, for 48 h Data are expressed as a percentage of the untreated control cell samples, and represent means ± SEMs for at least three independent experiments performed in triplicate.
Trang 4dependent apoptosis, caspase activation is upstream of
cytochrome c release [35,36] To examine the sequence
of the process of cytochrome c release and caspase
activation, we examined the effect of zVAD-fmk, a
broad-spectrum caspase inhibitor, on
morphine-induced cytochrome c release SH-SY5Y cells were
treated with morphine alone or concomitantly with
morphine and zVAD-fmk for 24 h As shown in
Fig 2D, cotreatment of cells with zVAD-fmk led to a
significant decrease in cleaved caspase-3 levels in
mor-phine-treated cells, but had no effect on the cytosolic
accumulation of cytochrome c, suggesting that active
caspases are not required for cytochrome c release in
morphine-induced cell death, and that cytochrome c
release is upstream of caspase activation during
mor-phine-induced apoptosis in SH-SY5Y cells Thus,
mitochondria may be direct targets of death signals
initiated by morphine The results are consistent with
previous studies showing that SH-SY5Y cells do not
express caspase-8 [37], an essential mediator of
CD95-triggered apoptosis [38]
ROS were generated in SH-SY5Y cells following morphine treatment
It has been reported that ROS are implicated in the mediation of caspase-dependent cell apoptosis by pro-moting cytochrome c release [39] Previous studies have also demonstrated that morphine-induced apoptosis requires the generation of ROS [40] To investigate whether ROS generation is one of the molecular events upstream of release of cytochrome c, we detected ROS generation in response to morphine treatment for vary-ing times Usvary-ing flow cytometry to assess ROS genera-tion with the fluorescent indicators hydroethidine (HE) and 2,7-dichlorodihydrofluorescein diacetate (DCFH2 -DA), to detect O2·) and H2O2, respectively, we found that treatment of cells with 4 mm morphine led to increases in O2·)and H2O2 levels An increase in ROS generation was detected as early as after 6 h of morphine treatment, and the maximal enhancements of
O2·) and H2O2 levels were detected after 24 h of morphine treatment (Fig 3A) N-Acetylcysteine (NAC)
A
B
D
C
Fig 2 Morphine treatment induced cytochrome c release and caspase-3 and caspase-9 activation (A–C) Morphine (Mor) dose-dependently activated caspase-3 and caspase-9, and released cytochrome c from mitochondria Cells were treated with increasing concentrations of morphine, as indicated, for 48 h Extracts from whole cells or the cytosol were subjected to 12% SDS ⁄ PAGE, and immunoblotted with antibodies against procaspase-3, procaspase-9, cleaved caspase-3, and cytochrome c (D) zVAD-fmk was able to inhibit morphine-induced caspase-3 activation, but unable to suppress cytochrome c release Cells were treated with 4 m M morphine for 24 h in the absence or presence of 12 l M zVAD-fmk, and then harvested for detection of cleaved caspase-3 and cytochrome c (a) A representative image of immunoblots for cleaved caspase-3 and cytochrome c (b) Densitometric analysis of changes in levels of cleaved caspase-3 and cytosolic cytochrome c All images are representative of three independent experiments yielding similar results Data are means ± SEMs for three independent experiments *P < 0.05 as compared with morphine.
