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Introduction
Autophagy is a dynamic process in which intracellular
membrane structures sequester proteins and organelles for
degradation in a lytic compartment. It is an evolutionarily,
conserved process that occurs in all eukaryotic cells, from
yeast to mammals[1,2]. Autophagy is mediated by double- or
multi-membrane autophagosomes and can be induced under
conditions of starvation or by certain
hormones[3]. In addition to its basic role in the turnover of proteins and organelles,
autophagy has multiple physiological and
pathophysiological functions including cell differentiation, immune defense,
and cell death[4]. Recent studies have found the activation
of autophagy under pathological conditions, such as
neurodegenerative diseases and hereditary
myopathies[5,6]. While autophagy can represent an independent mechanism
of cell self-destruction, named type II programmed cell death,
it is often associated closely with apoptosis otherwise known
as type I programmed cell death[7,8]. Autophagy and
apoptosis can occur in the same cell concurrently or
sequentially in response to the same stimulus. Autophagy
exerts multiple impacts on apoptotic cell death, ranging from
promoting to antagonizing apoptosis. In cells which have
integrated caspase-dependent apoptotic signaling systems,
autophagy can be triggered by mitochondrial membrane
potential collapse and cytochrome c (cyto-c) redistribution
following stimulation with apoptotic
inducers[9_11]. However, the relationship between autophagic activation and
caspase-independent apoptosis remains largely unknown. Moreover,
studying the role of autophagy in cancer is at a very early
stage[12], and even the most fundamental question __ whether
autophagy kills cancer cells or protects them from
unfavorable conditions __ has not been clearly answered.
Crotoxin (CrTX) is a cytotoxic PLA2 isolated from the
venom of the South American rattlesnake,
Crotalus durissus terrificus[13]. It is
a non-covalent complex formed by 2 non-identical subunits,
one acidic (subunit A) and one basic (subunit
B). Subunit B is a PLA2 formed by a single chain of
122 amino acid residues cross-linked by 7 disulfide
bonds[14]. Its cytotoxicity was independent of cell growth since both
quiescent and proliferating cells had similar sensitivities.
CrTX displays cytotoxic activity against a variety of
murine[15] and human tumor cell
lines in vitro[16] and appears to be
highly active toward cell lines expressing a high density of
epidermal growth factor receptors
(EGFR)[17]; however, the precise mechanism of the cytotoxicity remains to be
determined. It had previously been assumed that the toxin
simply induces lysis through the disruption of the cell
membrane.
Previous studies found that CrTX-induced death of
human leukemic K562 cells was associated with the collapse
of the mitochondrial membrane potential, the release of
cyto-c, and the activation of caspase-3. Caspase inhibitors
attenuated CrTX-induced K562 cell death. These studies provided
evidence that an apoptotic mechanism contributes to the
CrTX-induced death of K562 cells. CrTX also activated
autophagy in these cells; however, the inhibition of
autophagy potentiated the cytotoxicity of CrTX, suggesting
autophagy delayed apoptosis in K562[18].
The human breast cancer cell line, MCF-7, which lacks
procaspase-3 due to the functional deletion of the
caspase-3 (CASP-3) gene, has been widely used in studying the
importance of caspase-3 in apoptosis. Some studies found that
caspase-3-mediated events appeared to be dispensable in
determining the overall killing of cells, since 50 kb DNA
fragments, chromatin condensation, Rb, gelsolin, and poly
(ADP-ribose) polymerase (PARP) cleavage also occurred in
caspase-3-deficient MCF-7 cells[19_21]. When caspase-3 has
no contribution to cell death, cells can still die apoptotically
by non caspase-3-dependent or caspase-independent mechanisms due to the activation of other effective caspases
or the nuclear translocation of AIF[22].
To further investigate the relationship between
molecular mechanisms of apoptosis and autophagy in tumor cells,
this study focused on the role of autophagy in
CrTX-induced cytotoxicity in MCF-7 cells where apoptosis is
hampered due to the lack of caspase-3 activity.
Materials and methods
Drug preparation CrTX was supplied by Celtic Biotech
Ltd (Dublin, Ireland). It was purified from Crotalus
durissus terrificus venom by a combination of size exclusion and anion exchange. The identity of the protein was confirmed
through molecular weight, determined by mass
spectrometry (showing averaged signals at 9500 and 14500, in addition to
the presence of isoforms). Purity was determined by PAGE
and size exclusion was greater than 90%. Lethality in mice
was determined by an ip injection of 0.1 mg with death being
recorded within 3 h.
