TOXICOLOGICAL SCIENCES 117(1), 109–121 (2010)
doi:10.1093/toxsci/kfq172
Advance Access publication June 13, 2010
Azaspiracid-1 Inhibits Endocytosis of Plasma Membrane Proteins
in Epithelial Cells
Mirella Bellocci, Gian Luca Sala, Federica Callegari,1 and Gian Paolo Rossini2
Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, I-41125 Modena, Italy
1
Present address: Dipartimento Integrato di Medicina, Endocrinologia, Metabolismo e Geriatria, Università di Modena e Reggio Emilia,
Via P. Giardini 1355, 41126 Modena, Italy.
2
To whom correspondence should be addressed at Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia,
Via G. Campi 287, I-41125 Modena, Italy. Fax: þ39-059-205-5410. E-mail: [email protected].
Received February 25, 2010; accepted June 4, 2010
The effect of azaspiracid-1 (AZA-1) on the plasma membrane
proteins E-cadherin, Na1/K1-ATPase, and prolactin receptor
(Rprl) has been investigated in MCF-7 cells. Cell treatment for
24 h with 1nM AZA-1 induced the accumulation of a proteolytic
fragment of E-cadherin and significant increases in the levels of
Na1/K1-ATPase and Rprl at the level of membranous structures.
The effect induced by AZA-1 was mimicked by latrunculin A,
suggesting that the toxin might act by blocking the endocytosis
of plasma membrane proteins. The exposure of intact cells to
a biotinylation reagent that does not permeate the plasma
membrane provided data showing that AZA-1 treatment of
MCF-7 cells doubled the levels of total protein located on the
cell surface. The exposure of intact cells to exogenous proteases
(trypsin and proteinase K) showed that AZA-1 treatment of MCF7 cells modifies the availability of the three membrane protein
markers to proteolytic attacks, providing evidence that significant
portions of the protein pools are located in structures that are not
exposed to the cell surface after the treatment with AZA-1.
Distinct subcellular locations of the membrane protein markers
in MCF-7 cells exposed to AZA-1 were confirmed by immunofluorescence microscopy. Direct evidence that AZA-1 inhibits
endocytosis was obtained by showing that AZA-1 blocked the
intracellular transfer of E-cadherin-bound antibody in MCF-7
cells. The effects of AZA-1 on the E-cadherin system were
confirmed in Caco-2 and Madin Darby canine kidney epithelial
cell lines. We conclude that AZA-1 inhibits endocytosis of plasma
membrane proteins in epithelial cells.
Key Words: azaspiracid; E-cadherin; Na 1/K 1-ATPase;
prolactin receptor; endocytosis; yessotoxin; cytoskeleton.
Azaspiracids (AZAs) consist of polyether compounds chemically characterized by the presence of a spiral ring assembly
containing a heterocyclic amine and an aliphatic carboxylic acid
moiety (reviewed in Twiner et al., 2008a). AZA-group toxins
are produced by the microalga Azadinium spinosum (Tillmann
et al., 2009), and their chemical synthesis has been achieved
(Nicolaou et al., 2003a,b).
Mollusks have been originally found to contain AZA-group
toxins (McMahon and Silke, 1996), but other shellfish species
can accumulate these toxins as a consequence of their feeding
behavior (Twiner et al., 2008a). The presence of AZAs in
shellfish poses significant risks to consumers, as documented
by episodes of human intoxication recorded in recent years
(European Food Safety Authority, 2008). The toxicity of AZAgroup toxins has been studied by investigations in animal
models. For instance, AZA-1, that represents the reference
compound of AZA-group toxins, has been shown to cause
mouse death at doses of 200 and 250–700 lg/kg body weight,
when administered by ip injection (Satake et al., 1998) and the
oral route (Ito et al., 2000, 2002, 2006), respectively. The
symptoms recorded in these animal studies have shown that
AZA-group toxins cause severe damage to the gastrointestinal
(GI) tract, liver, lung, and thymus in the mouse (Ito et al., 2000,
2002, 2006). Furthermore, toxicokinetic data have shown that
AZA-group toxins are absorbed in the GI tract; thus, toxin
concentrations in the 1–10nM range can be found systemically
(European Food Safety Authority, 2008).
The toxicity of AZA-group toxins has stimulated considerable
efforts to clarify their mechanism of action, but the molecular
component(s) selectively targeted by these algal biotoxins
remain(s) unknown. The extensive tissue damage found in
animals exposed to AZA-group toxins are matched by the potent
toxic effects AZA-1 exerts on cellular systems in vitro (Kulagina
et al., 2006; Ronzitti et al., 2007; Twiner et al., 2005; Vale et al.,
2007; Vilariño et al., 2006). Multiple effects have been found to
be induced by 1–10nM AZA-1 in cultured cells, including
alterations of F-actin-based cytoskeletal structures (Twiner et al.,
2005), the inhibition of bioelectrical activity of spinal cord
neurons (Kulagina et al., 2006), the accumulation of an
intermediate proteolytic fragment of E-cadherin in epithelial
cells (Ronzitti et al., 2007), and the accumulation of activated
c-Jun-NH2-terminal protein kinase and the alteration of ion
homeostasis in cerebellar granule cells (Vale et al., 2007, 2010).
Ó The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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AZA-1 could also modify metabolic activities in cultured cells,
as it has been shown by studying the transcription profiles in
human lymphocytes (Twiner et al., 2008b) and the proteome of
neuroblastoma cells (Kellman et al., 2009). In this latter case,
AZA-1 was found to upregulate proteins involved in energy
metabolism and the functioning of the cytoskeleton. Furthermore, AZA-1 downregulates proteins involved in several cellular
functions, including transcription, translation, and protein
modification (Kellman et al., 2009).
The finding that AZA-1 treatment of epithelial cells
determines the accumulation of a 100-kDa proteolytic fragment
of E-cadherin (termed E-cadherin-related antigen, ECRA100)
has attracted our interest because the effect induced by the toxin
is part of a cytotoxic response (Ronzitti et al., 2007). Moreover,
the molecular alteration of E-cadherin caused by AZA-1 was
undistinguishable from that caused by yessotoxin (Pierotti et al.,
2003; Ronzitti et al., 2004), a marine biotoxin chemically and
toxicologically distinct from AZA-group toxins (Rossini and
Hess, 2010). Subsequent investigations have shown that
yessotoxin does not induce the proteolysis of E-cadherin but
inhibits its endocytosis (Callegari and Rossini, 2008). The
blockade of endocytic process then prevents the complete
proteolysis of the protein, and the altered E-cadherin structures
are accumulated in intracellular compartments (Callegari and
Rossini, 2008; Ronzitti and Rossini, 2008). Based on these
findings, we reasoned that the accumulation of ECRA100 caused
by AZA-1 could have been because of the inhibition of
endocytosis and complete degradation of E-cadherin. We have
then hypothesized that AZA-1 can alter the endocytosis of
E-cadherin and other proteins located at the level of the plasma
membrane, thereby determining severe alterations of organ
functions. The toxicological relevance of this hypothesis stems
from both general and specific considerations. In general terms,
endocytosis is a basic biological process playing roles in a variety
of cellular functions, whose alteration leads to pathological states
(Scita and Di Fiore, 2010; Sorkin and von Zastrow, 2009). In
more specific terms, the impairment of the intracellular transport
of extracellular materials, as well as the malfunctioning of
plasma membrane proteins, could represent major contributions
to alterations AZA-1 causes in animal epithelia (Ito et al., 2000,
2002, 2006).
