Molecular Cancer Therapeutics
Induction of endoplasmic reticulum stress by
ellipticine plant alkaloids
Maria Hägg, Maria Berndtsson,
Aleksandra Mandic, Rong Zhou,
Maria C. Shoshan, and Stig Linder
Department of Oncology and Pathology, Cancer Center
Karolinska, Karolinska Institute and Hospital, Stockholm, Sweden
Abstract
Anticancer drugs often show complex mechanisms of
action, including effects on multiple cellular targets.
Detailed understanding of these intricate effects is important for the understanding of cytotoxicity. In this study, we
examined apoptosis induction by ellipticines, a class of
cytotoxic plant alkaloids known to inhibit topoisomerase II.
The potent ellipticine derivative 6-propanamine ellipticine
(6-PA-ELL) induced rapid apoptosis in MDA-MB-231 breast
cancer cells, preceded by a conformational change in Bak
and cytochrome c release. Experiments using knock-out
mouse embryo fibroblasts established that Bak was of
particular importance for cytotoxicity. 6-PA-ELL increased
the expression of the endoplasmic reticulum chaperones
GRP78/BiP and GRP94, suggesting induction of endoplasmic reticulum stress. Induction of GRP78 expression was
dependent on the endoplasmic reticulum stress response
element (ERSE) of the GRP78 promoter. Examination of
different ellipticine derivatives revealed a correlation
between pro-apoptotic activity and the ability to induce
GRP78 expression. Furthermore, 6-PA-ELL was found to
induce splicing of the mRNA encoding the XBP1 transcription factor, characteristic of endoplasmic reticulum stress,
and to induce activation of the endoplasmic reticulumspecific caspase-12 in mouse colon cancer cells. We finally
demonstrate that 6-PA-ELL induces apoptotic signaling also
in enucleated cells, consistent with the existence of a
cytoplasmic target for this compound. Our data suggest
that induction of endoplasmic reticulum stress may
contribute to the cytotoxicity of ellipticines. [Mol Cancer
Ther. 2004;3(4):489 – 497]
Introduction
Some anticancer drugs in clinical use and various investigational agents have been reported to affect more than one
cellular target. Etoposide induces DNA damage at low
Received 11/17/03; revised 1/14/04; accepted 1/21/04.
Grant support: Swedish Cancer Society (Cancerfonden) (S. Linder and
M. Shoshan) and Gustav V Jubilee Foundation (S. Linder).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for Reprints: Stig Linder, Department of Oncology and Pathology,
Cancer Center Karolinska, Karolinska Hospital, S-171 76 Stockholm,
Sweden. Phone: 46-8-5177-2452; Fax: 46-8-33-90-31.
E-mail: [email protected]
concentrations, but has direct effects on mitochondria at
higher concentrations (1). Taxol directly affects mitochondrial function and interferes with the Ca2+ signal cascade
(2) and cisplatin induces endoplasmic reticulum stress
(3). These pleiotropic effects are not surprising, considering
that some of these compounds are used in micromolar
concentrations.
Nuclear DNA damage and triggering of death receptors
are well-established apoptotic stimuli. Cellular organelles
such as the endoplasmic reticulum and the lysosomes also
possess the capacity to sense stress and to transmit signals to
mitochondria, leading to mitochondrial pore formation and
apoptosis (reviewed in Ref. 4). Apoptosis via the endoplasmic reticulum may be elicited by accumulation of unfolded
proteins induced by inhibitors of protein glycosylation (5, 6).
The endoplasmic reticulum senses stress through chaperones such as GRP78 and GRP94, Ca2+-binding proteins, and
Ca2+ release channels, which might transmit endoplasmic
reticulum Ca2+ responses to mitochondria (7). The endoplasmic reticulum contains Bcl-2-family proteins, and Bcl-2
exerts part of its cytoprotective effect within the endoplasmic reticulum (8, 9). Lysosomal cathepsins have been
implicated in apoptosis induced by tumor necrosis factor
(TNF) and other stimuli (10 – 12). Finally, some anticancer
drugs, such as lonidamine, arsenite, and betulinic acid, act
directly on mitochondria (13).
Ellipticine [5,11-dimethyl-6H-pyrido(4,3-b)carbazole] is
a plant alkaloid with antitumor activity (14). Ellipticine
analogues are active against brain tumor cell lines (15 – 17)
and have showed promising results in treatment of
metastatic breast cancer (18). The principal mechanism of
cytotoxicity is believed to be inhibition of topoisomerase
II-h (19). In addition, ellipticine has been reported to bind
to proteins (20), to induce the generation of cytotoxic free
radicals (21), to inhibit cytochrome P4501A1 (22), and to
uncouple oxidative phosphorylation (23, 24). Interestingly,
it was recently reported that ellipticine has the ability to
activate the transcription function of mutant p53 (25).
The reports of complex biological activities by ellipticines
raise the question of whether these compounds induce
multifaceted biological responses. We here show that
apoptotic activity of ellipticine derivatives correlates to the
ability to induce expression of the endoplasmic reticulum
chaperone GRP78, and that the potent ellipticine derivative
6-propanamine-ellipticine (6-PA-ELL) induces splicing of
XBP1 mRNA. Our findings show that ellipticine derivatives
can induce endoplasmic reticulum stress, and suggest that
endoplasmic reticulum stress is a contributing factor to the
cytotoxic activity of this class of topoisomerase II inhibitors.
Materials and Methods
Materials
Ellipticine compounds were obtained from the Developmental Therapeutics Program at the National Cancer
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
489
490 Induction of Endoplasmic Reticulum Stress by Ellipticines
Institute (http://www.dtp.nci.nih.gov): NSC 176328, 6PA-ELL; NSC 176327, 9-methoxy-6-PA-ELL; NSC 162907,
6-(5-hexen-1-yl)ellipticine; NSC 98949, 9-methylellipticine;
NSC 338258, 9-dimethyl amino-ethoxy-ellipticine; NSC
69187, 9-methoxy-ellipticine; NSC 152731, 6H-pyrido{4,3-b}
carbazole, 5,11-dimethyl-6-(oxiranylmethyl); NSC 360319,
h-D-ribofuranoside, (5,6-dimethyl-6H-pyrido{3,4-b}carbaazol-11-yl)methyl; NSC 335142, 6H-pyrido{4,3-b}carbaazole-1-carboxamide, 5,11-dimethyl-, monohydrochloride.
