Robert A. Newman , Peiying Yang , Alison D. Pawlus and Keith I

14 Pages·2008·2.61 MB·English

The class of steroid-like compounds designated cardiac glycosides includes well-known drugs such as
digoxin, digitoxin, and ouabain. Their continued efficacy in treatment of congestive heart failure and
as anti-arrhythmic agents is well appreciated. Less well known, however, is the emerging role of this
category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New
findings within the past five years have revealed these compounds to be involved in complex cell-signal
transduction mechanisms, resulting in selective control of human tumor but not normal cellular prolif-
eration. As such, they represent a promising form of targeted cancer chemotherapy. New clinical studies
of their anticancer potential as single or adjuvant treatments may provide insight into these potentially
valuable therapeutic options. This review focuses on recent findings on cellular pharmacology of cardiac
glycosides as they relate to treatment of human cancer and attempts to explain why these agents have been
overlooked in the past.
Robert A. Newman1, Peiying Yang1, Alison D. Pawlus1,
and Keith I. Block2
1Department of Experimental Therapeutics and Pharmaceutical Development Center,
University of Texas, M. D. Anderson Cancer Center, Houston, TX 77054;
2Institute for Integrative Cancer Care, Evanston, IL 60201
36
Cardiac Glycosides as Novel Cancer Therapeutic Agents
History of the Use of Cardiac Glycosides toad, known to contain multiple bufodeninolides (another type
for Cancer of cardiac glycoside), including bufalin (Figure 1) (5–7). The
potential use of cardenolide-like compounds for the treatment
The use of cardiac glycoside containing plants for medicinal of cancer, initially investigated forty years ago, was abandoned
purposes was first reported in ancient texts more than 1500 because of the toxicity of these compounds (8, 9). It was only
years ago. They have been used traditionally as arrow poisons, recently, however, that Scandinavian oncologists suggested that the
abortifacients, emetics, diuretics, and heart tonics. It is the latter apoptosis produced by digitalis in human tumor cells occurred at
pharmacologic activity that cardiac glycosides are most com- concentrations that could be achieved without toxicity in humans
monly associated with, and after 200 years, compounds such and, therefore, this agent might be useful for treatment of cancer
as digitalis and digoxin are still prescribed by Western doctors (10–12). In 1979, Stenkvist et al. (13) noted that the altered mor-
for control of congestive heart failure. Their use began after a phology of breast cancer cells from women treated with digitalis
meticulous analysis of a local herbalist’s formula in 1775 by the (who had undergone mastectomy). Women receiving digitalis had
English physician and scientist William Withering. He found that tumor cells with more benign characteristics than those tumor
a patient with “dropsy” (congestive heart failure) improved after cells in patients not receiving this cardiac glycoside. Moreover,
administration of an extract containing foxglove (Digitalis purpurea the cancer recurrence rate of women taking digitalis was lower,
L.) (1). Compounds extracted
from foxglove and oleander
R=lactone ring
i1n)c, lsuudceh c aasr ddeingiotalildise,s d(iFgiogxuirne, 19 11H12 18131716 H Lactone Moiety OO O
and oleandrin, which increase 2 1 10 9 8 1415 Cardenolides R= O OO
cardiac contractility and act 3 5 H 7 OH
4 6 H
as antiarrythmic agents to O
O H
H
control atrial fibrillation (2, HO O
3). The mechanism of their Bufadienolides R= OO H OH
HO Steroid Bufalin
action for the treatment of OH HO
H
congestive heart failure arises
OO O
from the inhibition of Na+,K+- Glycone
ATPase, with a resulting OO O HO OH
H
increase in intracellular cal- H
HO H
cium concentrations. Cardiac H
H O
glycosides, however, have a H OH
narrow therapeutic index, H OH O O OH OO O
limiting their wider applica-
tion to the treatment of other HO O O H HO O Ouabain H
diseases, such as cancer. OCH Oleandrin HO OH H
Despite their potential 3
to cause serious side effects, H OH
application of plant extracts O O
O O H
containing cardiac glycosides O O
HO
for the treatment of malignant OH Digitoxin
disease may extend back to OH OH OO O
HO H
Arab physicians in the eighth OO O
century (4). It is not just H
O
H
plants, however, that con- HO
H
tribute to our appreciation of HHNN SS OH H H OH
O
cardiac glycosides possibly
O O
having a role in cancer man- H OH O O H
O O
agement. An ancient Chinese O OH HO OH Digioxin
OH
medicinal treatment of cancer
OH
still in use today involves UNBS-1450 O
application of an extract of Figure 1. Structures of cardiac glycosides with antiproliferative activity. Representative cardenolide and bufadieno-
secretions of the Bufo bufo lide cardiac glycoside compounds are presented.