We then developed an investigation aimed at probing
whether AZA-1 can alter the cellular dynamics and endocytosis
of plasma membrane proteins in epithelial cells. This investigation was carried out in both normal and transformed
cells, using the MCF-7 breast cancer cell line as our reference
model. This choice was made on the basis of two major
considerations. On the one hand, MCF-7 cells are considered
an excellent system for the study of plasma membrane protein
functioning and dynamics in epithelial cells (see, e.g.,
Birchmeier and Behrens, 1994; Fu and Roufogalis, 2007;
Gumbiner, 2000). Furthermore, MCF-7 cells are targeted by
AZA-1 (Ronzitti et al., 2007). In this report, we present the data
from our study and show that AZA-1 alters the intracellular
dynamics and inhibits the endocytosis of plasma membrane
proteins, leading to their accumulation in multiple membranous
subcellular compartments in epithelial cells.
MATERIALS AND METHODS
Materials. AZA-1 was obtained from the National Research Council of
Canada (Canada). Stock AZA-1 solutions were stored in glass vials protected
from light at 20°C. The mouse monoclonal antibody recognizing Naþ/KþATPase (sc-48345) and the rabbit polyclonal antibody recognizing E-cadherin
(sc-7870) were from Santa Cruz Biotechnology. The monoclonal anti-actin
antibody was obtained from Chemicon International. The monoclonal antiE-cadherin antibodies were from Alexis (clone HECD-1) and Sigma (clone
DECMA-1). The mouse monoclonal antibody recognizing prolactin receptor
(Rprl; 35-9200), the 4#,6-diamidino-2-phenylindole dilactate (DAPI, dilactate)
stain, fluorescent secondary antibodies Alexa Fluor 568–conjugated anti-mouse
IgG and anti-rat IgG and Alexa Fluor 468–conjugated anti-rabbit IgG, and
ProLong Gold mounting media were from Invitrogen. Peroxidase-linked antirat and anti-mouse IgG antibodies and the enhanced chemiluminescence (ECL)
detection reagents were from GE Healthcare. Latrunculin A, chloroquine,
biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (sodium salt),
avidin-peroxidase, the soybean trypsin inhibitor, and P2714 protease inhibitor
cocktail were from Sigma. The trypsin-EDTA solution was obtained from
Worthington. Proteinase K from Tritirachium album was from Boehringer
Mannheim GmbH. PMSF was purchased from Bio-Rad. The nitrocellulose
membrane Protran BA 83 was obtained from Schleicher & Schuell. All other
reagents were from Sigma.
Cell culture conditions and toxin treatments. MCF-7 cells were obtained
from the European Collection of Animal Cell Cultures (ECACC No. 86012803,
CB No. 2705) and were grown in 5% carbon dioxide in air at 37°C, in 90-mmdiameter Petri dishes, with a culture medium composed of Dulbecco’s modified
Eagle’s medium, containing 1% nonessential amino acids and 10% fetal calf
serum, as previously described (Ronzitti et al., 2007). Caco-2 cells were
obtained from the American Type Culture Collection (ATCC No. HTB-37),
and their culture medium was composed of minimum essential medium with
Earle’s balanced salt solution, containing 2mM glutamine, 1mM sodium
pyruvate, 1% nonessential amino acids, and 20% fetal calf serum. Madin Darby
canine kidney (MDCK) cells were obtained from the American Type Culture
Collection (ATCC No. CCL-34), and their culture medium was composed of
minimum essential medium with Earle’s salts, containing 2mM glutamine,
1mM sodium pyruvate, 1% nonessential amino acids, and 10% fetal calf serum.
If not stated otherwise, cells in logarithmic growth received either 1nM
AZA-1 or vehicle (control cells) and were then incubated for 24 h at 37°C.
Preparation of cell extracts. If not stated otherwise, at the end of
incubations, cells were harvested and processed to obtain extracts by procedures
that were performed at 4°C. Cells from every dish were washed with 5 ml of
20mM phosphate buffer, pH 7.4, 0.15M NaCl (PBS buffer). Cells were then
mechanically detached from culture plates with a scraper and were washed three
times by resuspension in 5 ml of PBS and centrifugation for 10 min at 800 3 g.
Cells were then lysed by resuspension in 125 ll per dish of PBS containing 1%
(vol/vol) Triton X-100 and 0.1 mg/ml PMSF. The presence of a mild detergent,
such as Triton X-100, in the lysis buffer was needed to solubilize the membrane
proteins studied in this report. After 20 min at 4°C the material was collected and
centrifuged for 30 min at 16,000 3 g. The supernatant of this centrifugation was
a cytosoluble extract that was saved; its protein content was determined with
bicinchoninic acid (Smith et al., 1985) and was then used for protein separation by
SDS polyacrylamide gel electrophoresis (PAGE).
Biotinylation of cell surface proteins. At the end of indicated treatments,
cells were harvested, resuspended in 150 ll per dish of PBS, and treated with
2.5 mg/ml final concentration of the biotinylation reagent (biotinamidohexanoic
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AZASPIRACID-1 INHIBITS ENDOCYTOSIS
acid 3-sulfo-N-hydroxysuccinimide ester, sodium salt). After 30 min of
incubation in gentle shaking at 4°C, the reaction was stopped by cell treatment
with 40mM lysine for 30 min at 4°C. Cells were washed three times by
resuspension in 1 ml of PBS and centrifugation for 10 min at 800 3 g, before
being processed to obtain cytosoluble extracts, as described previously.
Proteinase digestion of cell surface proteins. MCF-7 cells were subjected
to proteinase treatment at the end of their exposure to 1nM AZA-1. At the end
of the incubations, MCF-7 cells were washed with 5 ml PBS, harvested by
scraping, and transferred to centrifuge tubes. Cell suspensions were centrifuged
for 8 min at 800 3 g, dispersed in 2 ml PBS, subdivided into two equal
portions, and recovered by low-speed centrifugation. The first aliquot of each
sample was dispersed with 1 ml of PBS containing 1mM EDTA (PBS-EDTA),
whereas the second one received 1 ml of PBS-EDTA containing 0.1 mg/ml
trypsin. The samples were then incubated for 30 min at 4°C on an orbital shaker.
The treatment was then terminated by adding 0.5 mg/ml of soybean trypsin
inhibitor to PBS-EDTA. Cells were then centrifuged for 8 min at 2500 3 g,
washed with 1 ml PBS, and recovered by low-speed centrifugation, before being
processed to obtain cytosoluble extracts, using 83 ll per dish of lysis buffer
containing 1:20 P2714 protease inhibitor cocktail, as described previously.
The same protocol was used for the treatment of MCF-7 cells with
proteinase K. In this case, the final concentration of the enzyme used for cell
treatments was 7.5 lg/ml, and 0.9 mg/ml PMSF was used to block the protease
activity at the end of the incubation.