Other reagents: z-VAD-fmk (Enzyme Systems Products,
Livermore, CA); E-64d (10 AM, Sigma Aldrich, Sweden,
Stockholm, Sweden); carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma); Rhodamine 123 (Sigma); cisplatin
and doxorubicin (Karolinska Hospital Pharmacy, Stockholm, Sweden); BMH (Pierce, Rockford, IL); ellipticine
(Sigma); and thapsigargin (Molecular Probes, Leiden, The
Netherlands).
Cell Culture
MDA-MB-231 and MCF-7 human breast carcinoma
(generously provided by Dr. Bert Vogelstein), mouse
CT51 colon carcinoma cells (26) (kindly provided by Dr.
Björn Öbrink), and SV40 immortalized mouse embryo
fibroblast (MEF) lines defective in Bak and Bax (kindly
provided by Dr. Stanley Korsmeyer) were maintained at
+37jC in 5% CO2 in DMEM supplemented with FCS
(10%), L-glutamate, penicillin, and streptomycin. HCT116
colon carcinoma (p53wt and / ) (27) were maintained
in McCoy’s 5A modified medium/10% FCS.
Assessment of Apoptosis
Air-dried smears were fixed in acetone-methanol (2:1)
and stained with ethidium bromide (5 ng/ml) for 5 min.
After being rinsed in tap water, stained cells were
examined by UV microscopy. Cytokeratin-18 (CK18)
cleavage was measured using an ELISA (28). Cells were
seeded at 104/well in 96-well plates 6 h before
treatment and treated with drugs as indicated. At the
end of the incubation period, NP40 was added to the
tissue culture medium to 0.5% and 25 Al of the content
of each well (thus including activity released to the
medium from attached and floating cells and cell
fragments) were assayed for the CK18-Asp396 epitope
as recommended by the manufacturer (PEVIVA AB,
Bromma, Sweden).
Preparation of Cytoplasts
Cells were harvested, resuspended in 12.5% Ficoll
containing complete medium supplemented with cytochalasin B (10 Ag/ml), and incubated for 30 min at
+37jC. Three milliliters of cell suspension were layered
onto a density gradient prepared in ultracentrifuge tubes
with the following layers: 2 ml of 25%, 2 ml of 17%,
0.5 ml of 16%, and 0.5 ml of 15% Ficoll. The gradient was
prepared in complete medium supplemented with 10 Ag/ml
of cytochalasin B and pre-equilibrated in a CO2 incubator
overnight. The cells were centrifuged in a prewarmed
(+32jC) Beckman SW41 rotor for 60 min at 25,000 rpm.
The resulting enucleated cytoplasts were collected at
the 16 – 17% interface and were washed with medium
and allowed to recover for 2 h. The purity of the
cytoplast preparation was determined by flow cytometry
after resuspending it in PBS containing digitonin (50
Ag/ml) and labeling for 10 min with 5 Ag/ml propidium
iodide.
Isolation of Mitochondria and Cross-Linking of Bak
Cells were resuspended in 200 Al Buffer A [250 mM
mannitol, 70 mM sucrose, 0.5 mM EGTA, 5 mM HEPES (pH
7.2), 0.1 mM phenylmethylsulfonyl fluoride] and lysed in a
glass homogenizer on ice. All further steps were conducted at +4jC. Homogenates were centrifuged at 3400
rpm in an Eppendorf centrifuge for 10 min. The
supernatant was recentrifuged (13,000 rpm, 30 min) and
the pellet was resuspended in 60 Al Buffer B [250 mM
sucrose, 10 mM HEPES (pH 7.5), 2 mM KH2PO4, 5 mM Nasuccinate, 25 mM EGTA, 0.1 mM phenylmethylsulfonyl
fluoride, 4 mM MgCl2]. The sample was divided: 40 Al
were treated with BMH cross-linker (10 mM, Pierce) and
20 Al as a control with DMSO for 30 min at room temperature as described (29). Samples were analyzed by
SDS-PAGE and Western blotting.
Isolation of Mitochondria and Measurements of DW
Cells were resuspended in 500 Al 250 mM sucrose in
10 mM Tris (pH 7.2), and homogenized on ice. All further
steps were conducted at +4jC. After centrifugation at
600 g for 5 min, the supernatant was collected and centrifuged at 10,000 rpm for 10 min. The pellet was suspended in 200 Al 250 mM sucrose, 10 mM Tris (pH 7.2). To
assess changes in mitochondrial membrane potential,
Rhodamine 123 was added to 5 AM. Changes in fluorescence induced by 6-PA-ELL (5 AM), CCCP (100 AM), or
buffer only were monitored by flow cytometry.
Western Blot Analysis
Cell extract proteins were resolved by SDS-PAGE and
transferred onto a polyvinylidene difluoride membrane for
Western blotting. The following antibodies were used: antiBak (1:1000; Upstate Technologies, Lake Placid, NY); antimouse pro-caspase-12 (1:40; kindly provided by Dr. J.
Yuan); anti-GRP78 (1:1000; BD Biosciences Pharmingen,
San Diego, CA); anti-GRP94 (1:1000; Calbiochem, San
Diego, CA); and antitubulin (1:1000; Sigma Aldrich).
Tubulin was used as an internal standard for loading.
Flow Cytometric Analysis
Activating conformational changes leading to exposure
of otherwise inaccessible NH2-terminal epitopes of Bak and
Bax were detected by flow cytometry as previously
described (30). One antibody recognizing amino acids
1 – 52 of Bak (Oncogene Research Products, Boston, MA)
and one recognizing amino acids 12 – 24 of Bax (PharMingen) were used. CK18 cleavage was assessed using the
monoclonal antibody M30 (31). Cells or cytoplasts were
fixed with 0.25% paraformaldehyde for 5 min, washed
twice in PBS, and incubated for 30 min with primary
antibody in the presence of digitonin for permeabilization,
followed by incubation with the secondary FITC-conjugated antibody (DAKO, Glostrup, Denmark) for 1 h in the
absence of digitonin (32, 33). Analysis of GRP78 levels was
similarly performed using an antibody from BD Biosciences (used at a 1:50 dilution).
