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Experimental Therapeutics |
Departments of Cancer Biology [D. J. M., L. K. N.] and Experimental Therapeutics [Y. L., R. A. N.], University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Ozelle Pharmaceuticals, Inc., San Antonio, Texas 78230 [H. Z. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Oleandrin and oleandrigenin are cardiac glycosides derived from oleander (Nerium oleander) that have been used in the treatment of cardiac abnormalities in Russia and China for years (4) . Interestingly, anecdotal evidence has emerged from this experience suggesting that they may produce beneficial side effects in patients with leiomyosarcoma, Ewing’s sarcoma, prostate cancer, and breast cancer. Indeed, there are also scattered reports of breast tumor regression in Scandinavian patients taking other cardiac glycosides (5) . Therefore, there is growing interest in evaluating the oleander products and possibly other cardiac glycosides as antineoplastic agents. The first of these therapies to be developed in the United States is a patented, water-soluble oleander extract called Anvirzel. Preclinical studies have demonstrated that the extract has excellent activity against a variety of human solid tumor cell lines. In preparation for clinical trials within the United States, an Investigational New Drug application for Anvirzel has been submitted recently for Food and Drug Administration approval.2 %Disruption of intracellular Ca2+ homeostasis results in the induction of apoptosis in diverse cell types (reviewed in Ref. 6 ). The mechanisms involved have been particularly well studied in prostate cells. In the normal prostate, androgen withdrawal leads to a rapid wave of apoptosis (7) associated with Ca2+ influx (8) and Ca2+-dependent endonuclease activation (9, 10) . In addition, agents that directly stimulate an intracellular Ca2+ increase (Ca2+ ionophores, thapsigargin) trigger apoptosis in androgen-sensitive and -insensitive prostate cancer cells (11) . Given that cardiac glycosides cause increases in cytosolic Ca2+, we wondered whether Anvirzel, oleandrin, and/or other cardiac glycosides would induce apoptosis in prostate cancer cells. Such an effect might, in part, explain the antitumor effects of the drug in cancer patients.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The PC-3 human prostatic adenocarcinoma line was obtained from American Type Culture Collection (Rockville, MD). A metastatic subclone of PC-3 (PC-3 M) was isolated from a liver metastasis produced in nude mice after intrasplenic injection of PC-3 cells (12) . PC-3 M cells were then used to derive both PC-3 M-Pro4 and PC-3 M-LN4 by orthotopic "recycling" in nude mice (13) . Both subclones are highly tumorigenic and metastatic to lymph nodes, but the LN4 cells display increased metastasis to distant sites (13) . The cells were maintained in RPMI 1640 supplemented with 10% FCS, antibiotics, sodium pyruvate, and 10 mM HEPES (pH 7.4). Cell survival/cytostasis were quantified at 72 h using the tetrazolium dye MTT3 as described previously (14) . Each experiment was performed in triplicate.
DNA Fragmentation Analysis.
DNA fragmentation was quantified by propidium iodide staining and FACS
analysis as described previously (15)
. Cells were
resuspended in PBS containing 50 µg/ml propidium iodide, 0.1% Triton
X-100, and 0.1% sodium citrate. Samples were stored at 4°C for
16 h and vortexed prior to FACS analysis (Becton Dickinson
FACScan, Mountain View, CA; FL-3 channel). For qualitative analysis of
oligonucleosomal DNA fragmentation (DNA laddering), cells were lysed in
a hypotonic buffer containing 0.5% Triton X-100, 20 mM
EDTA, and 25 mM Tris (pH 8.0) and centrifuged for 20 min at
12,000 x g, and DNA fragments in the
supernatants were precipitated with isopropanol (overnight at
-20°C). The DNA fragments were treated with proteinase K (0.2 mg/ml
for 1 h at 37°C) and resolved by electrophoresis for 1 h at
75 V on 1.5% agarose gels preimpregnated with ethidium bromide.
PARP Cleavage.
Extraction of PARP and detection of specific
Mr 89,000 caspase-derived
cleavage product by immunoblotting was conducted as described
previously (16)
. Cells (1 x 106) were resuspended in 50 µl ice-cold PBS and
mixed with 200 µl of a buffer containing 6 M
urea, 2% SDS, 10% glycerol, 5 mM EDTA, 5%
2-mercaptoethanol, and 100 mM Tris (pH 6.8).