Fractionation of proteins by SDS-PAGE and immunoblotting. Samples
containing the same amount of protein were fractionated according to Laemmli
(1970), using a 10% separating gel and a 4% stacking gel. At the end of the
electrophoresis, the proteins in the gels were electrophoretically transferred
onto a nitrocellulose membrane (Protran BA 83), and the binding sites
remaining on the membrane were blocked by incubation of blots for 1 h at
room temperature with a solution composed of 20mM Tris-HCl, pH 7.5 at
25°C, 0.15M NaCl, and 0.05% (vol/vol) Tween 20 (immunoblotting buffer),
containing 3% nonfat dry milk. When immunoblotting was used to detect
E-cadherin, the immunoblotting buffers used for blocking unspecific sites on
the membrane and for the incubation with the primary antibody (mouse
monoclonal HECD-1 and rat monoclonal DECMA-1) did not include Tween
20 but contained 1mM CaCl2. After blocking the unspecific sites, the
membranes were incubated for 1 h at room temperature with immunoblotting
buffer, containing 1% nonfat dry milk and the primary antibody at a final
concentration ranging between 0.1 and 2 lg/ml, depending on the antigen to be
detected and according to the information sheet of the respective antibody.
After incubation, membranes were washed five times with immunoblotting
buffer and incubated for 1 h at room temperature with a peroxidase-linked
secondary antibody at a 1:3000 to 5000 dilution, depending on the antigen to be
detected in immunoblotting buffer containing 1% nonfat dry milk. After
washing, the membranes were processed using the ECL detection system, and
results were visualized by autoradiography.
In the case of biotinylated samples, membranes were blocked for 1 h at room
temperature using a 5% bovine serum albumin (BSA) solution in PBS and were
then incubated for 1 h at room temperature with blotting buffer containing 1%
nonfat dry milk and 1:50000 peroxidase-linked avidin. After washing,
membranes were processed using the ECL detection system, as described
previously.
Immunofluorescence microscopy. When immunofluorescence microscopy was used to characterize our experimental system, cells were grown in
35-mm Petri dishes containing one coverslip glass in each dish. Cells in
logarithmic growth were treated for 24 h with indicated AZA-1 concentrations
or vehicle as described previously. Cells were then fixed with 4% (wt/vol)
paraformaldehyde in PBS for 15 min at room temperature. After fixation, cells
were permeabilized by treatment with PBS buffer containing 3% (wt/vol) BSA
and 0.02% Triton X-100, for 1 h at room temperature. Samples were then
incubated overnight at 4°C with the primary antibodies used in immunoblotting
analyses, which were diluted in PBS containing 1% BSA, at the final
concentration indicated on the information sheet of the respective antibody. In
the case of double labeling, cells were incubated with a PBS solution
containing the rabbit polyclonal anti-E-cadherin and the mouse monoclonal
anti-Naþ/Kþ-ATPase antibodies. The incubations with fluorescent secondary
antibodies were performed for 45 min at room temperature in the dark, using
antibodies at a final 1:1000 dilution. In the case of double labeling, a mix of
anti-mouse and anti-rabbit fluorescent secondary antibodies, each of which at
a 1:1000 dilution, was used in our procedure. Labeling of nuclei was obtained
by cell incubation for 5 min with a 0.14 lg/ml DAPI solution in PBS. Cells
were then mounted on microscopic slides using ProLong Gold (Invitrogen)
mounting media and evaluated under Zeiss Axioskop-40 microscope. Images
were acquired by the AxioCam HRc camera (Carl Zeiss), using the Axiovision
3.1 software (Carl Zeiss).
Internalization assay. The procedure used was a slight modification of an
already published method (Paterson et al., 2003). MCF-7 cells cultured in the
presence of a coverslip received an equal volume of absolute ethanol or AZA-1
and were incubated for 24 h at 37°C. Cells were then exposed to the HECD-1
anti-E-cadherin antibody (4 lg/ml) and were subsequently incubated for 1 h at
4°C. At the end of this treatment, cells were washed with ice-cold PBS and
a second time either with an acidic solution (0.5M acetic acid, 0.5M sodium
chloride), which removed the antibody bound to E-cadherin on the cell surface,
or with PBS. Cells that had been washed with PBS were then incubated for 1 h
at 37°C with culture media. At the end of the incubation, coverslips containing
the cells that had been exposed to the acidic solution were washed with PBS and
those that had been washed with PBS were then exposed to the acidic solution.
Cells were then fixed with paraformaldehyde as described previously. The basal
state of E-cadherin at the surface of both control and AZA-1-treated cells was
ascertained by cell processing with paraformaldehyde directly after the
exposure to the anti-E-cadherin antibody and the first PBS wash. Immunofluorescence microscopy was carried out as described previously, without any
further exposure of cell samples to paraformaldehyde.
Statistical analysis. The Student’s t-test was used to evaluate the significance of experimental data.
Results
Effect of AZA-1, Latrunculin, and Chloroquine on the Levels
of Selected Plasma Membrane Proteins Solubilized from
MCF-7 Cells
Many proteins are located in plasma membranes at the
surface of the cells, comprising hormone and neurotransmitter
receptors, ion channels and pumps, transporters, adhesion
proteins, and so forth, playing key roles in cellular functioning
and homeostasis. Quantitative and/or qualitative alterations of
the protein pool on plasma membranes would then determine
profound impairing of cellular functioning. The working
hypotheses of this study were that the effect exerted by
AZA-1 on E-cadherin consisted in the inhibition of endocytosis
and the alteration of this cellular process could involve other
plasma membrane proteins. We then studied the AZA-1 effects
on cellular dynamics of plasma membrane proteins, by
analyzing components involved in three distinct cellular
functions. E-cadherin, which is responsible for cell-cell
adhesion in epithelia (Nollet et al., 2000), was the first plasma
membrane protein and represented the reference molecular
marker of the toxin’s effect in our experimental system
(Ronzitti et al., 2007). The Naþ/Kþ-ATPase, representing
a ubiquitous protein responsible for ion movement across the
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cell membrane (Kühlbrandt, 2004), was a second potential
target of toxin action. Rprl was the third plasma membrane
protein analyzed in this study, and it was chosen because it is
a transducer of an extracellular hormonal signal into an
intracellular response (Clevenger et al., 2009).
The experimental conditions chosen for our initial experiments involved the treatment of MCF-7 cells with 1nM AZA-1
for 24 h. Under these conditions, no major alterations of cell
morphology could be detected by microscopic examination of
MCF-7 cells (see Supplementary fig. 1). The choice to use
a concentration of 1nM was made on the basis of the doseresponse study we originally carried out (Ronzitti et al., 2007),
showing that maximal accumulation of ECRA100 was detected
after 24-h exposure of MCF-7 cells to toxin concentrations in
the 1–10nM range. This concentration range and a time frame
of 24 h for AZA-1 exposures are significant with regard to both
the toxicokinetics of this compound and the chance of human
exposure to materials contaminated with AZA-group toxins
(European Food Safety Authority, 2008).