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Cytochrome c Release
Cells were incubated for 30 min in medium containing
10 nM Mitotracker Red CMXRos (Molecular Probes) and
cytocentrifuged onto glass slides. Cells were fixed in 3%
paraformaldehyde for 20 min and permeabilized using
0.2% Triton X-100 in PBS for 10 min. Fixed cells were
incubated with anti-cytochrome c antibody (1:300; 65918A;
PharMingen) for 1 h, followed by incubation with a FITCconjugated anti-mouse antibody (DAKO). Slides were
mounted in Vectashield containing DAPI and images
recorded in a Zeiss Axioplan 2 fluorescence microscope.
RT-PCR
Total RNA was isolated from cells by the guanidineHCl method. RNA was reverse transcribed and cDNA
amplified using a GeneAmp kit from PE Biosystems (San
Francisco, CA). XBP1 primers have been described by
Yoshida et al. (34) (nucleotides 412 – 431; 834 – 853). XBP1
amplification products were separated by electrophoresis
in 2% agarose gels.
Reporter Assays
MDA-MB-231 cells were transfected by the calcium
phosphate co-precipitation technique. The GRP78-LUC
and GRP78mut-LUC reporters were kindly provided by
Dr. Kazutoshi Mori (35). We used 1 Ag of pGL3GRP78( 132)-LUC (GRP78-LUC) or GRP78mut-LUC, and
0.1 Ag pRL-SV40 plasmid (expressing the Renilla luciferase,
Promega, Madison, WI) for each transfection. In the
GRP78mut-LUC reporter, the ERSE sequences are mutated
(35). At 24 h after transfection, agents were added and cells
were harvested at the indicated time points.
Results
Induction of Apoptosis by Ellipticines in Human MDAMB-231Breast Cancer Cells
An assay measuring total accumulation of caspasecleaved CK18 fragments (CK18-Asp396) in cells and culture
media (28) was used to examine ellipticine-induced apoptosis in MDA-MB-231 breast carcinoma cells. Of 10 ellipticines
tested, 6-PA-ELL (NSC176328; 5 AM) and 9-methoxy-6-PAELL (NSC176327; 2 AM) elicited the strongest responses
(Fig. 1A). Compounds NSC162907, NSC360319, and
NSC335142 did not induce CK18 cleavage at 5 AM. Neither
did ellipticine (NSC71795); however, cleavage was observed
at 20 AM (not shown). 9-Methoxy-ellipticine (NSC69187) and
three additional compounds showed intermediate activity.
NSC176328 (6-PA-ELL) was studied in detail and was found
to induce a 5- to 20-fold increase in CK18 cleavage over
18 h in different experiments. The CK18-cleavage inducing
activity of 6-PA-ELL was inhibited by the caspase-inhibitor
z-VAD (Fig. 1B).
Induction of CK18-Asp396 cleavage by 6-PA-ELL was
rapid (Fig. 1C). After 6 h of treatment with 5 AM 6-PA-ELL,
CK18-Asp396 cleavage was 5-fold above background
levels. In contrast, 2 Ag/ml doxorubicin induced a 3-fold
increase at 9 h. Decreased CK18-Asp396 cleavage was
observed at higher doxorubicin concentrations. Nuclear
fragmentation, typical of apoptosis, was observed within
4.5 h of treatment with 5 AM 6-PA-ELL (Fig. 1D). Release of
cytochrome c was induced by 6-PA-ELL. Cytochrome c
staining was localized to mitochondria in untreated cells
[Fig. 2A; green staining overlapped with MitoTracker Red
staining (not shown)]. In contrast, after 4.5 h treatment, the
majority of the cells showed a diffuse cytoplasmic staining
indicative of released cytochrome c from mitochondria
(Fig. 2B). We conclude that 6-PA-ELL induces rapid
apoptosis in MDA-MB-231 cells.
Cytotoxic Activity of 6-PA-ELL on Human and Mouse
Carcinoma Cell Lines
MDA-MB-231 is a highly malignant and metastatic
breast cancer cell line (36). The non-metastatic breast
cancer cell line MCF-7 was less sensitive to 6-PA-ELL
(Table 1). HCT116 human colon cancer cells (p53wt) were
approximately 2-fold more sensitive than a HCT116
derivative with a disrupted p53 locus (27) (Table 1).
Mouse CT51 colon cancer cells showed a similar level of
sensitivity to 6-PA-ELL as the human cancer cell lines.
Involvement of Bak in 6-PA-ELL-Induced Apoptosis
The Bcl-2 family proteins Bak and Bax are instrumental
in outer mitochondrial membrane pore formation (37).
Here, MEF cell lines defective in Bak or Bax were
examined with regard to sensitivity to 6-PA-ELL. First,
the effect of each drug on the viability of MEF wild-type
cells was determined as an LD90 dose, that is, a dose
leading to 10% viability. In Bak / MEFs, this dose of
6-PA-ELL led to a viability of approximately 65% (Fig. 3A).
Although the Bak / cells showed reduced sensitivity also to doxorubicin or cisplatin, their resistance to
6-PA-ELL was significantly greater. Compared to the
Bak-deficient cells, Bax-deficient cells were less resistant to
the three drugs, and the difference between doxorubicin and
6-PA-ELL was insignificant (Fig. 3A). These results demonstrate that Bak is of particular importance for 6-PA-ELLinduced apoptosis.
During apoptosis, Bak and Bax undergo conformational
changes leading to exposure of otherwise inaccessible
NH2-terminal epitopes (32, 33, 38), believed to represent
activation of these proteins. Using a monoclonal antibody
recognizing the NH2 terminus of Bak, we observed an increase in Bak modulation already after 2 – 3 h of 6-PA-ELL
treatment of MDA-MB-231 cells (Fig. 3, B and C). Bak
conformation can also be studied using the cysteinreactive molecular cross-linker BMH (29). Bak has a
molecular weight of 25,000. In viable cells, Bak can be
intramolecularly cross-linked by BMH to a faster migrating species (M r 21,000) believed to represent an inactive
form (29). The M r 21,000 band was reduced in cells
exposed to 6-PA-ELL for 5 h, and was undetectable after
7.5 h (Fig. 3D).