February 2008
Volume 8, Issue 1 37
Review
Table 1. List of Plants and Animals with Cardiac Glycosides Having Antiproliferative Activities
Plant/Animal Species Cardiac Glycoside(s) In Vitro Cytotoxic Effect Reference
Apocynum cannabinum L. Apocannoside, cymarin Human nasopharynx carcinoma (KB) (66)
(Apocynaceae)
Asclepias curassavica L. Calotropin, 16α-acetoxycalotropin, Human lung carcinoma (A549), breast carcinomas (67)
(Asclepiadaceae) 15β-hydroxycalotropin, calactin, (MCF-7 and MDA-MB-231), and hepatoma (HepG2)
15β-hydroxycalactin, asclepin,
16α-hydroxyasclepin, uscharidin,
uscharin, uzarigenin
Beaumontia brevituba Digitoxigenin, oleandrigenin, digi- Human breast carcinoma (BC1), colon carcinoma (68)
Oliver (Apocynaceae) toxigenin, α-l-cymaroside, digitoxigenin (Col2), fibrosarcoma (HT-1080), nasopharyngeal
β-gentiobiosyl-α-l-cymaroside, Δ16- carcinoma (KB), vinblastine-resistant KB (KB-V1), lung
digitoxigenin β-d-glucosyl-α-l-cymaroside carcinoma (Lu1), and melanoma (Mel2)
Bufo bufo gargarizans L. Bufalin, cinobufagin Prostate carcinomas (LNCaP, DU145, PC3), and (69, 70)
hepatoma (PLC/PRF/5)
Calotropis procera (Ait.) Calotropin, calactin, uscharin, voruscha- Human non-small-cell lung carcinoma (A549), human (71, 72)
R. Br. (Asclepiadaceae) rin, 2’’-oxovoruscharin glioblastomas (Hs683 and U373), human colon
carcinomas (HCT-15 and LoVo), hepatoma (Huh7),
non-hepatoma (COS-1), and colorectal carcinoma
(COLO 320)
Cerbera odollam Gaertner 2′-O-Acetyl cerleaside A, 17α-neriifolin, Human oral epidermoid carcinoma (KB), breast (73)
(Apocynaceae) 17β-neriifolin, cerberin carcinoma (BC), and small-cell lung carcinoma
(NCI-H187)
Coronilla varia L. Hyrcanoside Human lymphocytic leukemia (P-388) and (74)
(Fabaceae) nasopharynx carcinomas (9KB)
Crossopetalum gau- Securigenin-3β-O-β-6-deoxyguloside, Human oral epidermoid carcinoma (KB) (75)
meri (Loes.) Lundell 19-hydroxy-sarmentogenin-3β-O-β-6-
(Celastraceae) deoxyguloside, sarmentogenin-3β-O-
(α-allosyl-(1→4)-β-6-deoxyalloside),
securigenin-3β-O-(α-allosyl-(1→4)-β-6-
deoxyalloside)
Digitalis purpurea L. Digoxin, digitoxin, gitoxin Human prostate carcinomas (LNCaP, DU145, PC3), (76, 77)
(Scrophulariaceae) renal adenocarcinoma (TK-10), breast adenocarci-
Digitalis lanata noma (MCF-7), malignant melanoma (UACC-62),
(Scrophulariaceae) and chronic myelogenous leukemia (K-562)
Elaeodendron sp. Elaeodendrosides Human ovarian carcinoma (A2780) (78)
Euonymus alata (Thunb.) Acovenosigenin A 3-O-α-l- Human oral epidermoid (KB), promyelocytic (79)
Sieb. (Celastraceae) ramnopyranoside, euonymoside A, euo- lymphoma (HL-60), non-small-cell lung carcinoma
nymusoside A (A549), and cervical carcinoma (Hela)
Euonymus sieboldianus Euonymoside A Human lung carcinoma (A549) and ovarian (80)
Blume (Celastraceae) adenocarcinoma (SK-OV- 3)
Maquira calophylla (P.&E.) Maquiroside A Human oral epidermoid carcinoma (KB) (81)
C.C. Berg (Moraceae)
Nerium oleander L. Oleander, oleandrin, cardenolide N-1, Human Jurkat leukaemia (T-cell), histiocytic lymphoma (82, 83)
(Apocynaceae) cardenolide N-4, 3β-O-(β-d-sarmentosyl)- (U-937), promyelocytic lymphoma (HL-60), cervical