Trang 5(5 mm), a well-characterized antioxidant, significantly
inhibited the generation of O2·)in SH-SY5Y cells
trea-ted with morphine for 6 h However, this effect of
morphine could not be suppressed by diphenylene
iodonium (DPI) (2 lm), an inhibitor of NADPH
oxi-dase, and allopurionol (ALP) (100 lm), an inhibitor of
xanthine oxidase (Fig 3B), suggesting that
mitochon-dria may be the major source of ROS In addition,
NAC (5 mm) also clearly inhibited the elevation of
intracellular O2·) and H2O2 levels after 24 h of
mor-phine treatment (Fig 3C) It has been suggested that
mitochondrial permeability transition pore (mPTP)
opening increases ROS production in vivo [41] and in
isolated mitochondria [42] To determine whether ROS
came from the mitochondria, we examined the effect of
the mPTP inhibitor cyclosporin A (CsA) on
morphine-induced ROS generation Treatment with CsA (1 lm)
alone had no effect on O2) generation, but it
signifi-cantly inhibited morphine-induced O2 ) generation
(Fig 3D), supporting the idea that the mitochondria
were the source of ROS generation
c-Jun N-terminal kinase (JNK) activation was upstream of ROS generation and was in turn regulated by ROS
Studies of JNK-induced neuronal apoptosis suggest that JNK-induced phosphorylation of the transcription factor c-Jun and the consequent expression of c-Jun-induced genes mediate JNK-c-Jun-induced apoptosis [43,44]
As ROS activate the JNK cascade [45,46], apoptosis induced by morphine may depend on activation of the JNK pathway To determine whether morphine-induced ROS generation leads to activation of JNK and its sub-strate target (c-Jun) in SH-SY5Y cells, we examined the effect of morphine treatment on the phosphorylation of JNK and c-Jun by western blot analysis, using phospho-specific antibodies As shown in Fig 4A, the phosphory-lation of JNK and the phosphoryphosphory-lation of c-Jun were both increased by morphine treatment (4 mm), starting
at 3 h and peaking at 24 h However, no differences in the phosphorylation of p38 were observed between controls and morphine-treated cells
A
B
C D
Fig 3 Morphine treatment increased intercellular O2·)and H2O2levels in SH-SY5Y cells (A) Morphine (Mor) time-dependently induced ROS generation Cells were treated with 4 m M morphine for the indicated time periods, and intracellular O2·)and H2O2levels were deter-mined by flow cytometry using 5 l M HE and 10 l M DCFH 2 -DA as fluorescent probes, as described in Experimental procedures The figure is representative of four independent experiments yielding similar results (B, C) NAC, but not DPI and ALP, suppressed morphine-induced ROS generation in SH-SY5Y cells Cells were pretreated with 5 m M NAC, 100 l M ALP and 2 l M DPI for 30 min prior to incubation with
4 m M morphine for 6 h, or pretreated with 5 m M NAC prior to incubation with 4 m M morphine for 24 h, and intracellular H 2 O 2 and O 2 ·) levels were then detected (a) A representative image of five independent experiments yielding similar results (b) Quantification of O2·)and
H2O2generation (D) CsA decreased morphine-induced enhancement of O2·)levels Cells were treated with either 4 m M morphine alone or with 4 m M morphine in combination with 1 l M CsA for 6 h, and intracellular O 2 ·)levels were then determined Values are means ± SEMs for
at least three independent experiments performed in triplicate **P < 0.01 as compared with morphine.
Trang 6The results showed that JNK was activated as early
as after 3 h of morphine treatment; this preceded ROS
generation, which was detected after 6 h of morphine
treatment, indicating that JNK activation was located
upstream of ROS generation (Fig 3A) To determine
whether JNK activation is involved in ROS
genera-tion, we detected the effect of SP600125, a selective
inhibitor of JNK that acts by binding to the
ATP-binding site, on O2) generation induced by morphine
treatment Pretreatment of cells with 20 lm SP600125
effectively attenuated O2)generation (Fig 4B),
indicat-ing that JNK activation is essential for ROS
genera-tion Consistent with the absence of activation of p38
by morphine, the p38 mitogen-activated protein kinase
inhibitor SB203580 had no effect on morphine-induced
ROS generation (Fig 4B) To obtain further evidence
that JNK activation precedes ROS generation, we
determined the effect of NAC on JNK activation
induced by 3 h of morphine treatment, because JNK activation started at 3 h NAC had no significant effect
on the phosphorylation of JNK induced by 3 h of morphine treatment (Fig 4C) These results support the idea that JNK activation precedes ROS generation, consistent with recent studies showing that ROS gener-ation was regulated by the JNK signaling pathway [47,48]
To determine whether ROS exert feedback regula-tion on JNK activaregula-tion, we next examined the effects
of the antioxidant NAC and the mPTP inhibitor CsA
on the phosphorylation of JNK and on its substrate transcription factor c-Jun following prolonged mor-phine treatment Figure 4D shows that, in the presence