As shown in Fig. 3E, modulation of Bax was observed at
18 h of 6-PA-ELL treatment, but not after 5 h. Because CK18
cleavage could be detected at 4 h in the majority of the cells
(Fig. 3F), we conclude that Bak, but not Bax, is modulated
before the onset of the execution phase of 6-PA-ELLinduced apoptosis, in accordance with a greater dependence on Bak than on Bax.
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
491
492 Induction of Endoplasmic Reticulum Stress by Ellipticines
Figure 1. Induction of apoptosis by
ellipticine analogues. A, MDA-MB231 cells were treated with different
ellipticines and the levels of the apoptosis-specific CK18-Asp396 epitope
in media and cell extracts were
assessed by ELISA after 18 h (for
compounds/NSC numbers, see Materials and Methods). All compounds
were used at 5 AM except NSC176327
which was used at 2 AM (rapid membrane blebbing and high side scattering in flow cytometry were observed
at higher concentrations, suggesting
general toxicity). B, MDA-MB-231
cells were treated with 5 AM 6-PAELL or 5 AM 6-PA-ELL + 50 AM
z-VAD-fmk for 18 h and the levels
of the CK18-Asp396 epitope were
determined by ELISA. C, caspasespecific cleavage of CK18-Asp396
was measured at the indicated time
points. MDA-MB-231 cells were treated with 2 AM 6-PA-ELL (open circles),
5 AM 6-PA-ELL (filled circles), 1 Ag/
ml doxorubicin (open squares), or 2
Ag/ml doxorubicin (filled squares). D,
nuclear morphology of untreated
MDA-MB-231 cells (left) and 6-PAELL-treated cells which show nuclear
fragmentation typical of apoptosis
(right). Cells were treated for 4.5 h
with 5 AM 6-PA-ELL, fixed and stained
with ethidium bromide.
6-PA-ELL Does Not Act Directly on Mitochondria to
Induce Loss of DW
A number of experimental anticancer drugs induce
apoptosis by acting directly on mitochondrial membranes
(13); uncoupling agents such as the protonophore CCCP
Figure 2. Cytochrome c is released from mitochondria in 6-PA-ELLtreated cells. MDA-MB-231 cells were treated with 5 AM 6-PA-ELL, fixed
and stained for cytochrome c . A, untreated cells. B, cells treated with
6-PA-ELL for 4.5 h. Note mitochondrial localization of cytochrome c in
untreated cells. After 4.5 h of treatment, cells showed cytoplasmic
localization of cytochrome c .
induce apoptosis (39). Ellipticines accumulate in mitochondria (23) and uncouple oxidative phosphorylation (24). We
tested whether 6-PA-ELL has a direct effect on the
mitochondrial transmembrane potential in MDA-MB-231
cells by incubating isolated mitochondria with the cationic
lipophilic dye Rhodamine 123. A rapid increase in
Rhodamine 123 fluorescence, indicating decreased quenching of the dye due to release from the mitochondrial matrix,
was observed after addition of CCCP. By contrast, a slow
increase in fluorescence, similar to that observed in the
control, was observed after addition of 5 AM 6-PA-ELL
(Fig. 4A).
Some apoptotic stimuli depend on the activity of
lysosomal enzymes such as cathepsin B, and apoptosis
may be blocked by inhibitors of lysosomal enzymes (11). We
examined the effect of the broad-spectrum inhibitor E-64d,
and did not observe any inhibition of apoptosis induction
(Fig. 4B).
6-PA-ELL Induces Endoplasmic Reticulum Chaperone
Proteins and Splicing of XBP1MRNA
Endoplasmic reticulum stress induces expression of the
endoplasmic reticulum-specific chaperones GRP78 and
GRP94 (7). Induction of GRP78 and GRP94 expression was
observed in 6-PA-ELL-treated MDA-MB-231 cells using
Western blotting (Fig. 5A). Thapsigargin, an inhibitor of the
endoplasmic reticulum Ca2+-ATPase, also induced GRP78
and GRP94. Induction of GRP78 was observed using flow
cytometry (Fig. 5B) and in HCT116 cells and 224 melanoma
cells (not shown). In addition to thapsigargin, DTT induced
GRP78 in MDA-MB-231 cells. Importantly, both of the
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Table 1. Effect of 6-PA-ELL (NSC176328) on human and mouse
tumor cell lines
Cell Line
MDA-MB-231
MCF-7
HCT116 p53+/+
HCT116 p53 /
CT51
Type
LD90 (AM)a
Breast cancer (p53mut; ER b)
Breast cancer (p53wt; ER+b)
Colon cancer
Colon cancer
Mouse colon cancer
0.06
0.09
0.06
0.11
0.15
a
Dose required to elicit a 90% reduction in colony formation ability. Cells were
treated with 6-propanamine-ellipticine for 4 h, and clonogenic outgrowth was
determined.
b
ER, estrogen receptor.
pro-apoptotic ellipticines [NCS176328 (6-PA-ELL) and
NCS176327 (9-methoxy-6-PA-ELL)] induced expression of
GRP78, whereas GRP78 was not induced by three ellipticines that did not induce apoptosis (Fig. 5B). Induction
of GRP78 was observed at time points preceding apoptosis;
a 2-fold increase was observed within 3 h (Fig. 5C).
During endoplasmic reticulum stress, the mRNA encoding the transcription factor XBP1 is spliced by the
endoribonuclease IRE1 (34, 40). Splicing induces a frame
shift and translation of a 376-amino acid form of XBP1 with
enhanced activity as transcriptional activator (34) (Fig. 5D).
6-PA-ELL and thapsigargin were found to induce splicing
of XBP1 mRNA in HCT116 cells (Fig. 5D).
6-PA-ELL Induction of GRP78 Depends on the Endoplasmic Reticulum Stress Response Element
The GRP78 promoter contains a cis-acting endoplasmic
reticulum stress response element (ERSE). To determine
whether induction of GRP78 expression by 6-PA-ELL is
dependent on the ERSE, MDA-MB-231 cells were transfected with GRP78-promoter reporter constructs. As shown
in Fig. 5E 6-PA-ELL induced increased activity of a GRP78LUC reporter (filled bars), but did not induce expression of
a GRP78-LUC reporter with deletions of the ERSEs
(GRP78mut-LUC; open bars). These results show that 6-PAELL-induced GRP78 expression is mediated by the ERSE.