When we analyzed E-cadherin in cell extracts obtained from
AZA-1-treated cells, we confirmed our original observations
(Ronzitti et al., 2007) that ECRA100 is not detected in extracts from
control cells but represents a conspicuous band in samples
prepared from MCF-7 cells exposed to this toxin (Fig. 1). The
increase in total immunoreactivity detected by our antiE-cadherin antibody (intact E-cadherin þ ECRA100) was
about fourfold (4.10 ± 1.95, n ¼ 8; p ¼ 0.0005). Similarly,
increased levels of Naþ/Kþ-ATPase and Rprl were found in
extracts obtained from AZA-1-treated cells, as compared with
controls. In these cases, the extracts from cells exposed to the
toxin contained Rprl at levels that were about threefold higher
(2.82 ± 1.86, n ¼ 8; p ¼ 0.015; Fig. 1) than those detected in
samples from control cells. A smaller effect was exerted by
AZA-1 on Naþ/Kþ-ATPase, whose levels were increased by
about 50% (1.43 ± 0.37, n ¼ 8; p ¼ 0.0055; Fig. 1) in the
samples obtained in these experiments. No significant change,
in turn, could be detected in the levels of actin, comparing
samples from control and toxin-treated cells (Fig. 1). Some
variability was observed in the intensity of responses in
different experiments (Supplementary fig. 2 and table 1), but
the statistical analysis of our results showed that AZA-1
treatment of MCF-7 cells causes significant increases in the
levels of E-cadherin, Naþ/Kþ-ATPase, and Rprl immunoreactivity in cellular membranous structures.
In a previous study, we showed that ECRA100 represents an
intermediate proteolytic fragment of E-cadherin, which is
accumulated in MCF-7 cells as a consequence of impairment
of E-cadherin endocytosis and complete protein disposal
(Callegari and Rossini, 2008). In that study, the accumulation
of ECRA100 could be induced by several agents known to
inhibit endocytosis and lysosomal protein degradation. We then
repeated those experiments by analyzing the effect of latrunculin
A and chloroquine on the levels of E-cadherin, Naþ/Kþ-ATPase,
and Rprl immunoreactivity, under our experimental conditions
(Fig. 1; Supplementary figs. 3 and 4, and tables 2 and 3). When
cells were exposed to latrunculin A, we confirmed that inhibition
of actin-based endocytosis (Ayscough, 2005; Lamaze et al.,
1997) results in the accumulation of ECRA100 in MCF-7 cells
(Callegari and Rossini, 2008). Increased levels of Naþ/KþATPase and Rprl immunoreactivity were also detected in extracts
FIG. 1. Effect of AZA-1, latrunculin A, and chloroquine on the levels of selected plasma membrane proteins in MCF-7 cells. Cells were incubated with 1nM
AZA-1, 2.4lM latrunculin A, 0.1mM chloroquine or vehicle for 24 h at 37°C. At the end of the incubation, cells were processed to prepare cytosoluble extracts,
which were subjected to SDS-PAGE and immunoblotting, using the antibodies for the indicated proteins. The detection of actin has been included as a loading
control for our procedure. The electrophoretic mobilities of b-galactosidase (116 kDa), lactoferrin (90 kDa), and lactate dehydrogenase (36.5 kDa) subunits, used as
marker proteins running in a parallel lane, are indicated on the left.
AZASPIRACID-1 INHIBITS ENDOCYTOSIS
113
obtained from cells exposed to latrunculin A (Fig. 1). Similar
findings were obtained when lysosomal protein degradation was
inhibited by MCF-7 cell treatment with chloroquine (Fig. 1). The
observation that inhibitors of endocytosis and lysosomal protein
degradation caused an effect similar to that induced by AZA-1
provided a first indication that the cell exposure to this toxin
could alter the intracellular trafficking and turnover of the
selected plasma membrane proteins in our model system.
Effect of AZA-1 on the Levels of Total Protein in the Plasma
Membrane of MCF-7 Cells
Taking into consideration that the biomarkers of AZA-1
effect we have chosen play different cellular functions, our
initial findings led us to check whether the toxin could induce
a generalized accumulation of proteins on plasma membrane. To
probe this hypothesis, we exposed control and AZA-1-treated
MCF-7 cells to a plasma membrane–impermeant biotinylation
reagent (Elia, 2008), leading to tagging of proteins that are
exposed on the cell surface with biotin. The levels of biotinylated
proteins in extracts from control and AZA-1-treated MCF-7 cells
were then analyzed by subjecting the cytosoluble extracts to
SDS-PAGE, the transfer of proteins on a nitrocellulose membrane, and the detection of biotinylated proteins by horseradish
peroxidase–conjugated avidin (see the ‘‘Materials and Methods’’
section). The inspection of autoradiographs showed that AZA-1
treatment of MCF-7 cells caused an increase in the levels of total
protein located on the cell membrane and available to
a biotinylation reagent that does not permeate the plasma
membrane itself (Fig. 2, top panel; Supplementary fig. 5). Under
our experimental conditions, AZA-1 treatment of MCF-7 cells
caused an almost doubling (1.70 ± 0.48; p ¼ 0.012) of the
levels of biotinylated proteins in the five separate experiments we performed, as compared with controls (Supplementary table 4). The levels of the selected biomarkers of AZA-1
effects (E-cadherin, Naþ/Kþ-ATPase, and Rprl) were also
evaluated by paired immunoblotting analyses, and our findings
confirmed that the toxin caused an increase in the levels of
those protein markers in membranous structures of MCF-7 cells
(Fig. 2, bottom panel).
Effect of AZA-1 on Sensitivity of Plasma Membrane Proteins
to Exogenous Proteases in Intact MCF-7 Cells
The experiments involving the biotinylation of proteins on
the surface of MCF-7 cells showed that AZA-1 causes
a generalized accumulation of components on plasma membrane. We then examined whether the effect of the toxin is
confined to proteins located on the cell surface, or if it also
includes some sequestration of a portion of that protein pool in
intracellular membranous structures. To distinguish between
the two possibilities, we obtained cell suspensions and treated
them with proteolytic enzymes, which would degrade components exposed to the surface of the cell but should not attack
those located inside the cell (see the ‘‘Materials and Methods’’
FIG. 2. Effect of AZA-1 on the levels of biotinylated proteins in the plasma
membrane of MCF-7 cells. Cells were incubated with either 1nM AZA-1 or
vehicle for 24 h at 37°C. At the end of the incubation, cells were harvested and
were subjected to biotinylation, as described in the ‘‘Materials and Methods’’
section. Cells were then processed to prepare cytosoluble extracts, which were
subjected to SDS-PAGE. Biotinylated proteins were detected by immunoblotting, using peroxidase-linked avidin (top panel), and selected antigens were
detected by immunoblotting using the antibodies for the indicated proteins.
The detection of actin has been included as a loading control for our procedure.
The electrophoretic mobilities of a2-macroglobulin (180 kDa), b-galactosidase
(116 kDa), lactoferrin (90 kDa), pyruvate kinase (58 kDa), fumarase (48.5 kDa),
lactate dehydrogenase (36.5 kDa), and triosephosphate isomerase (26.6 kDa)
subunits, used as marker proteins running in a parallel lane, are indicated on the left.
section for the detailed procedure). We then analyzed the state
of proteins solubilized from cell membranes of cells exposed to
proteases, by subjecting extracts to immunoblotting.
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A first series of experiments was carried out by treating
control and AZA-1-treated cells with trypsin. When proteins in
cell extracts were subjected to immunoblotting, the analysis
with anti-E-cadherin antibody showed that trypsin treatment
of cell suspensions strongly decreased the levels of intact
E-cadherin in both control and AZA-1-treated cells (Fig. 3).