Cell culture Human breast cancer, MCF-7 cells were
purchased from the Shanghai Institute of Cell Biology,
Chinese Academy of Sciences (Shanghai, China). The cells were
maintained in DMEM medium (Gibco, Rockville, MD, USA)
supplemented with 10% heat-inactivated fetal bovine serum
(FBS) (Hangzhou Sijiqing Biological Engineering Material
Co Ltd, Hangzhou, China), 0.03% l-glutamine (Sigma, St Louis,
MO, USA), and 200 U/L insulin incubated in a 5%
CO2 atmosphere at 37 °C and routinely subcultured every 2 or 3 d.
Cells in a mid-log phase were used in the experiments. To
determine the dose- and time-response of MCF-7cells to
CrTX, the MCF-7 cells were plated into 96-well microplates
(1.5×104 cells/well) and cultured for 14 h. CrTX (12.5, 25, 50,
and 100 µg/mL) was added to the culture medium, and cell
viability was assessed with an MTT assay 24, 48, and 72 h
after drug treatment.
Determination of cell viability Cell viability was assessed
by MTT assay[23] as described before. Briefly, when the
MCF-7 cells confluenced by 70% in the 96-well microplates,
different concentrations of CrTX were added in the absence
or presence of the pan-caspase inhibitor, Z-Vad-fmk
(Calbio-chem, La Jolla, CA, USA) ; 12.5, 25, and
50 μmol/L), for the indicated time. The MTT solution (5 mg/mL, Sigma) was
added 4 h before the end of incubation and the reaction was
stopped by 10% acidified SDS. The absorbance value (A)
per well at 570 nm was read using an automatic multiwell
spectrophotometer (Bio-Rad, Hercules, CA, USA). The
ratio of cell death was calculated as follows: cell death
(%)=(1-A of experiment well/A of control well )×100%.
Cytotoxicity assays Cytotoxicity was measured by LDH
leakage using the LDH detection kit (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China). The samples of
the supernatants and lysates were prepared as follows: when
the MCF-7 cells confluenced by 70% in the 24-well
micro-plates, the autophagy inhibitors 3-MA (10 mmol/L) or
NH4Cl (10 mmol/L) were added 1 h before CrTX treatment. After
being incubated for 24 h, the supernatants of the culture
were collected following centrifugation. The cell pellets were
lysed in 1% Triton X-100 at 37 °C for 45 min. Then the samples
of supernatants and lysates were processed according to
the instructions of the manufacturer. The absorbance value
(A) at 400 nm was quantified by a spectrophotometer. LDH
leakage was calculated as follows: LDH leakage (%)=(A
positive_A positive blank)/(A negative-A negative blank)×100%.
Visualization of MDC-labeled vacuoles Autophagic
vacuoles were labeled with monodansylcadaverin (MDC,
Sigma)[24]. Briefly, the cells were seeded into microplates
pre-exposed to 0.01% polylysine and incubated with CrTX
(100 µg/mL) for 3, 6, and 12 h. Fresh DMEM containing 0.05
mmol/L MDC was added and the cells were incubated at
37 °C for another10 min. Then the cells were washed with
phosphate buffered saline (PBS) 3 times and the microplates
were inverted onto a slide and analyzed by fluorescence
microscopy using an inverted microscope (Nikon Eclipse TE
300, Tokyo, Japan) equipped with a filter system (V-2A
excitation filter: 380-420 nm, barrier filter: 450 nm).
Microphotographs were taken with a Charge Coupled Device (CCD)
camera.
Transmission electron microscope
examination[25]
After treatment with CrTX, the cells were fixed in ice-cold
2.5% glutaraldehyde in 0.1 mol/L PBS and preserved at 4
°C for further processing. When processing resumed, the cells
were post fixed in 1% osmium tetroxide in the same buffer,
dehydrated in graded alcohols, embedded in Epon 812,
sectioned with ultramicrotome, stained with uranyl acetate and
lead citrate, followed by an examination with a transmission
electron microscope (Philips Tecnai 10, Eindhoven, the
Netherlands).