Involvement of Caspase-12
In murine cells, endoplasmic reticulum stress results in
the activation of the endoplasmic reticulum-resident procaspase-12 (41). 6-PA-ELL was cytotoxic to normal mouse
fibroblasts (Fig. 3A) and to mouse colon cancer CT51 cells
Figure 3. 6-PA-ELL-induced apoptosis involves Bak. A, mouse embryo fibroblast (MEF ) cell lines were treated with different doses of doxorubicin (DOX ),
6-PA-ELL, or cisplatin (CISPL ). The viability of Bak / and Bax / MEF at doses of each compound, which produced a 90% loss of viability of a wild-type
MEF cell line, are shown. The numbers of viable cells were determined 24 h after treatment, and data are based on three independent experiments. Note that
Bak / MEF cells show a viability of 66 + 2.6% at a dose of 6-PA-ELL which generates a viability of 10% on wild-type MEF cells (i.e. , an LD90 dose). B – D,
conformational modulation of Bak. B, MDA-MB-231 cells were treated with 5 AM 6-PA-ELL for 3 h. Cells were fixed, permeabilized, and incubated with an
antibody recognizing an epitope in the unstructured NH2-terminal loop of Bak followed by analysis by flow cytometry (30). C, conformational modulation of
Bak at different times of treatment with 5 AM 6-PA-ELL. D, MDA-MB-231 cells were treated with 5 AM 6-PA-ELL for the indicated times. Mitochondria were
isolated and incubated with the cross-linker BMH (upper panel ). Proteins were solubilized and subjected to Western blot analysis for Bak. E,
conformational modulation of Bax in 6-PA-ELL-treated MDA-MB-231 cells. Cells were fixed after 5 and 18 h of treatment, incubated with an antibody
against the NH2 terminus of Bax and analyzed by flow cytometry (untreated cells shown). F, caspase-mediated cleavage of CK18 in MDA-MB-231 after 4 h of
6-PA-ELL treatment. The intracellular levels of the CK18-Asp396 epitope were examined by staining with the epitope-specific M30 antibody and analysis by
flow cytometry. Note that the CK18-Asp396 epitope is exposed in most cells at 4 h, when no conformational activation of Bax can be detected.
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
493
494 Induction of Endoplasmic Reticulum Stress by Ellipticines
Modulation of Bak and Caspase Cleavage Does Not
Depend on the Cell Nucleus
An endoplasmic reticulum stress-mediated cytotoxic
insult would occur in parallel with DNA damage. To
determine whether 6-PA-ELL induces apoptotic signaling
independent of damage to nuclear DNA, MDA-MB-231
cells were enucleated by isopyknic density centrifugation
in the presence of cytochalasin B. Fractions containing
nucleus-free cytoplasts and some contaminating intact cells
were treated with 6-PA-ELL for 4 h. Cell preparations
were stained with propidium iodide to enable the identification of enucleated cells by electronic gating during
Figure 4. Examination of potential mitochondrial and lysosomal targets.
A, 6-PA-ELL does not induce loss of mitochondrial DC . Mitochondria were
isolated from MDA-MB-231 cells. The cationic lipophilic dye Rhodamine
123 was added and the fluorescence measured (I ). Compounds or buffer
were added at (II), and increases in fluorescence (due to decreased
quenching of Rhodamine 123 after release from mitochondria) were
measured. Open squares, CCCP; open circles, 6-PA-ELL; closed circles,
buffer. Note that the effect of 5 AM 6-PA-ELL does not differ from the control,
whereas CCCP induces rapid release of Rhodamine 123, characteristic of
depolarization. B, a broad-spectrum lysosomal protease inhibitor does not
inhibit 6-PA-ELL-induced apoptosis. MDA-MB-231 cells were incubated
with 5 AM 6-PA-ELL in the presence or absence of 30 AM E-64d. The levels of
caspase-cleaved CK18-Asp396 were examined by ELISA at 18 h.
(Table 1). 6-PA-ELL and 9-methoxy-6-PA-ELL induced
cleavage of caspase-12 in CT51 cells (Fig. 6A). A M r 60,000
protein is detected by caspase-12 antibodies in human cells,
including MDA-MB-231 (42, 43) (Fig. 6B). 6-PA-ELL
treatment reduced the intensity of this band. Caspase-12
is cleaved by calpain during endoplasmic reticulum stress
(44), and the calpain inhibitor calpeptin inhibited 6-PAELL-induced caspase cleavage (Fig. 6B). Interestingly, 6PA-ELL-induced apoptosis was inhibited by two different
calpain inhibitors (Fig. 6C), suggesting a role of calpain in
the apoptosis process.
Figure 5. Induction of ER stress by pro-apoptotic ellipticines. A, 6-PA-ELL
treatment induces expression of the ER chaperones GRP78 and GRP94.
MDA-MB-231 cells were incubated with 5 AM 6-PA-ELL for the times
indicated or with 300 nM thapsigargin for 12 h. Total cell extracts were
analyzed by Western blotting. B, MDA-MB-231 cells were incubated with
the indicated ellipticines for 5 h (at 5 AM; NSC176327 at 2 AM), 300 nM
thapsigargin for 5 h, or 5 mM DTT for 5 h. The levels of GRP78 were
determined by flow cytometry. The levels of GRP78 were determined by
flow cytometry (see Materials and Methods for NSC numbers/compounds). C, time course of induction of GRP78 by 6-PA-ELL, measured
by flow cytometry. D, induction of XBP1 splicing in HCT116 cells treated
with 6-PA-ELL for 8 h or thapsigargin for 16 h. The 416-bp form appearing
in treated cells represents the spliced form of the mRNA. PCR primers were
identical to those described in Ref. (34). E, induction of the GRP78-promoter by 6-PA-ELL. MDA-MB-231 cells were transfected with a GRP78LUC reporter (closed bars), or a mutant reporter where the ERSE elements
in the GRP78 promoter are deleted (open bars ) (35). A control plasmid
expressing the Renilla luciferase was cotransfected as an internal
standard. Agents were used at the same concentrations and times as in
B. Results are from four independent experiments. The difference between
6-PA-ELL-treated cells transfected with GRP78-LUC and GRP78mut-LUC
was statistically significant (P = 0.042).