of 5 mm NAC, morphine treatment for 12 h failed to induce increases in the phosphorylation of JNK and c-Jun Additionally, in the presence of 1 lm CsA, morphine treatment for 12 h also failed to induce JNK
A
E
C
D
B
Fig 4 JNK activation induced by morphine (Mor) was upstream of ROS generation and was in turn regulated by ROS (A) Morphine acti-vated the JNK ⁄ c-Jun, but not the p38, signaling pathway Cells were treated with 4 m M morphine for increasing time periods, as indicated Extracts from whole cells were subjected to 12% SDS ⁄ PAGE and immunoblotted with antibodies against JNK, JNK, p38, phospho-p38, c-Jun, phospho-c-Jun and actin (B) SP600125, but not SB203580, blocked morphine-induced O2)generation Cells were treated with
4 m M morphine alone, or treated with 4 m M morphine in the presence of 20 l M SP600125 or 20 l M SB203580 for 6 h, and intracellular O 2 ·) levels were then determined by flow cytometry Values are means ± SEMs for at least three independent experiments performed in tripli-cate **P < 0.01 as compared with morphine (C) NAC failed to suppress phosphorylation of JNK induced by 3 h of morphine treatment Cells were either treated with 4 m M morphine alone or treated with 4 m M morphine in combination with 5 m M NAC for 3 h (D) NAC and SP600125 were able to inhibit phosphorylation of JNK and c-Jun induced by 12 h of morphine treatment Cells were treated with 4 m M mor-phine for 12 h in the absence or presence of 5 m M NAC or 20 l M SP600125 (E) CsA abolished phosphorylation of JNK induced by 12 h of morphine treatment Cells were treated with 4 m M morphine for 12 h in the absence or presence of 1 l M CsA.
Trang 7and c-Jun phosphorylation (Fig 4E) The effect of
morphine on the phosphorylation of JNK and c-Jun
could also be inhibited by SP600125, an inhibitor of
JNK activation These results support the idea that
ROS exert a positive feedback effect on JNK
activa-tion after prolonged morphine treatment Altogether,
the findings suggest that morphine induces ROS
accu-mulation by activation of the JNK pathway, and that
ROS can, in turn, activate JNK in a positive feedback
fashion
Morphine treatment differentially regulated the
expression of Bcl-2 family members
Bcl-2 family members are major regulators of
mito-chondrial integrity and mitochondria-initiated
cyto-chrome c release and caspase activation The Bcl-2
family includes antiapoptotic members such as Bcl-2
and Bcl-XL, and proapoptotic members such as Bax,
Bak, and Bim Bax and Bak are potent regulators of
cytochrome c release from mitochondria under a
vari-ety of stress conditions JNK⁄ c-Jun signaling has been
implicated in the induction of the BH3-only Bcl-2
fam-ily member Bim (Bcl-interacting mediator of cell
death), a key mediator of Bax-dependent cytochrome c
release during neuronal apoptosis [49,50] Next, we
investigated whether morphine treatment caused
induc-tion of Bim in SH-SY5Y cells As was expected, in parallel with JNK activation, treatment with morphine resulted in enhancement of Bim expression in a time-dependent and dose-time-dependent manner (Fig 5A,B) Bcl-2 prevents release of cytochrome c by heterodimer-izing with Bax [51,52] Therefore, the effects of treat-ment with morphine on the protein levels of Bcl-2 and Bax were assessed As shown in Fig 5C, treatment of cells with morphine induced a robust decrease in the protein level of Bcl-2, but failed to produce a signifi-cant change in the protein level of Bax As a result, the Bcl-2⁄ Bax ratio was decreased, which is indicative
of an increase in mitochondrial permeability
Inhibitors of JNK and antioxidant suppressed morphine-induced changes in Bim and Bcl-2 expression, cytochrome c release, and caspase-3 activation
The data presented above show that a positive feed-back cycle operates in morphine-treated SH-SY5Y cells, in which JNK activation causes ROS generation, which, in turn, leads to further activation of JNK We thus hypothesized that JNK activation might play a crucial role in morphine-induced cytochrome c release and consequent caspase-3 activation by increasing the expression of the BH3-only protein Bim and decreasing
A
B
C
Fig 5 Morphine (Mor) treatment differentially regulated the expression of Bcl-2 family members (A, B) Morphine dose-dependently and time-dependently increased the expression of Bim Cells were exposed to various concentrations of morphine for 24 h or 4 m M morphine for various times, as indicated Extracts from cells were subjected to 12% SDS ⁄ PAGE and immunoblotted with antibody against Bim (C) Morphine decreased Bcl-2 levels in a dose-dependent manner Cells were treated with various concentrations of morphine for 48 h, and extracts from cells were subjected to 12% SDS ⁄ PAGE and immunoblotted with antibodies against Bax and Bcl-2 (a) Representative immunoblots for Bcl-2 and Bax (b) Densitometric analysis of changes in levels of Bcl-2 ⁄ Bax All images are representative of at least three independent experiments yielding similar results Values are means ± SEMs for at least three independent experiments.