Samples were disrupted by pipetting through a 1-ml tip and sonicated
for 20 s at high power. Lysates were then incubated for 15 min at
65°C and resolved on 8% SDS-PAGE gels. Proteins were transferred to
nitrocellulose membranes (0.2 µm; Schleicher & Scheull, Keene, NH),
and PARP was detected with a mouse monoclonal antibody (C2–10;
generously provided by Dr. Scott H. Kaufmann, Department of Oncology
Research, Mayo Clinic, Rochester, MN). Immunoreactive bands were
detected by enhanced chemiluminescence (West Pico; Pierce, Inc.,
Rockford, IL).
Caspase Processing and Activation.
Cells were lysed in a buffer containing 1% Triton X-100, 150
mM NaCl, 25 mM Tris (pH 7.5), and protease
inhibitors (Complete Mini tablet; Boehringer-Mannheim). Proteins were
resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes,
and caspases were detected by immunoblotting using polyclonal
antibodies to human caspase-3, caspase-7, caspase-8, or caspase-9
(PharMingen, San Diego, CA). Caspase activation was measured in
cytosolic extracts using the peptide substrate, DEVD-pNA (Alexis
Biochemicals, San Diego, TX; Ref. 17
). Cells were lysed in
a buffer containing 150 mM NaCl, 25 mM HEPES
(pH 7.5), 5 mM EDTA, 5 mM DTT, and 10
µM digitonin for 15 min on ice. Samples were centrifuged
for 10 min at 12,000 x g, and protein
content in the supernatants was measured using the Bradford reagent
(purchased from Bio-Rad, Hercules, CA). Extracts were diluted to 1
mg/ml in lysis buffer and incubated in triplicate for 60 min at 37°C
with 50 µM peptide. Blanks were prepared by
incubating peptide in lysis buffer alone. Absorbance was measured at
405 nm on a microtiter plate reader. Results are expressed as
absorbance units/µg protein.
Cytochrome c Release Measurements.
Release of cytochrome c from mitochondria was measured by
immunoblotting essentially as described previously (18)
.
Cells were gently lysed (30 s) in an ice-cold buffer containing 250
mM sucrose, 1 mM EDTA,
0.1% digitonin, and 25 mM Tris (pH 6.8). Lysates
were centrifuged for 2 min at 12,000 x g,
supernatants were mixed with 2x Laemmli’s reducing SDS-PAGE sample
buffer, and extracts from equal numbers of cells (10–20 x 106) were resolved by 15% SDS-PAGE.
Polypeptides were transferred to nitrocellulose membranes, and
cytochrome c was detected by immunoblotting with a
monoclonal antibody (clone 7H8.2C12; PharMingen, San Diego, CA).
Quantification of Intracellular Ca2+ in Prostate
Cancer Cells.
Cells were plated on a 22 x 30-mm glass
coverslip. On culture day 2, cells were loaded with fura-2
acetoxymethyl ester (10 µM; Molecular Probes) for 1 h at 37°C with 5% C02. The coverslips were
washed thoroughly afterward with PBS and mounted on a 1.5-ml volume
chamber (cells facing upward). The chamber was placed on an
epifluorescence/phase contrast microscope for
Ca2+ imaging and quantification. Cells were
bathed in 1 ml of HBSS with 1 mM Ca2+
at room temperature. After a baseline
[Ca2+]i was established,
cells were then treated with oleandrin (10 µg/ml), ouabain (1
µM), or thapsigargin (5 µM). An INCA
workstation (Intracellular Imaging, Inc.) was used to measure
[Ca2+]I levels.
Fluorescence was monitored using a x20 fluorescence objective. Cells
were illuminated alternately at excitation wavelengths of 340 and 380
nm using a xenon arc lamp. The emitted fluorescence was monitored at
511 nm with a video camera, and the calculated free
[Ca2+]i was determined
using the cell-free calibration curve. The data were collected with
INCA software (Win 3.1 version).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incubation of Pro4 cells with oleander extract (Anvirzel) or purified
oleandrin resulted in concentration-dependent growth arrest,
measured by mitochondrial reduction of MTT (Fig. 1
). Comparison of the IC50 concentrations of
Anvirzel and oleandrin demonstrated that oleandrin was at least 50-fold
more potent than Anvirzel.