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
Figure 6. Induction of caspase12 cleavage. A, mouse CT51 cells
were treated with the indicated
concentrations (AM) of 6-PA-ELL or
9-methoxy-6-PA-ELL (NSC176327 )
for 8 h. Thapsigargin (Thaps ) was
used at 0.3 AM for 8 or 16 h (#).
Cleavage of mouse caspase-12 was
assayed by Western blotting, note
the appearance of a cleaved product
in treated cells. B, MDA-MB-231
cells were treated with 5 AM 6-PAELL for the indicated times in the
presence or absence of the calpain
inhibitor calpeptin (10 AM). The
levels of a M r 60,000 protein reacting with a caspase-12 antibody were
determined by Western blotting.
C, MDA-MB-231 cells were treated
with 5 AM 6-PA-ELL for 16 h in the
presence or absence of two different calpain inhibitors, calpeptin
(10 AM) or PD150606 (20 AM). The
amount of caspase-cleaved CK18
was determined by ELISA.
flow cytometry. Significant CK18 cleavage as well as conformational modulation of Bak were detected in cytoplasts
after 4 h of treatment, that is, at a time point when apoptosis was well under way also in intact cells (Fig. 7).
Discussion
Many anticancer drugs are chemically reactive and used in
micromolar concentrations. Not surprisingly, in addition to
their postulated main mechanisms of action, various side
activities have been attributed to such drugs. It is as yet
unclear to which extent such additional effects contribute to
the cytotoxicity. Etoposide and paclitaxel have direct effects
on mitochondria (1, 2), and cisplatin has been shown to
induce endoplasmic reticulum stress (3). We have here
studied the ellipticines, a class of plant alkaloids known to
inhibit topoisomerase II (19), but also reported to activate the
transcription function of mutant p53 (25) and to directly affect
mitochondria (24). We demonstrate that the ellipticine
derivative 6-PA-ELL induces endoplasmic reticulum stress,
a previously not described activity of this class of compounds.
Known endoplasmic reticulum stresses include calcium
store depletion, inhibition of glycosylation, reduction of
disulfide bonds, and overexpression of mutant proteins
(45 – 47). Ellipticine interacts with proteins (20, 48), and
bind to and prevent folding of proteins in the endoplasmic
reticulum. We have previously observed that cisplatin
induces GRP78 expression and caspase-12 cleavage in
human melanoma cells (3), and have identified other
cytotoxic drugs in the NCI mechanistic set which induce
cleavage of mouse caspase-12 in concentrations of 5 AM,
suggesting that induction of endoplasmic reticulum stress
by cytotoxic compounds is not uncommon.2 It is conceiv-
2
Unpublished observations.
able that the protein folding compartment of the endoplasmic reticulum may be quite sensitive to disturbances, or
that the Ca2+ stores of the endoplasmic reticulum may be
released by some of these drugs.
The association between the ability of different ellipticines to induce endoplasmic reticulum stress and apoptosis
suggests that endoplasmic reticulum stress may be of
importance for the cytotoxic activity of ellipticines. However, it is possible that endoplasmic reticulum active
compounds are highly cell permeable, or that that these
compounds share activities required for both topoisomerase II inhibition and endoplasmic reticulum stress. Increased expression of the endoplasmic reticulum chaperone
GRP78 was detected within a few hours in 6-PA-ELLtreated cells, before release of cytochrome c from mitochondria and caspase cleavage of CK18. GRP78 then
accumulated in the cells. Endoplasmic reticulum stress is
therefore likely to be an early event, preceding apoptosis.
6-PA-ELL induction of pro-caspase-12 cleavage is consistent with the hypothesis that apoptotic signaling is
triggered by endoplasmic reticulum stress. These findings
suggest that endoplasmic reticulum stress contributes to
the cytotoxicity of 6-PA-ELL, but the relative importance of
endoplasmic reticulum stress induction is uncertain. We
were able to demonstrate, however, that 6-PA-ELL induced
Bak activation and CK18 caspase cleavage in enucleated
cells, directly demonstrating a non-nuclear apoptotic sensor. The cytoplasmic target may be important for the anticancer activity of the compound, and/or may be involved
in producing toxic side effects in normal tissues.
The endoplasmic reticulum chaperones GRP78 and
GRP94 are generally regarded as reliable markers for
endoplasmic reticulum stress (7, 49, 50). The transcription
factor ATF6 is an endoplasmic reticulum transmembrane
protein which binds GRP78 in unstressed cells, but translocates and is cleaved in the Golgi following endoplasmic
reticulum stress induction (51). ATF6 then binds the ERSEs
of the GRP78 and GRP94 promoters. Studies of induction of
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
495
496 Induction of Endoplasmic Reticulum Stress by Ellipticines
Acknowledgments
We thank the Developmental Therapeutics Program of the National Cancer
Institute for distribution of compounds. We are grateful for the kind gift of
Bak and Bax defective MEF cells from Dr. Stanley Korsmeyer, and thank
Dr. Bert Vogelstein for the HCT116 cell lines. The GRP78-LUC reporter
was kindly provided by Dr. Kazutoshi Mori.
References
1. Robertson JD, Gogvadze V, Zhivotovsky B, Orrenius S. Distinct
pathways for stimulation of cytochrome c release by etoposide. J Biol
Chem, 2000;275:32438 – 43.
2. Kidd JF, Pilkington MF, Schell MJ, et al. Paclitaxel affects cytosolic
calcium signals by opening the mitochondrial permeability transition pore.
J Biol Chem, 2002;277:6504 – 10.
3. Mandic A, Hansson J, Linder S, Shoshan MC. Cisplatin induces
endoplasmic reticulum stress and nucleus-independent apoptotic signaling. J Biol Chem, 2003;278:9100 – 6.
4. Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol, 2001;3:E255 – 63.
5. Ma Y, Hendershot LM. The unfolding tale of the unfolded protein
response. Cell, 2001;107:827 – 30.
6. Hampton RY. Endoplasmic reticulum stress response: getting the UPR
hand on misfolded proteins. Curr Biol, 2000;10:R518 – 21.
7. Lee AS. The glucose-regulated proteins: stress induction and clinical
applications. Trends Biochem Sci, 2001;26:504 – 10.