Trang 8Bcl-2 levels To test this hypothesis, we determined
whether the selective inhibitor of JNK SP600125 and
the antioxidant NAC suppressed morphine-induced
changes in Bim and Bcl-2 expression, cytochrome c
release, and caspase-3 activation As shown in Fig 6,
pretreatment of cells with 20 lm SP600125 or 5 mm
NAC alone had no effects on Bim and Bcl-2 expression,
cytochrome c release, and caspase-3 activation
However, in the presence of SP600125 or NAC,
treat-ment of cells with 4 mm morphine for 24 h failed to
increase Bim expression, decrease Bcl-2 levels, increase
cytochrome c release, or activate caspase-3, indicating
that the positive feedback amplification of JNK
activation by ROS and the consequent changes in the
expression of Bim and Bcl-2 may contribute to
morphine-induced cytochrome c release and caspase-3
activation
SP600125, small interfering RNA (siRNA) against
JNK and NAC attenuated morphine-induced
apoptosis
Accumulating evidence shows that activation of the
JNK⁄ c-Jun pathway with subsequent enhancement of
cytochrome c release via induction of Bim expression
and reduction of Bcl-2 expression are key events
required for neuronal apoptosis [50,53] In this study,
we found that SP600125 could inhibit or prevent the morphine-induced occurrence of these molecular events Therefore, the last set of experiments addressed the question of whether JNK-mediated cytochrome c release and caspase-3 activation via alteration of Bim and Bcl-2 was correlated with morphine-induced apop-tosis in SH-SY5Y cells SH-SY5Y cells were treated with 4 mm morphine for 48 h in the absence or pres-ence of 20 lm SP600125 or SB203580, which were added 30 min before morphine Apoptotic cells were measured by flow cytometry and phase contrast microscopy Morphine at 4 mm caused drastic apopto-sis, and this apoptosis was substantially inhibited by preincubation of the cells with the JNK inhibitor SP600125 (20 lm; Fig 7A) The involvement of JNK
in morphine-induced apoptosis was further investigated
in SH-SY5Y cells that had been transiently transfected with siRNA against JNK mRNA Transfection of JNK siRNAs resulted in a decrease in the basal pro-tein level of JNK and markedly suppressed the apop-tosis of cells in response to morphine treatment (Fig 7B), indicating a pivotal role of the JNK⁄ c-Jun pathway in morphine-induced apoptosis Given the role of ROS in feedback amplification of JNK activa-tion, we next investigated the effect of the antioxidant NAC on morphine-induced apoptosis As shown in Fig 7C, treatment of cells with 4 mm morphine for
A B
Fig 6 SP600125 and NAC suppressed morphine-induced changes in Bim and Bcl-2 expression, cytochrome c release and caspase-3 activa-tion in SH-SY5Y cells Cells were either left untreated or treated with 4 m M morphine for 24 h, in the presence or absence of 20 l M SP600125 or 5 m M NAC Extracts from whole cells or the cytosol were subjected to 12% SDS ⁄ PAGE and immunoblotted with antibodies against Bim, Bcl-2, cytochrome c, and cleaved caspase-3 (A) A representative image of immunoblots for Bim, Bcl-2, cytochrome c, and cleaved caspase-3 (B) Densitometric analysis of changes in levels of Bim, Bcl-2, cytochrome c, and cleaved caspase-3 All images are repre-sentative of three independent experiments yielding similar results Data are means ± SEMs for three independent experiments *P < 0.05
as compared with morphine.