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). Both Anvirzel and oleandrin induced
concentration-dependent DNA fragmentation in the Pro4 and LN4 cells
(Fig. 2B
). Chromatin cleavage was first detectable at
24 h and reached a maximum at 48 h (Fig. 2B
). The
nonmetastatic Pro4 cells were significantly (P < 0.01) more sensitive to oleandrin compared with the metastatic
LN4 cells at each concentration of oleandrin tested. Qualitative
analysis of DNA fragmentation by agarose gel electrophoresis confirmed
the presence of oligonucleosomal DNA fragments (DNA ladders)
characteristic of apoptosis (Fig. 3
). Oleandrin was at least 50-fold more potent than Anvirzel in promoting
DNA fragmentation (data not shown), consistent with the MTT analyses.
Oleandrin also caused marked accumulation of cells in the
G2-M phase of the cell cycle (Fig. 2
A,
arrow), indicating that cell cycle arrest also contributes to the
response. Importantly, the extent of cell cycle arrest did not appear
to vary significantly in the Pro4 and LN4 cells.
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). Concentration-response analyses demonstrated that 100 nM
concentrations of ouabain and digoxin stimulated maximal responses in
both cell lines. In this respect, all of the cardiac glycosides were
very similar (compare Figs. 2B
and 4).
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). Direct measurement of caspase activation using a
synthetic peptide substrate (DEVD-pNA) confirmed that oleandrin
stimulated protease activation more rapidly in the Pro4 cells (Fig. 5B
). These data are consistent with the DNA fragmentation
results and indicate that the LN4 cells are refractory to
oleandrin-mediated caspase activation.
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). Processing of procaspase-8 was also detectable in the LN4 cells by
36 h (Fig. 6A
). Interestingly, processing of
procaspase-8 appeared to precede processing of procaspase-3 in the Pro4
cells (Fig. 6)
. Furthermore, procaspase-8 appeared as a doublet, and
levels of both species declined in oleandrin-treated cells. Although we
do not have an explanation for this phenomenon at present, it could be
attributable to posttranslational modification. The doublet can also be
detected in human LNCaP prostate adenocarcinoma cells and a variety of
human breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-435; data
not shown), indicating that its presence is not specific to
PC-3-derived cells. Caspase processing appeared to coincide with DNA
fragmentation, consistent with work demonstrating that caspases can
directly stimulate endonuclease activation (21)
. This was
confirmed in experiments with a pan caspase inhibitor (zVADfmk), which
blocked DNA fragmentation in Pro4 cells treated with the cardiac
glycosides (Fig. 6C
).
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18 h (Fig. 7
). This release appeared to precede activation of caspase-3 and
caspase-8 (compare Figs. 6 and 7
). Importantly, oleandrin-induced
cytochrome c release in the LN4 cells was only obvious after
36 h (Fig. 7)
. Therefore, the slower kinetics of apoptosis in the
LN4 cells treated with cardiac glycosides are probably attributable to
impaired cytochrome c release.
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). Importantly, drug-induced Ca2+ increases were comparable
in the Pro4 and LN4 cells, indicating that the block in the apoptotic
pathway in the LN4 cells is downstream of Ca2+.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here we provide the first evidence that cardiac glycosides are potent
inducers of apoptosis in cells derived from the PC-3 human prostate
adenocarcinoma. Because these cells lack functional androgen receptor
and p53, apoptosis is by definition independent of these pathways. The
data indicate that oleandrin-induced apoptosis involves an early,
sustained Ca2+ increase that precedes release of
cytochrome c from mitochondria and caspase activation, and
the involvement of Ca2+ in the pathway is
consistent with the known mechanism of action of cardiac glycosides in
the heart. Importantly, we have confirmed that prostate cancer cells
express the Na+/K+- ATPase that serves as the
molecular target for the drug and that cardiac glycosides inhibit its
activity.4
The relative potency of oleandrin compared with the Anvirzel crude
oleander extract (50-fold higher) strongly suggests that much of the
proapoptotic activity of Anvirzel is directly attributable to
oleandrin, which makes up 0.25% of the extract. Their high
activities and water solubility make Anvirzel and possibly oleandrin
attractive choices for future prostate cancer therapy. In ongoing
studies, we are directly investigating the effects of Anvirzel and the
cardiac glycosides on orthotopic PC-3 tumors in nude mice
(13)
. Importantly, serum oleandrin concentrations reached
therapeutic levels in a cancer patient administered 15 mg of Anvirzel
via i.m. injection [14 nM (8 ng/ml) at 3 h,
with sustained levels above 10 nM (5 ng/ml) for
an additional 3 h, as quantified using a validated LC/MS
analytical assay5
].