Subcellular fractionation The isolation of mitochondria
was performed as described by Qin et
al[26]. Cells treated with CrTX for different times were harvested and rinsed with
ice-cold PBS twice. The cells were suspended in buffer A
(250 mmol/L sucrose, 1 mmol/L EDTA, 50 mmol/L Tris-HCl,
1 mmol/L DL-Dithiothreitol (DTT), 1 mmol/L phenylmethyl
sulfonylfluoride (PMSF), 1 mmol/L benzamidine, 0.28 u/mL
aprotinin, 50 µg/mL leupeptin, and 7 µg/mL pepstatin A, pH
7.4) and homogenated with a glass Pyrex microhomogenizer
(30 strokes). The homogenates were centrifuged at
1000×g at 4 °C for 10 min, and the resultant supernatants were
transferred to a new Eppendorf tube and centrifuged at
10000×g at 4 °C for 20 min to obtain the mitochondrial pellets and
supernatants. The supernatants were transferred to a new
tube and centrifuged at 100 000×g for 1 h at 4 °C to generate
the cytosolic fraction. The mitochondrial pellet was washed
3 times in buffer B [250 mmol/L sucrose, 1 mmol/L
ethylene-glycol bis(2-aminoethyl ether)tetraacetic acid (EGTA), 10
mmol/L Tris-HCl, 1 mmol/L DTT, 1 mmol/L PMSF, 1 mmol/L
benzamidine, 0.28 u/mL aprotinin, 50 µg/mL leupeptin, and 7
µg/mL pepstatin A, pH 7.4] at 10 000 g for 10 min at 4 °C and
then lysed in Western blot lysing buffer.
Protein preparation and immunoblotting The cells were
harvested and rinsed with ice-cold PBS twice. Five volumes
of Western blot lysing buffer for each volume of cell pellets
was added and the mixture was sonicated on ice (1 s/mL per
sonicate, resting wait for 30 s between intervals, 5 times),
microcentrifugated at 10 000 rpm for 10 min, and the
supernatants were preserved at -70 °C. Before immunoblotting, the
protein content of each sample was adjusted by a
bicin-choninic acid (BCA) protein assay kit (Pierce, Rockford, IL,
USA). Proteins were separated on 12% SDS-PAGE gel,
transferred to a nitrocellulose membrane and immunoblotted with
goat polyclonal cathepsin B antibody (1:100; sc-6493, Santa
Cruz, Santa Cruz, CA, USA), goat polyclonal cathepsin D
antibody (1:100; sc-6486, Santa Cruz) or goat polyclonal
cathepsin L antibody (1:100; sc-6499, Santa Cruz), and mouse
monoclonal cyto-c antibody (1:1000; 7H8.2C12, PharMingen,
San Diego, CA, USA) at 4 °C overnight. The primary
antibodies were detected using horseradish
peroxidase-conjugated anti-goat or anti-mouse antibody (Sigma) at a ratio of
1:5000 in blocking solution for 1 h at room temperature.
Immunoreactivity was detected by enhanced
chemiluminescence (ECL kit, Amersham Pharmacia Biotech, Piscataway,
NJ, USA ) and visualized by autoradiography. Protein
β-actin (1:5000, A441, Sigma) and heat shock protein (HSP) 60
(1:2000, H3524, Sigma) were used as loading controls.
Immunofluorescence[27] The MCF-7 cells were seeded
onto microplates and treated with CrTX (100
µg/mL). After fixation (methanol) and blocking (1% bovine serum albumin
(BSA) dissolved in 0.1% Triton X-100), the cells were
incubated with mouse polyclonal AIF antibody (1:100; PM-0215,
Beijing Zhongshan Co Ltd, Beijing, China) at 4
°C overnight. A fluorescent fluorescein isothiocyanate (FITC) antibody
(Sigma) was used to visualize the binding sites of the
primary antibody. The cells were examined with a laser
confocal microscope (Leica DMIRE2, Wetzlar, Germany).
Statistical analysis All data were presented as mean±
SEM. Statistical analysis was carried out by ANOVA
followed by Dunnett's t-test, with P<0.05 considered
signi-ficant.
Results
Inhibition of cell viability of MCF-7 cells by CrTX
The cells were exposed to various concentrations of CrTX and
various lengths of time. The cytotoxicity of CrTX was
determined using the MTT assay. CrTX inhibited MCF-7 cell
viability in a time- and dose-dependent manner. The MTT
assay revealed that following 24 h of treatment, the
inhibitory rate of CrTX (100 µg/mL) on MCF-7 cells was
28.85%± 0.01%, and when the incubation time was prolonged
to 48 h, the inhibitory rate increased to 51.61%±0.03%, and
then it reached 57.04%±0.04% after 72 h of treatment. At the
dose of 50 μg/mL crotoxin, the inhibitory ratio was only
33.82%±0.04% with 72 h of treatment (Figure 1). Based on its
effectiveness in inhibiting cell viability of MCF-7 cells, the
CrTX concentration of 100 µg/mL was chosen for the
subsequent experi-ments.
Vesicular accumulation of MDC after CrTX treatment
The autofluorescent substance MDC has recently been
shown to be a specific marker for autophagic vacuoles (AVs).