Trang 948 h in the presence of 5 mm NAC greatly reduced
morphine-induced apoptosis, by 35% (from 57% in
the absence of NAC to 22% in the presence of NAC),
supporting its role in the amplification of JNK
acti-vation
Discussion
In the present study, we demonstrated that chronic
high-dose morphine treatment was able to cause
apop-totic cell death of SH-SY5Y cells in an opioid
recep-tor-independent manner, consistent with previous
studies [9,32] Moreover, we found that JNK signaling
played a central role in the regulation of SH-SY5Y cell apoptosis via activation of the mitochondria-dependent pathway We also demonstrated that a positive feed-back cycle operates in SH-SY5Y cells, in which morphine-mediated JNK activation caused ROS generation, which in turn led to further activation of JNK
The activation of JNK by morphine (starting after
3 h of treatment) preceded ROS generation (starting after 6 h of treatment), indicating that the JNK activa-tion occurred upstream of the ROS generaactiva-tion SP600125 almost completely inhibited the accumula-tion of morphine-induced intracellular O2 ), further
Mock –
– –
– +
+ SiRNA
JNK
80
60
40
20
0
80
60
40
20
0
0 Concentration (m M )
Concentration (m M )
4
0 4
80
Mor Mor + Mor SiRNA JNK + Mor
Mor
SP600125 + Mor
SP203580 + Mor
*
*
* *
*
60
40
20
0
Con NAC Mor NAC
+ Mor
Action 1.2 1.0 0.8 0.6 0.4 0.2 0.0
Con Mock SiRNA
(b)
(b)
C
Fig 7 SP600125, siRNA against JNK and NAC attenuated morphine-induced apoptosis (A) SP600125, but not SB203580, attenuated morphine-induced apoptosis Cells were treated with 4 m M morphine for 48 h in the absence or presence of 20 l M SP600125 or 20 l M SB203580 (a) Representative phase contrast microscopy images (b) The apoptotic cells were detected by flow cytometry (B) JNK siRNA markedly decreased morphine-induced apoptosis (a) Cells were transfected with 200 n M JNK1 ⁄ 2 siRNA or Mock, and the protein levels of JNK were determined by western blot after transfection for 24 h, and densitometric analysis of changes in the basal protein levels of JNK, with *P < 0.05 in comparison with both untransfected and mock-transfected cells (b) Cells were transfected with 200 n M JNK1 ⁄ 2 siRNA or Mock for 24 h, and then treated with 4 m M morphine for 48 h The apoptotic cells were detected by flow cytometry as described in Experi-mental procedures (C) NAC inhibited morphine-induced apoptosis Cells were treated with 4 m M morphine for 48 h in the absence or pres-ence of 5 m M NAC, and the apoptotic cells were detected by flow cytometry All images are representative of at least three independent experiments yielding similar results Values are means ± SEMs for at least three independent experiments performed in triplicate.
*P < 0.05, **P < 0.01 as compared with morphine.
Trang 10indicating that ROS generation was JNK-dependent.