Previous work has established the importance of intracellular ion
fluxes in the regulation of apoptosis. For example, many investigators
have shown that intracellular Ca2+ alterations often
precede cell death, and that the antiapoptotic BCL-2 protein acts (at
least in part) to prevent these changes (6)
. More recent
work has shown that caspase activation and DNA fragmentation are
preceded by a drop in intracellular K+ levels
(23, 24)
, and that inhibition of this drop blocks caspase
activation (20, 24) and cell death (24)
.
Importantly, cardiac glycosides induce both an increase in
Ca2+ and a decrease in K+.
In addition, parallel studies have shown that oleandrin suppresses
nuclear factor-
B activation (25)
, which could also
contribute to cell death induction (26)
. However, the cell
death-promoting activity of cardiac glycosides appears cell type
specific, because other work has shown that they inhibit multiple
pathways of apoptosis in vascular smooth muscle cells
(27)
. Whether this antiapoptotic activity will be observed
in all excitable tissues (including the heart) remains to be
determined.
Our results demonstrate that the apoptotic response of the metastatic PC-3 M-LN4 cells is attenuated compared with the response observed in the PC-3 M-Pro4 cells. This apoptosis resistance is manifest as a delay in cytochrome c release and caspase activation and is not attributable to defect(s) in early Ca2+ signaling. The uncoupling of early signaling from cytochrome c release is consistent with a role for BCL-2 family member(s) in apoptosis resistance, because these polypeptides are thought to act at the level of the mitochondrion to inhibit caspase activation (18) . Importantly, the LN4 cells are cross-resistant to several other triggers of apoptosis (staurosporine, doxorubicin, and proteasome inhibitors6 ), indicating that the mechanism(s) of apoptosis resistance is not directly linked to the biochemical resistance mechanism of oleandrin action.
Although apoptosis was a prominent factor in oleandrin-induced growth
suppression, the drug is also capable of promoting arrest at the
G2-M phase of the cell cycle (Fig. 2A
). Interestingly, the effects of oleandrin on cell cycle
arrest appear to be much more pronounced than those of
thapsigargin,7
suggesting that the oleandrin-induced intracellular Ca2+
elevation is not principally responsible for the effect and that
alterations in K+ and/or
Na+ may also be involved. Inhibition of
proliferation by Anvirzel may contribute substantially to the drug’s
antitumor effects in vivo.
Recent work indicates that caspase activation in apoptosis occurs via two general pathways (28) . In the first, so-called "death receptors," such as Fas and the type I receptor for tumor necrosis factor, can directly interact with caspase 8 and caspase 10, and ligand-induced multimerization leads directly to caspase activation. In the second, apoptotic stimuli disrupt mitochondrial homeostasis, resulting in release of cytochrome c (29) and assembly of a molecular complex known as the "apoptosome" (30) . Formation of the apoptosome catalyzes the proteolytic activation of procaspase-9, which in turn activates downstream ("effector") caspases such as caspase-3 and caspase-7. Importantly, caspase-8 activation can also lead to mitochondrial cytochrome c release via cleavage of the BCL-2 family polypeptide, BID (31) . Our observation that caspase-8 activation occurs coincidentally with cytochrome c release and precedes caspase-3 activation argues that a death receptor pathway may be involved. Alternatively, oleandrin may directly promote caspase-8 activation via a novel mechanism. The availability of bid-/- mice (32) should help define the role of BID in the mitochondrial alterations observed in this and a variety of other apoptotic model systems.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Department of Cancer Biology, University of Texas M. D.
Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.
Phone: (713) 792-8591; Fax: (713) 792-8747; E-mail: dmcconke{at}mdanderson.org 
2 R. A. Newman, personal communication.
3 The abbreviations used are: MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FACS,
fluorescence-activated cell sorter; PARP, poly(ADP-ribose)
polymerase.
4 Y. Lin, unpublished observations.
5 R. A. Newman, unpublished observations.
6 L. K. Nutt, unpublished observations.
7 Y. Lin, unpublished observations.
Received 11/24/99. Accepted 5/17/00.
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