16β-acetoxy-14-hydroxy-5β,14β-card- carcinoma (Hela), breast carcinoma (MCF-7), pros-
20-(22)-enolide, 16β-acetoxy-3β,14- tate carcinomas (LNCap, DU145, PC3), malignant
dihydroxy-5β,14β-card-20-(22)-enolide fibroblast (VA-13), and liver carcinoma (HepG2)
38
Cardiac Glycosides as Novel Cancer Therapeutic Agents
Table 1. continued
Nierembergia aristata D. 17-epi-11α-hydroxy-6,7- Human breast carcinoma (BC1), fibrosarcoma (HT), (84)
Don (Solanaceae) dehydrostrophanthidin-3- lung cancer (LU1), melanoma (Mel2), colon carci-
O-β-boivinopyranoside; noma (Col2), oral epidermoid (KB), drug resistant KB
6,7-dehydrostrophanthidin- with and without vinblastine, epidermoid carcinoma
3-O-β-boivinopyranoside; (A-431), prostate carcinoma (LNCaP), hormone-
6,7-dehydrostrophanthidin-3-O-β- dependent breast carcinoma (ZR-75-1), and glioma
oleandropyranoside (U373)
Ornithogalum umbellatum Convallatoxin Human oral epidermoid carcinoma (KB) (85)
L. (Hyacinthaceae)
Pergularia tomentosa L. 3′-O-β-d-glucopyranosylcalactin, 12-dehy- Kaposi’s sarcoma (KS) (86)
(Asclepiadaceae) droxyghalakinoside, 6′-dehydroxygha-
lakinoside, ghalakinoside, calactin
Periploca graeca L. Periplocin isomers Human prostate carcinoma (PC-3) (87)
(Asclepiadaceae)
Rhodea japonica (Thunb.) Rhodexin A Human leukemia (K562) (88)
Roth. (Liliaceae)
Saussurea stella Maxim. 3-O-β-d-fucopyranosylstrophanthidin, Human gastric cancer (BGC-823) and hepatoma (89)
(Asteraceae) 3-O-β-d-quinovopyranosylperiplogenin, (Bel-7402)
3-O-β-d-glucopyranosyl-(1→4)-α-l-
rhamnopyranosylcannogenin, 3-O-β-d-
xylopyranosylperiplogenin, 3-O-β-d-
quinovopyranosylstrophanthidin,
3-O-β-d-xylopyranosylstrophanthidin,
3-O-β-d-fucopyranosylperiplogenin,
3-O-α-l-rhamnopyranosylcannogenol,
convallatoxin, 3-O-α-l-
rhamnpyranosylacovenosigenin A
Streblus asper Lour. Stebloside, mansonin Oral human epidermoid carcinoma (KB) (90)
(Moraceae)
Streptocaulon juven- Periplogenin digitoxoside, Human fibrosarcoma (HT-1080) (91)
tas (Lour.) Merr. Periplocymarin, digitoxigenin
(Asclepiadaceae) 3-O-(O-β-d-glucopyranosyl-(1→6)-
O-β-d-glucopyranosyl-(1→4)-β-d-
digitoxopyranoside, echujin, corchoru-
soside C
Streptocaulon griffithii 3-O-(β-glucopyranosyl)acovenosigenin A Human gastrointestinal cancer (HCG-27), lung carci- (92)
Hook.f. (Asclepiadaceae) noma (A549), breast carcinoma (MCF-7), and cervi-
cal carcinoma (HeLa)
Strophanthus gratus Ouabain Human prostate carcinomas (LNCaP, DU145, PC3) (76)
Thevetia ahouia (L.) A. Neriifolin, 3′-O-methylevomonoside, National Cancer Institute’s human disease oriented (93)
DC. (Apocynaceae) 2′-acetylneriifolin 60-cell line tumor screening panel
Thevetia peruviana Thevetin A and B, thevetoside Human hepatoma (SMMC-7721), gastric carcinoma (93)
(Pers.) K. Schum. (SGC-7901), and cervical carcinoma (HeLa)
(Apocynaceae)
Urginea maritime (L.) Proscillaridin A, scillaren A Human breast carcinoma (MCF-7) (94–96)
Baker (Liliaceae)
February 2008
Volume 8, Issue 1 39
Review
suggesting an important beneficial anticancer effect of this cardiac 1). The purpose of the present review, therefore, is to examine