ECRA100, in turn, was detected at high levels only in extracts
from cells exposed to AZA-1, as expected, and its cellular
levels were not affected by trypsin treatment of intact cells
(Fig. 3). Considering the fact that ECRA100 represents the
extracellular domain of E-cadherin (Ronzitti et al., 2004),
whose sensitivity to trypsin treatment is established (see
above), the trypsin resistance of ECRA100 in intact cells may
not be because of a lack of the amino acid sequences attacked
by this protease. These results, therefore, showed that most of
the intact E-cadherin is exposed at the cell surface, whereas
most of its proteolytic fragment ECRA100 is not exposed at the
cell surface and is not accessible to the exogenous proteolytic
enzyme. If the results obtained by immunoblotting analysis
with anti-Naþ/Kþ-ATPase and anti-Rprl antibodies are considered, the quantitative increases of respective antigens caused by
AZA-1 treatment in MCF-7 cells are confirmed. With regard to
the trypsin sensitivity of the two proteins, in turn, our results
showed that most of Naþ/Kþ-ATPase is trypsin resistant in
FIG. 3. Effect of trypsin treatment of intact cells on selected plasma
membrane proteins in MCF-7 cells exposed to AZA-1. Cells were treated with
either 1nM AZA-1 or vehicle for 24 h at 37°C. At the end of treatments, cells
were incubated with (þ) or without () 100 lg/ml trypsin, for 30 min at 4°C,
as described in the ‘‘Materials and Methods’’ section. Proteolysis was
terminated by the addition of 0.5 mg/ml of soybean trypsin inhibitor and the
cells were processed to obtain cytosoluble extracts, which were subjected to
SDS-PAGE and immunoblotting, using the antibodies for the indicated
proteins. The detection of actin has been included as a loading control for
our procedure. The electrophoretic mobilities of b-galactosidase (116 kDa),
lactoferrin (90 kDa), and lactate dehydrogenase (36.5 kDa) subunits, used as
marker proteins running in a parallel lane, are indicated on the right.
both control and AZA-1-treated cells, whereas Rprl was trypsin
sensitive in control but not in AZA-1-treated MCF-7 cells
(Fig. 3).
Although the trypsin sensitivity of our antigens provides
a direct proof that the protease treatment we used targeted
proteins located at the cell surface, the detection of trypsin
resistance would be compatible with two possible interpretations. On the one hand, membrane proteins might not have been
degraded by the protease added to the buffer of cell suspensions
because of an intracellular location of the membrane structures
containing these proteins, making them unavailable to the
proteolytic attack. A possible alternative, however, is that the
structural arrangement of intact proteins within the plasma
membrane at the surface of the cell could keep the amino acid
sequences attacked by the enzyme inaccessible to the protease.
This condition has been described in many systems, when the
sensitivity of plasma membrane proteins to proteases has been
shown to depend on structural changes induced by exogenous as
well as endogenous factors (see, e.g., Hoe and Rebeck, 2005;
Hyafil et al., 1981; van Tetering et al., 2009).
To identify the conditions that could apply to the selected
antigens in MCF-7 cells, we modified our experimental
conditions and treated intact cells with proteinase K, which
catalyzes the proteolytic attack at sites of the amino acid chains
differing from those processed by trypsin (Ebeling et al.,
1974). We then found that virtually the entire pools of intact
E-cadherin and Naþ/Kþ-ATPase are sensitive to proteinase K
in both control and AZA-1-treated MCF-7 cells, showing that
these proteins are on the surface of the cell and are available to
proteolytic attack, independent of the cell exposure to the toxin
(Fig. 4). ECRA100, in turn, was mostly resistant to proteinase
K, confirming that this proteolytic fragment of E-cadherin is
not accessible to proteases added to the exterior of the cell
membrane in AZA-1-treated cells (Fig. 4). When we analyzed
Rprl in extracts from cells that had been exposed to proteinase
K, we found that most of this protein is proteinase K sensitive
in control but not in AZA-1-treated cells (Fig. 4), confirming
the observations made with trypsin (Fig. 3). The results we had
obtained with extracts from MCF-7 cells that were exposed to
proteinase K, therefore, indicated that most of Rprl is not
accessible to proteolytic attack in cells that have been exposed
to AZA-1.
Effect of AZA-1 on Subcellular Location of Plasma
Membrane Proteins in MCF-7 Cells
The treatment of intact cells with exogenous proteases
showed that the proteins selected for our analyses display
component-specific alterations, suggesting different subcellular
locations and/or ultrastructural arrangements of the amino acid
chains in the plasma membrane of cells treated with AZA-1. To
probe the subcellular location of selected plasma membrane
proteins in MCF-7 cells after exposure to AZA-1, we used
immunofluorescence microscopy. Cells were then incubated
with specific antibodies, whose location was visualized using
AZASPIRACID-1 INHIBITS ENDOCYTOSIS
FIG. 4. Effect of proteinase K treatment of intact cells on selected plasma
membrane proteins in MCF-7 cells exposed to AZA-1. Cells were treated with
either 1nM AZA-1 or vehicle for 24 h at 37°C. At the end of treatments, cells
were incubated with (þ) or without () 7.5 lg/ml proteinase K, for 30 min at
4°C, as described in the ‘‘Material and Methods’’ section. Proteolysis was
terminated by the addition of 0.9 mg/ml PMSF and the cells were processed to
obtain cytosoluble extracts, which were subjected to SDS-PAGE and
immunoblotting, using the antibodies for the indicated proteins. The detection
of actin has been included as a loading control for our procedure. The
electrophoretic mobilities of b-galactosidase (116 kDa), lactoferrin (90 kDa),
pyruvate kinase (58 kDa), fumarase (48.5 kDa), and lactate dehydrogenase
(36.5 kDa) subunits, used as marker proteins running in a parallel lane, are
indicated on the right.
secondary antibodies tagged with a fluorescent moiety. The
merging of the DAPI nuclear staining and the red emission of
fluorescent secondary antibodies were then used to detect
individual antigens in fields representative of MCF-7 cells,
under our experimental conditions. In control cells, most
E-cadherin colocalized with the plasma membrane along cellcell contacts (Fig. 5A; Supplementary fig. 6), as expected for
this cell-cell adhesion protein (Hirano et al, 1987; Nollet et al.,
2000; Paterson et al., 2003; Vestweber and Kemler, 1985). A
diffuse distribution, in turn, was observed for Naþ/Kþ-ATPase,
Rprl, and actin (panels C, E, and G; Supplementary figs. 7–9).
The treatment of MCF-7 cells with AZA-1 caused an extensive
change in the subcellular distribution of proteins immunoreactive
to the anti-E-cadherin and anti-Naþ/Kþ-ATPase antibodies. In
both cases, the immunofluorescence was found mostly concen-
115
FIG. 5. Effect of AZA-1 on the cellular distribution of selected proteins
detected by immunofluorescent staining of MCF-7 cells. Cells were incubated
with either 1nM AZA-1 or vehicle for 24 h at 37°C, as indicated. At the end of
the incubation, cells were processed for analysis by immunofluorescence
microscopy, as described in the ‘‘Materials and Methods’’ section. Micrographs
show the merging of the nuclear DAPI staining with the emissions of fluorescent
secondary antibodies in cells that had been previously exposed to primary
antibodies recognizing E-cadherin (panels A and B), Naþ/Kþ-ATPase (panels C
and D), Rprl (panels E and F), and actin (panels G and H), as indicated. The scale
bar indicated in panel A corresponds to 20 lm and applies to all panels.
trated in some areas of the cell (Figs. 5B and 5D), and the antigen
distributions observed in control cells were lost. The immunostaining detected with anti-Rprl and anti-actin antibodies, in turn,
was not extensively changed in cells treated with the toxin, and
a homogeneous distribution of immunoreactivity was observed
in both control (Figs. 5E and 5G) and AZA-1-treated (Figs. 5F
and 5H) MCF-7 cells.