When the cells were viewed with a fluorescence microscope,
MDC-labeled AVs appeared as distinct dot-like structures
distributing in the cytoplasm or in the perinuclear regions. It
was found that there was no notable difference in the
number of vesicles after 0.5 h of CrTX treatment (data not shown).
When the duration of CrTX treatment was extended to 3 h,
an increase in the MDC-labeled vesicles was observed as
indicated by the appearance of punctuate MDC fluorescence,
suggesting an induction of AVs formation after CrTX
treatment. Increased vacuolar accumulation of MDC
continued during the 12 h of the CrTX treatment (Figure 2).
Ultrastructural examination of autophagy after CrTX
treatment MCF-7 cells are very useful in the study of
apoptotic-autophagic relationships because they may
enter both the apoptotic and the autophagic pathways of
cell death. The ultrastructural changes in MCF-7 cells
during CrTX treatment were evaluated with the use of a
transmission electron microscope (TEM). The untreated cells
exhibited normal ultrastructural morphology of cytoplasm,
cell organelles, and nuclei (Figure 3A). CrTX treatment for 6
h resulted in the formation of many autophagosomes and
the sequestration of cytoplasm portions and organelles by
double membranes, possibly derived from the endoplasmic
reticulum. Double-membraned, giant autophagosomes filled
with organelles were observed. Mitochondrial swelling and
autolysosomes were also frequently observed. These
processes preceded apoptotic morphology including nuclear
envelope umbilication and chromatin compaction and
pyknosis, features of caspase-independent apoptosis (Figure
3B_3F).
Increases in protein levels of cathepsin B, D, and L after
CrTX treatment Since the lysosomal proteases are involved
in both apoptosis and autophagy, they (or their targets) may
serve as molecular links between both programmed cell death
(PCD) types in tumor cells. Western blotting results revealed
that a transient increase in cathepsin B levels was observed
within 3 h following CrTX treatment. Cathepsin L decreased
initially following CrTX treatment, but markedly increased
12 h later; pro-cathepsin D showed little change after CrTX
treatment, but decreased dramatically 24 h later. Processed
cathepsin D (active cathepsin D) was maintained at a high
level 6 h after CrTX treatment (Figure 4).
Redistribution of cyto-c and AIF with CrTX treatment
As mitochondrial swelling induced by CrTX was observed
by TEM, immunoblotting and immunofluorescence were used
to investigate the localization of cyto-c and AIF after CrTX
treatment. Cyto-c was released from the mitochondria into
the cytoplasm 3 h after CrTX treatment (Figure 5). Confocal
microscopy was employed to evaluate the changes of AIF
cellular localization. AIF was released from the mitochondria
and transferred into the nucleus as early as 1.5 h after CrTX
treatment; AIF aggregated in the nucleus and displayed a
tendency for uneven distribution (Figure 6).
Effects of autophagy and caspase inhibitors on the
cytotoxicity of CrTX It has been reported that LDH leakage not
only occurs during necrosis, but also during the process
of apoptosis[28,29]. Since 3-MA interferes with the MTT
assay, LDH leakage was measured as an index of cell death
after co-treatment with CrTX and the autophagic
inhibitors 3-MA (10 mmol/L) and NH4Cl (10 mmol/L). 3-MA, or
NH4Cl individually had no significant effect on LDH
leakage; however, pretreatment of MCF-7 cells with 3-MA
and NH4Cl significantly decreased the LDH leakage
induced by CrTX (Figure 7A).
As caspases other than caspase-3 may contribute to
apoptotic death in MCF-7 cells after CrTX treatment, the
effects of pretreatment with the pan-caspase inhibitor,
Z-Vad-fmk, were assessed. As shown in Figure 7B,
Z-Vad-fmk slightly, but significantly inhibited the cytotoxicity of
CrTX.
Discussion
CrTX is a potent antitumor agent and displays cytotoxic
activity against a variety of murine and human tumor cell
lines in vitro[15,16]. It
requires both the PLA2 activity of
subunit B[15] and the ability of the complex to dissociate into
their subunits[15,29].
CrTX-induced cytotoxic effects appear to be highly active in
cell lines expressing a high density of
EGFR[17], thus suggesting that
EGFR, or a receptor function, play a role in targeting.
Antitumor efficacy in vivo, using a
daily im administration of CrTX, has been demonstrated on
Lewis lung carcinoma (83% growth inhibition) and MX-1
human mammary carcinoma (69% growth
inhibition). A lower activity (44% growth inhibition) was observed with
HL-60 leukemia cells, suggesting that CrTX may have a
certain specificity toward solid
tumors[30].