glycoside (14). the hypothesis already expressed by some (10, 14–20), that use of
Within the past ten years, there has been a substantial selected cardiac glycosides may represent a worthwhile approach
increase in the number of studies observing the effects of cardiac toward control of malignant cell proliferation even despite their
glycosides on the growth of human malignant tumor cells. A narrow therapeutic index. This is all the more timely because
review of the literature indicates a surprising variety of plants and promising clinical trials of cardiac glycosides and extracts contain-
even animals whose extracts and isolated cardiac glycoside com- ing them have recently been initiated.
pounds have been cited for their antiproliferative effects (Table
Table 2. Reported Mechanisms of Cardiac Glycoside–Mediated Inhibition of Tumor Cell Proliferation
Compound Proposed antiproliferative mechanism(s) Reference
Oleandrin Alteration of membrane fluidity (15, 35, 97, 100)
Decreased activation of nuclear transcription factors NF-κB, (19, 98)
JNK, and AP-1
Increased intracellular calcium (17, 50)
Increased expression of FasL (99)
Increased ROS production, oxidative injury, and mitochondrial injury (50, 51, 58)
Decreased phosphorylation of Akt (17, 36, 57)
Inhibition of cellular transport of tumor growth factors (FGF-2) (100)
Down regulation of IL-8 receptors (99)
Initiates Apo2L/TRAIL apoptosis via increased expression of death receptors 4 and 5 (18, 48)
Activation of calcineurin and nuclear transcription factor NF-AT (99)
Bufalin Increased activation of MAPKs (101–103)
Decreased cAMP content (5)
Inhibition of topoisomerases I and II (17, 104, 105)
Induction of differentiation in human myeloid leukemia (106, 107)
Downregulation of cyclin A, Bcl-2 and Bcl-xL; Increased expression of p21 and Bax (7, 104, 108)
Ouabain, Digitoxin Loss of mitochondrial membrane potential; increase Par-4 expression (17, 109)
Increased Ca2+ uptake (15, 16, 110, 111)
Acts as an estrogen receptor antagonist (15, 112)
Sustained ROS production (29, 31, 111)
Regulates expression of cell tight junctions and adhesion molecules (16, 17, 55)
Selective protein kinase C activation leading to differentiation (17, 113)
Increased activation of MAPKs (16, 113, 114)
Reduction in anti-apoptotic proteins Bcl-xL and Bcl-2 (114–116)
Increased cytochrome c release and caspase activation (28, 114, 116)
Inhibition of topoisomerase I (28, 61, 117, 118)
Block activation of the TNF-α/NF-κB signaling pathway (59, 119)
UNBS1450 Decreased heat shock protein (Hsp70) (59, 120)
Increased permeabilization of lyososomal membrane (120)
Block activation of the TNF-α/NF-κB signaling pathway (59)
NF-κB, Nuclear Factor-kappaB; JNK, c-Jun NH-terminal kinase; AP-1, Activator Protein-1; FasL, Fas ligand; ROS, reactive oxygen species;
2
FGF-2, Fibroblast Growth Factor 2; IL-8, Interleukin-8; TNF-α, Tumor Necrosis Factor–α; TRAIL, (TNF)-related apoptosis-inducing ligand; NF-AT,
Nuclear Factor of Activated T cells; MAPKs, mitogen-activated protein kinases.