The immunofluorescence detected with anti-E-cadherin and
anti-Naþ/Kþ-ATPase antibodies within a portion of the cell in
AZA-1-treated cells showed patterns that were not apparently
consistent with an identical subcellular location of those proteins
(Figs. 5B and 5D). We then checked their possible colocalization
in AZA-1-treated MCF-7 cells, by incubating our samples with
both anti-E-cadherin and anti-Naþ/Kþ-ATPase antibodies, and
double labeling the cells using Alexa Fluor 568–conjugated antimouse and Alexa Fluor 468–conjugated anti-rabbit IgG
secondary antibodies (see the ‘‘Materials and Methods’’ section).
These experiments (Fig. 6) confirmed the changes in the
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BELLOCCI ET AL.
subcellular distribution of immunoreactivity induced by AZA-1
in MCF-7 cells. Furthermore, the merging of the immunostaining
of the two secondary antibodies showed that the subcellular
distribution of the immunoreactivity to anti-E-cadherin and antiNaþ, Kþ-ATPase antibodies did not coincide in both control and
AZA-1-treated MCF-7 cells. In control cells, the distribution
of E-cadherin was mostly at the cell-cell contacts, whereas that
of Naþ/Kþ-ATPase was homogeneous (Fig. 6), as already
observed (Figs. 5A and 5C). The two antibodies, in turn, were
mostly localized in different areas of the toxin-treated MCF-7
cells, as indicated by very limited yellow staining (Fig. 6).
Overall, the immunostaining experiments showed that AZA-1
differently affects the subcellular location of the three selected
biomarker proteins, and individual patterns of subcellular
distribution are observed for each component in toxin-treated
MCF-7 cells.
Effect of AZA-1 on Cellular Internalization of E-cadherin in
MCF-7 Cells
The accumulation of E-cadherin immunoreactivity in granular
structures in MCF-7 cells exposed to AZA-1 is compatible with
two distinct subcellular localization. On the one hand, granular
structures might represent subdomains of plasma membrane
concentrating E-cadherin at the surface of the cells. On the other
hand, granular structures might be located inside the cells, where
the accumulation of E-cadherin immunoreactivity would result
from inhibition of endocytosis and, hence, the blockade of
lysosomal protein disposal. To distinguish between these two
possibilities, we analyzed the E-cadherin immunoreactivity
existing on the surface of MCF-7 cells. Control and AZA-1treated cells were then exposed to the HECD-1 anti-E-cadherin
antibody, which binds to the extracellular domain of the protein
(Shimoyama et al., 1989). Cells were not subjected to any
permeabilization step before the incubation with the antibody, to
restrict its binding to antigens exposing their epitopes at the cell
surface, and the treatment was carried out at low temperature, to
prevent any intracellular transfer of material. Under these
conditions, the HECD-1 anti-E-cadherin antibody was distributed at the surface of MCF-7 cells (Fig. 7, panels A and B). A
clear difference was detected between control and AZA-1treated cells, inasmuch as strong immunostaining was found in
control cells, particularly at cell-cell contacts, as already
observed (Figs. 5 and 6), whereas a more limited staining was
detected in AZA-1-treated cells, indicating a decrease in the
levels of E-cadherin accessible to the HECD-1 antibody on the
surface of MCF-7 cells exposed to the toxin. The location of Ecadherin immunoreactivity at the surface of MCF-7 cells was
ascertained by cell washing with an acidic hypertonic solution,
which removes the antibody bound to its antigen (Paterson et al.,
2003). When cells that had been exposed to the HECD-1
antibody at low temperature were subjected to the acid wash (see
the ‘‘Materials and Methods’’ section), no E-cadherin immunoreactivity could be detected (Fig. 7, panels C and D), confirming
that the antigen detected under these experimental conditions
(panels A and B) is actually located at the cell surface.
Furthermore, no accumulation of E-cadherin immunoreactivity
could be detected in granular structures at the surface of AZA-1treated MCF-7 cells (Fig. 7, panel B). Therefore, when cells were
exposed to anti-E-cadherin antibody after fixation and permeabilization (Figs. 5 and 6), the detected immunostaining was
bound to materials located in intracellular structures.
We next analyzed the endocytosis of E-cadherin located at
the surface of MCF-7 cells by paired cell cultures, which were
exposed to HECD-1 anti-E-cadherin antibody, washed with
FIG. 6. Analysis of colocalization of E-cadherin and Naþ/Kþ-ATPase in MCF-7 cells. Cells were incubated with either 1nM AZA-1 or vehicle for 24 h at
37°C, as indicated. At the end of the incubation, cells were processed for analysis by immunofluorescence microscopy, as described in the ‘‘Materials and
Methods’’ section. Micrographs show the nuclear DAPI staining, the emissions of fluorescent secondary antibodies bound to either the rabbit polyclonal antiE-cadherin or the mouse monoclonal anti-Naþ/Kþ-ATPase antibodies, and the merging of the immunostaining of the two secondary antibodies, as indicated. The
scale bar indicated in the top left panel corresponds to 20 lm and applies to all panels.
117
AZASPIRACID-1 INHIBITS ENDOCYTOSIS
FIG. 7. Effect of AZA-1 on cellular internalization of E-cadherin in MCF-7 cells. Cells were incubated with either 5nM AZA-1 or vehicle for 24 h at 37°C, as
indicated. At the end of the incubation, cells were processed for analysis of cellular internalization of E-cadherin by immunofluorescence microscopy, as described
in the ‘‘Materials and Methods’’ section. Micrographs show the merging of the nuclear DAPI staining with the emissions of fluorescent secondary antibody in cells
that had been previously exposed to primary antibody recognizing E-cadherin. The basal state of E-cadherin at the surface of control and AZA-1-treated cells is
reported in panels A and B, respectively. The subcellular distribution of immunostaining was also ascertained in cells that had been exposed to an acid wash after
exposure to the anti-E-cadherin antibody (panels C and D), and in cells that were exposed to the anti-E-cadherin antibody and were next incubated for 1 h at 37°C
before being subjected to acid wash (panels E and F). The scale bar indicated in panel A corresponds to 10 lm and applies to all panels.
PBS to remove excess antibody, and further incubated for 1 h
at 37°C. The exposure of cells to physiological temperature
allows the endocytosis of E-cadherin and of the antibody
bound to it under these experimental conditions (Paterson
et al., 2003). At the end of the incubation at 37°C, MCF-7 cells
were subjected to the acid wash, to remove the antibody that
might have remained associated with plasma membrane
structures, and were next processed to detect the E-cadherin
immunoreactivity that had been internalized (see the ‘‘Materials
and Methods’’ section). Microscopic examination showed that
the immunostaining was associated with localized structures
in control cells (Fig. 7, panel E), but no signal was found in
those treated with the toxin (Fig. 7, panel F). These experiments, therefore, provided direct proof that the endocytosis
of E-cadherin at the surface of MCF-7 cells is blocked in
AZA-1-treated cells.