Our previous studies have revealed that the mechanism
of the cytocidal action of CrTX may be more complex than
what has been previously thought. It has been demonstrated
that both autophagy and apoptosis were activated during
the CrTX-induced death of K562 leukemia cells, but
autophagy seemed to delay the onset of
apoptosis[18].
Autophagy is biochemically and morphologically distinct
from apoptosis, and is controlled by a different set of genes.
Malignant transformation is frequently associated with the
suppression of autophagy[31]. It has also been revealed that
the silencing of the pro-apoptotic gene BID in MCF-7 cells
led to the inhibition of apoptosis and a shift of cell death
towards autophagy[32]. The ability to induce autophagy in
cancer cells could be especially important in
apoptosis-resistant cell lines, where pro-apoptotic genes were mutated.
It is postulated that autophagic cell death induced by some
anticancer agents underlines their potential as a new cancer
therapy modality[31]. The present observations using
electron microscopy revealed many isolated membranes with
double or multi-membrane autophagosomes engulfing cytoplasma fractions and organelles 3 h after CrTX treatment.
Simultaneously, there was a notable increase in MDC-stained
vesicles following 3 h of CrTX treatment, which continued
even after 12 h of CrTX exposure. The blockage of
autophagy by the autophagic inhibitor, 3-MA, partially inhibited
CrTX-induced cell death. These studies suggest that
autophagy plays an important role in the CrTX-induced death
of MCF-7 cells.
Increasing evidence suggests that lysosomes are
important mediators of PCD. In autophagic cell death, lysosomes
fuse with autophagosomes to form autophagolysosomes in
which the cell contents are degraded[33]
. Cathepsins trigger apoptosis via a number of pathways including directly
activating caspases, and cleave
BID[34,35]. Recently, studies also
showed that if cathepsins were inhibited by a cathepsins
inhibitor E64d in MCF-7 cells treated with camptothecin
(CPT), CPT-induced BAX and BID aggregation on
mitochondria were prevented and apoptosis was significantly reduced.
This was accompanied by an increase in autophagosome
formation[32]. The present observation, using Western blot
analysis, showed a progressive increase in cathepsin B
activity 3 h after CrTX treatment, whereas cathepsin L
decreased initially, but markedly increased 12 h following
CrTX treatment. Pro-cathepsin D showed no change with
CrTX treatment, but processed cathepsin D (active
cathepsin D) was maintained at a very high level 6 h after CrTX
treatment. The activation of the lysosomal enzyme could be
blocked by NH4Cl. The effects of
NH4Cl further added evidence supporting an involvement of autophagy in the
antitumor action of CrTX.
Recently, several lines of evidence demonstrated that
the mitochondrion was the centre in cross-talk between
autophagy and caspase-independent
apoptosis[36]. The present results demonstrated that cyto-c release occurred
as early as 3 h after CrTX treatment. However, when the
treatment time prolonged to 6 h, the release of cyto-c
decreased suggesting that the effects of CrTX on
mitochondria were transient. MCF-7 cells are deficient in caspase-3
activity. Since caspase-3 is a major executioner caspase and
plays very important roles in apoptosis, it is speculated that
caspase-dependent apoptosis in MCF-7 cells would be
seriously hampered. Some studies showed that MCF-7 cells
underwent caspase-independent cell death. Autophagy thus
plays a role in MCF-7 death[32,37]. The present study found
that AIF was relocated from the mitochondrion into the
nuclei 1.5 h after CrTX treatment. AIF is a phylogenetically
conserved, mitochondrial intermembrane flavoprotein that
has the ability to induce apoptosis via a
caspase-independent pathway. AIF in the cytoplasm positively feedbacks to
mitochondria, leading to a drop in mitochondrial membrane
potential and the release of cyto-c[38,
39]. In the present study, the features of caspase-independent apoptosis including
nuclear envelope umbilication and chromatin compaction
were also observed after 6 h of CrTX treatment.
Pretreatment of MCF-7 cells with the pan-caspase inhibitor,
Z-Vad-fmk, slightly but significantly inhibited the cytotoxicity of
CrTX, suggesting that an apoptotic mechanism also
contributed to cell death induced by CrTX.
In summary, both autophagy and caspase-independent
apoptosis participated in cell death in the caspase-3-deficient
MCF-7 cells following treatment with CrTX. Autophagy (PCD
type II) may be the dominant mechanism of MCF-7 cell death
induced by CrTX. The precise mechanisms underlying the
different roles of autophagy in cell death remain to be studied.
It is important to investigate the interactions between the
autophagy and apoptotic signal pathways. Such studies
will help us understand the role of autophagy in cell death
and survival and provide new insights into antitumor
chemotherapy.
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