40
Cardiac Glycosides as Novel Cancer Therapeutic Agents
Na+,K+-ATPase: Beyond Cell Membrane cinogen 1,2-dimethyldrazine (24). There have also been reports
Exchange of Na+ and K+ of increased expression of particular subunits of Na+,K+-ATPase
in gastric (25) and bladder cancers (26). In addition, alterations
Na+,K+-ATPase, as an energy-transducing ion pump, has been in overall Na+,K+-ATPase activity and relative subunit abundance
studied extensively since its discovery in 1957 (21). This enzyme were observed in a highly invasive form of human renal carci-
consists of two types of subunits, designated α and β, in addition noma cells (27), non-small cell lung cancer (28), and carcinoma
to a single-transmembrane-spanning protein, FXYD––named for cell lines obtained from a number of other tissues (29). It would
the conserved amino acids in its signature motif: (Phe-Xxx-Tyr- appear, however, that simply looking at enzyme subunit content
Asp). The α subunit, responsible for binding of Mg2+, ATP, Na+, K+, or relative activity in malignant and non-malignant tissue may not
and cardiac glycosides, is considered the catalytic subunit of the provide adequate insight into the role of this enzyme in cancer.
enzyme. The β subunit is a glycoprotein that seems to act as an This can now be interpreted in the light of newly proposed conse-
adhesion molecule that regulates gap junction proteins; is involved quences of cardiac glycoside binding to Na+,K+-ATPase.
in structural and functional maturation of the holoenzyme; facili-
tates transport of the α subunit to the plasma membrane and main- Proposed Mechanism(s) of
tenance of the enzyme in the lateral membrane of epithelial cells Cardiac Glycoside–Mediated
(15–17). The function of the FXYD protein involves regulation of Antiproliferative Effects
the enzyme function, thus adapting the kinetic properties of active
Na+ and K+ transport to the specific needs of different cells (22, 23). An explanation of the role of Na+,K+-ATPase in complex cell
Four α subunit variants, as well as three β, and seven FXYD sub- signaling pathways, many of which are of critical importance
unit variants have been identified (17). The well-established func- to malignant cell proliferation, has been put forth by Xie and
tion of Na+,K+-ATPase is to use ATP as an energy source to drive colleagues (30–32). They have shown that binding of cardiac
excess Na+ out of cells in exchange for K+, thereby maintaining an glycosides (e.g., ouabain) to Na+,K+-ATPase triggers a complex
essential ionic and osmotic
Apo2/TRAIL
balance. Binding of certain α (J)
Upregulation of death receptors
subunits by cardiac glycosides (A) Altered membrane
inhibits ATP binding and dis- DR4 DR5 Oleandrin, TNF-(cid:65) fluidty
bufalin,
rupts the ability of the enzyme Caveolin digitoxin TNFR
to perform this exchange in an FGF-2 (I)
efficient manner. This, in turn, FADD EGFR Export inhibited
Caspase (H)
results in an enhanced entry Activation (cid:66) (cid:66)
TRADD Lysosomal membrane
of calcium into cells, which, Apoptosis PLC (cid:65) (cid:65) Caveolin RIP/TRAF2 permeabilization
Apoptosis(N)
in the event of failing cardiac ER/SC IP3 PI3K Src (F)
Ca2+ (G) JNK
myofibrils, helps produce a
Ras
more efficient myocardial con- Raf ASK-1 Cytochrome c release
traction and improves cardiac (B) Ca2+ i Akt NF-(cid:43)B(E) MEK (K) (M) LC3
pump activity. PKC PAkt (D) ROS
What, then, is the evi- Apoptosis EpErkr k1 /12/2 (L) Mitochondria Minjiutorcyh aonnddrial Autophagy
dence supporting the hypoth- Nuclear mpoetmenbtriaalne condensation
esis that Na+,K+-ATPase may AP-1 membrane Gene activation Bcl-XL, BcI-2
(C) differentiation topoisomerase I and IIa
be an important target for
cancer therapy? For the past Figure 2. The Na+,K+-ATPase signalosome complex. The binding of selected cardiac glycosides (CGs)––such as ole-
ten years, published stud- andrin, bufalin, and digitoxin––to Na+,K+-ATPase results in complex but well-documented changes in cell signaling events.