Figure 7. 6-PA-ELL-induced apoptotic signaling does not require the cell
nucleus. MDA-MB-231 or 224 cells were enucleated as described in
Materials and Methods, and fractions containing nucleus-free cytoplasts
and contaminating intact cells were treated with 5 AM 6-PA-ELL for 4 h.
Cells/cytoplasts were stained with propidium iodide to facilitate the
identification and electronic gating of enucleated cells. A, quantification of
the CK18-Asp396 epitope by flow cytometry. B, assessment of Bak
modulation in enucleated MDA-MB-231 cells using an antibody recognizing the NH2 terminus of Bak, as described in Fig. 3B.
8. Lam M, Dubyak G, Chen L, Nunez G, Miesfeld RL, Distelhorst CW.
Evidence that BCL-2 represses apoptosis by regulating endoplasmic
reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci USA, 1994;91:
6569 – 73.
9. Scorrano L, Oakes SA, Opferman JT, et al. BAX and BAK regulation
of endoplasmic reticulum Ca2+: a control point for apoptosis. Science,
2003;300:135 – 9.
10. Foghsgaard L, Wissing D, Mauch D, et al. Cathepsin B acts as a
dominant execution protease in tumor cell apoptosis induced by tumor
necrosis factor. J Cell Biol, 2001;153:999 – 1010.
11. Leist M, Jaattela M. Triggering of apoptosis by cathepsins. Cell Death
& Differ, 2001;8:324 – 6.
12. Kagedal K, Zhao M, Svensson I, Brunk UT. Sphingosine-induced
apoptosis is dependent on lysosomal proteases. Biochem J, 2001;359:
335 – 43.
the GRP78 promoter by 6-PA-ELL in MDA-MB-231 showed
that induction was dependent on the ERSE, providing
direct evidence for an 6-PA-ELL-induced endoplasmic
reticulum stress response. Increased levels of endoplasmic
reticulum chaperones have been associated with resistance
to chemotherapeutic agents (52, 53). It was recently reported that cell lines made to overexpress GRP78 show
resistance to both topoisomerase I and II inhibitors (54), an
effect attributed to binding of GRP78 to caspase-7 (54).
These findings suggest that the endoplasmic reticulum is
indeed involved in the response to chemotherapeutic
agents.
In summary, we report induction of endoplasmic reticulum stress as a novel property of a derivative of the
alkaloid ellipticine, 6-PA-ELL. The ellipticine induced rapid
apoptosis in MDA-MB-231 breast cancer cells with a strong
dependence on Bak. Together with our previous finding of
endoplasmic reticulum stress induction by cisplatin (3), the
present study raises the question of whether endoplasmic
reticulum stress may be induced by a variety of anticancer
drugs. To what extent these different activities contribute to
the antitumorigenic activity of each compound, and to
what extent they trigger various toxic side effects remains
to be established.
13. Costantini P, Jacotot E, Decaudin D, Kroemer G. Mitochondrion as
a novel target of anticancer chemotherapy. J Natl Cancer Inst, 2000;
92:1042 – 53.
14. Dalton LK, Demerac S, Elmes BC, Lorder JW, Swan JM, Teitel T.
Synthesis of the tumor-inhibitory alkaloids, ellipticine, 9-methoxyellipticine,
and related pyrido[4,3-b ]carbazoles. Aust J Chem, 1967;20:2715 – 27.
15. Acton EM, Narayanan VL, Risbood PA, Shoemaker RH, Vistica DT,
Boyd MR. Anticancer specificity of some ellipticinium salts against human
brain tumors in vitro. J Med Chem, 1994;37:2185 – 9.
16. Anderson WK, Gopalsamy A, Reddy PS. Design, synthesis, and study
of 9-substituted ellipticine and 2-methylellipticinium analogues as potential CNS-selective antitumor agents. J Med Chem, 1994;37:1955 – 63.
17. Jurayj J, Haugwitz RD, Varma RK, Paull KD, Barrett JF, Cushman M.
Design and synthesis of ellipticinium salts and 1,2-dihydroellipticines with
high selectivities against human CNS cancers in vitro. J Med Chem,
1994;37:2190 – 7.
18. Rouesse JG, Le Chevalier T, Caille P, et al. Phase II study of elliptinium
in advanced breast cancer. Cancer Treat Rep, 1985;69:707 – 8.
19. Tewey KM, Chen GL, Nelson EM, Liu LF. Intercalative antitumor
drugs interfere with the breakage-reunion reaction of mammalian DNA
topoisomerase II. J Biol Chem, 1984;259:9182 – 7.
20. Dodin G, Andrieux M, al Kabbani H. Binding of ellipticine to h-lactoglobulin. A physico-chemical study of the specific interaction of an antitumor drug with a transport protein. Eur J Biochem, 1990;193: 697 – 700.
21. Kovacic P, Ames JR, Lumme P, et al. Charge transfer-oxy radical mechanism for anti-cancer agents. Anticancer Drug Des, 1986;1: 197 – 214.
22. Lesca P, Rafidinarivo E, Lecointe P, Mansuy D. A class of strong
inhibitors of microsomal monooxygenases: the ellipticines. Chem Biol
Interact, 1979;24:189 – 97.
23. Sureau F, Moreau F, Millot JM, et al. Microspectrofluorometry of the
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
Molecular Cancer Therapeutics
protonation state of ellipticine, an antitumor alkaloid, in single cells.
Biophys J, 1993;65:1767 – 74.
24. Schwaller MA, Allard B, Lescot E, Moreau F. Protonophoric activity
of ellipticine and isomers across the energy-transducing membrane of
mitochondria. J Biol Chem, 1995;270:22709 – 13.
40. Lee K, Tirasophon W, Shen X, et al. IRE1-mediated unconventional
mRNA splicing and S2P-mediated ATF6 cleavage merge to regulate
XPB1 in signaling the unfolded protein response. Genes Dev, 2002;16:
452 – 66.
25. Peng Y, Li C, Chen L, Sebti S, Chen J. Rescue of mutant p53
transcription function by ellipticine. Oncogene, 2003;22:4478 – 87.
41. Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates
endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-h.
Nature, 2000;403:98 – 103.
26. Kunath T, Ordonez-Garcia C, Turbide C, Beauchemin N. Inhibition of
colonic tumor cell growth by biliary glycoprotein. Oncogene, 1995;11:
2375 – 82.