Effect of AZA-1 on E-cadherin in Epithelial Cells
The potential of AZA-1 to alter the cellular dynamics of
plasma membrane proteins in epithelial cells other than the
MCF-7 breast cancer cell line was next examined, by monitoring
the effect of the toxin on the E-cadherin system. The experiments were carried out in Caco-2 and MDCK cells, which
represent a transformed intestinal cell line of human origin
(Fogh et al., 1977) and a normal cell line isolated from dog
kidney (Gaush et al., 1966), respectively. We then found that
cell treatment with AZA-1 concentrations in the 1–10nM range
caused the accumulation of ECRA100 and an overall increase in
immunoreactivity detected by anti-E-cadherin antibodies in the
three epithelial cell lines (Fig. 8, left panel). Interestingly, the
increase in E-cadherin immunoreactivity we detected in extracts
from MDCK cells treated with AZA-1 comprised the 135-kDa
immature precursor of the protein (Shore and Nelson, 1991), in
addition to intact E-cadherin and its proteolytic fragment
ECRA100 (Fig. 8, left panel). The subcellular distribution of
immunoreactivity detected by anti-E-cadherin antibodies was
next analyzed in the three cell lines (Fig. 8, right panel), and we
found that AZA-1 treatment caused the subcellular redistribution of this plasma membrane protein. Under our experimental
conditions, most of E-cadherin immunoreactivity in AZA1-treated cells did not colocalize with cell-cell contacts but
was detected as strong immunostained granular structures that
were accumulated far from the cell contours in the three
epithelial cell lines we examined (Fig. 8, right panel).
DISCUSSION
In this study, we found that the treatment of MCF-7 cells
with AZA-1 determines a net increase in the content of proteins
located on plasma membrane, including the three components
that play distinct functional roles in the cell, consisting of
E-cadherin, Naþ/Kþ-ATPase, and Rprl (Figs. 1 and 2). Based
on the results obtained in this and other previous studies by our
group (Callegari and Rossini, 2008; Ronzitti et al., 2007), we
conclude that AZA-1 inhibits endocytosis in epithelial cells and
that this effect contributes to the accumulation of proteins in
cell membranes of sensitive cells. Several lines of evidence
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BELLOCCI ET AL.
FIG. 8. Effect of AZA-1 on the E-cadherin system in different epithelial cells. Culture dishes of MCF-7, Caco-2, and MDCK cells were incubated with AZA-1
at either the indicated concentrations (left panel) or at a 5nM concentration (right panel) for 24 h at 37°C. The experimental conditions for the data shown in the left
panel are as described in Figure 1 and those for the data shown in the right panel are as described in Figure 5. The scale bar indicated in the top left micrograph
corresponds to 10 lm and applies to all micrographs. The primary antibody used in the experiments differed according to the cell line. The HECD-1 antibody,
which does not recognize canine E-cadherin (Ronzitti et al., 2004), was used for analysis of antigens from MCF-7 and Caco-2 cells, and the DECMA-1 antibody
was used to detect the antigen in the MDCK cell system.
support this conclusion. In the first instance, the effect of
AZA-1 is characterized by molecular features observed when
endocytosis is inhibited in MCF-7 cells (Callegari and Rossini,
2008, Fig. 1). Another line of evidence supporting the
conclusion that AZA-1 inhibits endocytosis stems from the
results obtained by treatment of intact cells with exogenous
proteases, showing that a significant portion of the proteins
accumulated at the level of membranous structures in AZA1-treated cells is not accessible to the attack of enzymes added
to the medium bathing the cells. Thus, a considerable portion
of membrane proteins is not exposed on the cell surface but is
sequestered in some intracellular compartment of AZA1-treated cells (Figs. 3 and 4). In keeping with these findings,
changes in subcellular distribution of E-cadherin and Naþ/KþATPase are found in MCF-7 cells exposed to AZA-1, where
these proteins are not located on the cell surface, including the
cell-cell contacts, but concentrate in granular structures
detected in the cell body (Figs. 5–7). Furthermore, we obtained
direct evidence that endocytosis of E-cadherin exposed on the
cell surface is blocked in AZA-1-treated MCF-7 cells (Fig. 7).
The MCF-7 breast cancer cell line, however, does not represent
a unique experimental model targeted by AZA-1 because the
results we obtained on the E-cadherin system in Caco-2 and
MDCK cells (Fig. 8) show that this toxin alters the dynamics of
this plasma membrane protein in other epithelial cells. Thus,
AZA-1 affects both normal (MDCK) and transformed (MCF-7,
Caco-2) cells, independent of the organ (mammary gland,
colon, kidney) and the species of origin (human, dog). It seems
likely, therefore, that plasma membrane proteins are a target of
AZA-1 in different organs. The contention that AZA-1 inhibits
the endocytosis of plasma membrane proteins, for instance, is
in line with evidence recently obtained by Kellman et al.
(2009) in SH-SY5Y human neuroblastoma cells. By analyses
of the effects exerted by AZA-1 on the proteome of this cell
line, they found that two components involved in vesicle
transport are downregulated by the toxin (Kellman et al.,
2009). Furthermore, by electron microscopy examination of
cells, it was also found that AZA-1 induced the accumulation
of early endosomes in the proximity of the plasma membrane,
before alterations of the actin cytoskeleton could be detected
(Kellman et al., 2009). The different sensitivities of plasma
membrane proteins when intact AZA-1-treated cells are
exposed to exogenous proteases (Figs. 3 and 4) would be in
line with these observations.
Our contention that AZA-1 inhibits endocytosis of plasma
membrane proteins may not exclude that other processes
involving intracellular trafficking and dynamics of plasma
membrane proteins can be also affected by AZA-1. In
particular, the accumulation of the E-cadherin precursor
induced by the toxin in MDCK cells (Fig. 8) might be an
indication that the maturation of plasma membrane protein
precursors is another process altered by AZA-1 in sensitive
cells. Other experiments, however, are needed to establish
whether the accumulation of membrane protein precursors in
MDCK cells is either a component- or a cell-specific response
to AZA-1. In MCF-7 cells, in fact, the accumulation of proteins
AZASPIRACID-1 INHIBITS ENDOCYTOSIS
on the cell surface induced by AZA-1 involves a large portion
of plasma membrane proteins (Fig. 2) but would not represent
a generalized increase in protein synthesis, as the cellular levels
of actin are essentially unmodified (Figs. 1–4). The inhibition
of endocytosis of plasma membrane proteins by AZA-1 might
then represent only one aspect of the toxin’s capacity to alter
the cellular dynamics of membrane proteins. If this interpretation is correct, the subcellular distribution of membrane
proteins (and their precursors) in AZA-1-treated cells might
be a component-related phenomenon, which could involve
multiple ultrastructural arrangements and/or heterogeneous
subcellular distribution of different membrane proteins. This
interpretation would be supported by the different sensitivity of
E-cadherin, Naþ/Kþ-ATPase, and Rprl to proteases added to
the medium bathing MCF-7 cells (Figs. 3 and 4), taking into
consideration their subcellular location, as detected by
immunofluorescence microscopy (Figs. 5 and 6). Under our
experimental conditions, three major rearrangements of our
protein markers in MCF-7 cells exposed to AZA-1 would be
indicated (Figs. 3–5). A first rearrangement would involve the
sequestration of components within sites that, based on their
sensitivity to the attack of extracellular proteases, are not
exposed to the cell surface and do not concentrate in some
cellular compartment, as in the case of Rprl. The second type of
rearrangement was observed with Naþ/Kþ-ATPase and intact
E-cadherin, which are destroyed by extracellular proteases and
should be concentrated in some cellular compartment that
either remains on or is connected with the surface of the cell.