Additional evidence to support JNK activation prior
to ROS generation was that NAC, a well-characterized
antioxidant, failed to prevent JNK activation induced
by 3 h of morphine treatment Regulation of ROS
gen-eration by JNK has also been observed recently in the
fibroblast and PC3 prostate carcinoma cell lines
[47,48] Our study further demonstrated that ROS were
released from mitochondria through opening of the
mPTP because the mPTP inhibitor CsA, but not DPI
and ALP, robustly attenuated morphine-induced ROS
generation These results are consistent with the
find-ings reported by previous studies, showing that mPTP
opening increases ROS production in vivo [41] and in
isolated mitochondria [42,54], and that mPTP is the
target downstream of JNK activation [55], and is
inhibited by SP600125 [56] Moreover, we also
demon-strated that ROS could in turn activate JNK in a
posi-tive feedback fashion, because NAC significantly
attenuated prolonged JNK activation induced by 12 h
of morphine treatment In addition, CsA also
attenu-ated prolonged JNK activation induced by 12 h of
morphine treatment Therefore, this study clearly
shows a route from JNK activation by morphine to
initiation of ROS release from mitochondria, the ROS,
in turn, activating JNK in a positive feedback fashion
This novel route is not consistent with that reported
by previous studies, in which ROS generation initiated
JNK activation [45,57,58]
JNK can be activated by various stress stimuli In
the present study, we showed that morphine treatment
resulted in sustained activation of JNK, consistent
with previous studies showing that prolonged exposure
to morphine activates JNK in neurons and other cell
lines [59,60] We further demonstrated that activation
of JNK was necessary for the morphine-induced
apop-tosis by using the pharmacological inhibitor SP600125
Inhibition of JNK activity by SP600125 led to decrease
of apoptotic cell death in SH-SY5Y cells Additionally,
downregulation of endogenous JNK with transfected
specific siRNA resulted in resistance to the
proapop-totic effect of morphine Moreover, NAC, which was
shown to suppress ROS-amplified JNK activation, also
robustly attenuated morphine-induced apoptosis in
SH-SY5Y cells These results strongly suggest that
JNK signaling plays a pivotal role in
morphine-induced apoptosis
JNK is thought to induce apoptosis via
transcription-dependent and transcription-intranscription-dependent mechanisms
We found that JNK-mediated morphine-induced
apop-tosis appeared to be transcription-dependent
Activa-tion of JNK by morphine led to a robust increase
in the expression of the BH3-only protein Bim via
activation of the transcription factor c-Jun, and this effect could be suppressed by SP600125 In addi-tion, NAC attenuated morphine-induced Bim expres-sion, whereas it inhibited morphine-induced phosphorylation of JNK and c-Jun It has been shown that, in sympathetic neurons, activation of the JNK⁄ c-Jun pathway and increased expression of Bim are key events required for cytochrome c release and apop-tosis following nerve growth factor withdrawal [50] There is strong evidence that Bax and Bak are potent regulators of cytochrome c release from mitochondria under a variety of stress conditions Recent evidence has shown that BH3-only proteins (e.g Bim, Bad, HRK, and Bid) are essential for Bax and Bak activa-tion Bim has been shown to potentiate the proapop-totic effects of Bax and Bak while concomitantly suppressing the prosurvival function of Bcl-2 [61,62]
In agreement with these findings, we also demonstrated that activation of the JNK⁄ c-Jun pathway by mor-phine caused dramatic enhancement of cytochrome c release and caspase-3 activation, both of which were inhibited by SP600125 NAC also suppressed mor-phine-induced cytochrome c release and caspase-3 acti-vation, through its inhibition of the morphine-activated JNK⁄ c-Jun pathway Our findings thus indi-cate that Bim may be involved in the release of cyto-chrome c and initiation of the intrinsic death pathway
in response to prolonged morphine treatment
In addition to enhancement of the expression of the proapoptotic protein Bim, prolonged morphine treat-ment also dose-dependently decreased the expression
of the antiapoptotic protein Bcl-2 in a JNK-dependent manner Activation of JNK has been reported to induce the phosphorylation of Bcl-2, leading to Bcl-2 degradation through the proteasome pathway [63,64] SP60025 could inhibit the morphine-induced reduction
of Bcl-2 expression Decreases in Bcl-2 expression by prolonged morphine treatment have been observed in cell lines and in rat brain in previous in vitro and
in vivostudies [13,14,65] However, Bax expression was not significantly altered following prolonged morphine treatment As a result, the Bcl-2⁄ Bax ratio was decreased Bcl-2 can block cytochrome c release by heterodimerizing with Bax [51,52] Conversely, Bim promotes cytochrome c release by freeing Bax to be incorporated into the mitochondrial membrane [62] Also, Bim can interact with Bax and induce a confor-mational change that facilitates the formation of chan-nels for release of cytochrome c [61] Thus, morphine seems to promote the proapoptotic effect of Bax by both enhancement of Bim expression and downregula-tion of Bcl-2 expression, leading to cytochrome c release, caspase-3 activation, and apoptosis