42. Bitko V, Barik S. An endoplasmic reticulum-specific stress-activated
caspase (caspase-12) is implicated in the apoptosis of A549 epithelial cells
by respiratory syncytial virus. J Cell Biochem, 2001;80:441 – 54.
27. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21
to sustain G2 arrest after DNA damage. Science, 1998;282:1497 – 501.
43. Rao RV, Peel A, Logvinova A, et al. Coupling endoplasmic reticulum
stress to the cell death program: role of the endoplasmic reticulum
chaperone GRP78. FEBS Lett, 2002;514:122 – 8.
28. Hägg M, Biven K, Ueno T, et al. A novel high-through-put assay for
screening of pro-apoptotic drugs. Invest New Drugs, 2002;20:253 – 9.
29. Wei MC, Lindsten T, Mootha VK, et al. tBID, a membrane-targeted
death ligand, oligomerizes BAK to release cytochrome c. Genes Dev,
2000;14:2060 – 71.
30. Mandic A, Viktorsson K, Molin M, et al. Cisplatin induces the
proapoptotic conformation of Bak in a yMEKK1-dependent manner. Mol
Cell Biol, 2001;21:3684 – 91.
31. Leers MP, Kolgen W, Björklund V, et al. Immunocytochemical
detection and mapping of a cytokeratin 18 neo-epitope exposed during
early apoptosis. J Pathol, 1999;187:567 – 72.
32. Griffiths GJ, Dubrez L, Morgan CP, et al. Cell damage-induced
conformational changes of the pro-apoptotic protein Bak in vivo precede
the onset of apoptosis. J Cell Biol, 1999;144:903 – 14.
33. Nechushtan A, Smith CL, Hsu YT, Youle RJ. Conformation of the Bax
C-terminus regulates subcellular location and cell death. EMBO J, 1999;
18:2330 – 41.
34. Yoshida H, Matsui T, Yamamoto A, Okada T, Mori K. XBP1 mRNA is
induced by ATF6 and spliced by IRE1 in response to endoplasmic reticulum
stress to produce a highly active transcription factor. Cell, 2001;107:
881 – 91.
35. Yoshida H, Haze K, Yanagi H, Yura T, Mori K. Identification of the cis acting endoplasmic reticulum stress response element responsible for
transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J Biol Chem, 1998;
273:33741 – 9.
36. Runnebaum IB, Nagarajan M, Bowman M, Soto D, Sukumar S.
Mutations in p53 as potential molecular markers for human breast cancer.
Proc Natl Acad Sci USA, 1991;88:10657 – 61.
37. Wei MC, Zong WX, Cheng EH, et al. Proapoptotic BAX and BAK: a
requisite gateway to mitochondrial dysfunction and death. Science, 2001;
292:727 – 30.
38. Eskes R, Desagher S, Antonsson B, Martinou JC. Bid induces the
oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol, 2000;20:929 – 35.
39. Armstrong JS, Steinauer KK, French J, et al. Bcl-2 inhibits apoptosis
induced by mitochondrial uncoupling but does not prevent mitochondrial
transmembrane depolarization. Exp Cell Res, 2001;262:170 – 9.
44. Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J Cell Biol, 2000;
150:887 – 94.
45. Perez-Sala D, Mollinedo F. Inhibition of N-linked glycosylation induces
early apoptosis in human promyelocytic HL-60 cells. J Cell Physiol,
1995;163:523 – 31.
46. Jiang S, Chow SC, Nicotera P, Orrenius S. Intracellular Ca2+ signals
activate apoptosis in thymocytes: studies using the Ca(2+)-ATPase
inhibitor thapsigargin. Exp Cell Res, 1994;212:84 – 92.
47. Kaufman RJ. Stress signaling from the lumen of the endoplasmic
reticulum: coordination of gene transcriptional and translational controls.
Genes Dev, 1999;13:1211 – 33.
48. Froelich-Ammon SJ, Patchan MW, Osheroff N, Thompson RB.
Topoisomerase II binds to ellipticine in the absence or presence of DNA.
Characterization of enzyme-drug interactions by fluorescence spectroscopy. J Biol Chem, 1995;270:14998 – 5004.
49. Shiu RP, Pouyssegur J, Pastan I. Glucose depletion accounts for the
induction of two transformation-sensitive membrane proteinsin Rous
sarcoma virus-transformed chick embryo fibroblasts. Proc Natl Acad Sci
USA, 1977;74:3840 – 4.
50. Li WW, Alexandre S, Cao X, Lee AS. Transactivation of the grp78
promoter by Ca2+ depletion. A comparative analysis with A23187 and
the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. J Biol
Chem, 1993;268:12003 – 9.
51. Shen J, Chen X, Hendershot LM, Prywes R. endoplasmic reticulum
stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding
and unmasking of Golgi localization signals. Dev Cell, 2002;3: 99 – 111.
52. Shen J, Hughes C, Chao C, et al. Coinduction of glucose-regulated
proteins and doxorubicin resistance in Chinese hamster cells. Proc Natl
Acad Sci USA, 1987;84:3278 – 82.
53. Hughes CS, Shen JW, Subjeck JR. Resistance to etoposide induced
by three glucose-regulated stresses in Chinese hamster ovary cells. Cancer
Res, 1989;49:4452 – 4.
54. Reddy RK, Mao C, Baumeister P, Austin RC, Kaufman RJ, Lee AS.
Endoplasmic reticulum chaperone protein GRP78 protects cells from
apoptosis induced by topoisomerase inhibitors: role of ATP binding site
in suppression of caspase-7 activation. J Biol Chem, 2003;278:209
15 – 24.
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.
497
Induction of endoplasmic reticulum stress by ellipticine plant
alkaloids
Maria Hägg, Maria Berndtsson, Aleksandra Mandic, et al.
Mol Cancer Ther 2004;3:489-497.
Updated version
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://mct.aacrjournals.org/content/3/4/489
This article cites 54 articles, 27 of which you can access for free at:
http://mct.aacrjournals.org/content/3/4/489.full.html#ref-list-1
This article has been cited by 4 HighWire-hosted articles. Access the articles at:
/content/3/4/489.full.html#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from mct.aacrjournals.org on June 16, 2017. © 2004 American Association for Cancer Research.