A third rearrangement can be exemplified by ECRA100, the
proteolytic fragment of E-cadherin, which is resistant to the
attack of proteases added to the medium bathing the cells
and should be compartmentalized in membranous structures
that are not exposed on the cell surface. A further support to
our contention that multiple ultrastructural arrangements are
induced by AZA-1 is provided by the results of double labeling
experiments (Fig. 6), which complemented those obtained by
cell treatment with trypsin and proteinase K (Figs. 3 and 4) and
confirmed that different subcellular compartments are involved
in the sequestration of Naþ/Kþ-ATPase and ECRA100. AZA-1,
therefore, would cause a widespread impairment of intracellular trafficking of plasma membrane proteins in MCF-7 and
other epithelial cells.
The key molecular defect(s) induced by AZA-1 in MCF-7
and other epithelial cells is (are) presently undetermined, but
the observation that a large part of the cellular pool of plasma
membrane proteins is involved (Fig. 2) and the distinct features
characterizing the cellular response of individual proteins
(Figs. 3–6) indicate that some event(s) shared by intracellular
trafficking of membrane proteins could be targeted by the
toxin. Although the effects of latrunculin A could mimic those
of AZA-1 on the three plasma membrane proteins analyzed in
MCF-7 cells (Fig. 1), the response induced by the toxin should
not depend on a major perturbation of actin-based cytoskeleton
caused by AZA-1 in MCF-7 cells under our experimental
119
conditions. The concentrations of AZA-1 used in our
investigations, in fact, did not significantly alter the cellular
levels of F-actin, as measured by biochemical analyses
(Ronzitti et al., 2007), as well as their overall structural
organization in MCF-7 cells (Fig. 5 and Supplementary fig. 9).
The lack of extensive changes in the actin-based cytoskeleton
of MCF-7 cells following exposure to AZA-1 under the
conditions of the present study is relevant because the toxin has
been shown to perturb both actin-based cytoskeleton (Ronzitti
et al., 2007; Twiner et al., 2005; Vilariño et al., 2006, 2007)
and cytoskeleton-regulating proteins (Kellman et al., 2009) in
biological systems. Cell-specific factors are most likely
involved in the differential responses of actin-based cytoskeleton to cell exposure to AZA-1, as is apparent by paired
analyses of different cell lines treated with AZA-1 (Ronzitti
et al., 2007). The experimental conditions of different studies,
such as different AZA-1 concentrations and times of exposure
to the toxin, could also contribute to detection of different
experimental findings (Ronzitti et al., 2007; Twiner et al.,
2005; Vilariño et al., 2006). Indeed, the AZA-1 concentration
used in most of our experiments (1nM) is lower than the
effective doses employed by Twiner et al. (2005, 10nM) and
Vilariño et al. (2006, 50nM). Further investigations are then
needed to clarify the different sensitivities of actin-based
cytoskeleton to AZA-1 in distinct cell lines.
Independent of the proximal molecular event triggering the
cellular responses to AZA-1, which remains to be identified, the
results obtained with neuroblastoma (Kellman et al. 2009) and
epithelial cells (this study) support the general conclusion that
AZA-1 inhibits endocytosis in biological systems. Our working
hypothesis is that cell death represents only a secondary
response ensuing as a consequence of altered plasma membrane
dynamics induced by the toxin. The impairment of intracellular
trafficking and endocytosis exerted by AZA-1 could provide
a mechanistic explanation of several effects exerted by this
toxin in vitro and in vivo. The accumulation of fat in the liver of
mice exposed to AZA-1 (Ito et al., 2000, 2002) and the
upregulation of transcription of genes coding for enzymes
responsible for fatty acid and cholesterol synthesis (Twiner
et al., 2008b) represent two such effects. The impairment of
lipoprotein endocytosis, in fact, is a recognized key factor
leading to increased cholesterol synthesis in the liver (Goldstein
and Brown, 2009). The inhibition of intracellular vesicle
trafficking, in turn, leads to increased triglycerides and lipid
droplets in vitro and in vivo (Singh et al., 2009). With regard to
the results obtained by Twiner et al. (2008b), in particular, our
findings would support their proposals regarding a mechanistic
link between the cellular levels of cholesterol, the upregulation
of genes coding for enzymes of the pathway responsible for
cholesterol synthesis, and the membrane levels of light-density
lipoprotein receptors (LDLR) found in cells exposed to AZA-1.
Furthermore, our conclusion that AZA-1 inhibits the endocytosis of plasma membrane proteins would explain both the
increased levels of LDLR they found in T lymphocytes, as we
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BELLOCCI ET AL.
detected in the case of Rprl, and a decreased cellular uptake of
cholesterol associated to LDLR. These conditions would lead
to a lowering of the cellular content of cholesterol and a
consequent increased transcription of genes coding for enzymes
involved in cholesterol biosynthesis (Goldstein and Brown,
2009; Twiner et al., 2008b).
Other effects of AZA-1 could be explained by the impairment
of intracellular trafficking and endocytosis. The extensive
damage of the intestinal epithelium induced by AZA-1 (Ito
et al., 2000, 2002, 2006), for instance, could represent
a consequence of the altered turnover of E-cadherin we have
detected (Ronzitti et al., 2007; this study). Moreover, the altered
levels of proteins responsible for ion transport across the plasma
membrane induced by AZA-1 would contribute to the
impairment of ion homeostasis induced by this toxin in cultured
neurons (Vale et al., 2010). Interestingly, the involvement of
Naþ/Kþ-ATPase in the AZA-1 response has been found by both
Vale et al. (2010) and by us (this study). The capacity of AZA-1
to activate caspases (Cao et al., forthcoming; Vilariño et al.,
2007) might be another consequence of the inhibition of
intracellular vesicle trafficking (Kaushal et al., 2008; Yang
et al., 2009). In the light of the fact that endocytosis plays key
roles in the regulatory mechanisms of normal cell functioning
(Scita and Di Fiore, 2010; Sorkin and von Zastrow, 2009), the
demonstration that AZA-1 inhibits endocytosis could provide
a mechanistic frame for the characterization of the molecular
bases of responses induced by AZAs in biological systems.
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FUNDING
Italian Ministero dell’Istruzione, dell’Università e della
Ricerca (2004053150); Italian Ministero della Salute (IZSAM
09/06 RC).
ACKNOWLEDGMENTS
We thank Micol Marchetti for her excellent and kind
technical assistance in the development of procedures we used
for analyses by immunofluorescence microscopy.
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