The “signalosome” complex includes the enzyme, Na+,K+-ATPase as well as Src, phosphoinositide-3 kinase (PI3K), and
ies have suggested a role for
phospholipase C each of which, in turn, sets into action complex signaling events that can result in tumor cell death
Na+,K+-ATPase in regulation through either apoptosis or autophagy-related mechanisms. Administration of CGs can (A) increase the (cell surface)
of cell growth and expres- expression of death receptors (DR4, DR5) and activate caspase activity; (B) result in increased intracellular calcium con-
centrations, which, in turn, (C) decreases the expression of transcription factors such as Activator Protein-1 (AP-1). CG
sion of various genes beyond
treatment can also (D) inhibit activation (i.e., block the phosphorylation) of Akt, which normally blocks apoptosis; (E) inhibit
that of ion transport. Davies activation of the transcription factor Nuclear Factor-kappaB (NF-κB); (F) activate the Ras pathway, leading to increase
et al., for example, observed activity of Raf–MAPK pathway; (G) activate Src; (H) inhibit tumor necrosis factor (TNF)-mediated activation of NF-κB by
inhibiting the binding of tumor necrosis factor receptor 1–associated death domain protein (TRADD) to the cellular mem-
altered Na+,K+-ATPase activ-
brane; (I) inhibit extracellular transport of tumor growth factors, such as fibroblast growth factor-2 (FGF-2); (J) alter mem-
ity in premalignant mucosa brane fluidity which, in turn, may inhibit Fas-related signaling; (K) lead to the production of reactive oxygen species (ROS)
months before tumor devel- with subsequent injury to mitochondria; (L) produce a decrease in mitochondria membrane potential and a decrease in
quantity of anti-apoptotic proteins Bcl-XL and Bcl-2 and topoisomerases I and II; and (M) cause mitochondrial condensa-
opment induced by the car-
tion and loss of function, that, in turn, can lead to autophagic processes and cell death (N). Adapted from (30).
February 2008
Volume 8, Issue 1 41
Review
A 120
tion has been compiled (Table 2 and Figure 2), and there are
Panc-02
several excellent reviews on this subject (15–17). There are uni-
BxPC3
100
MiaPaca fying themes that link mechanisms involving the water-soluble
h PANC-1
wt (ouabain) and relatively lipid-soluble (oleandrin, bufalin, and
gro 80 digitoxin) cardiac glycosides, including activation of ERK1/2;
cell increased expression of the cell cycle inhibitor p21Cip1 and con-
ntrol 60 15.6 nM 210 nM IC sequent inhibition of cell cycle progression (through decreased
o 50
c expression of cyclin proteins); inhibition of transcription factors,
nt of 40 5.6 nM such as Nuclear Factor-kappaB (NF-κB) and Activator Protein-1
e
erc (AP-1); inhibition of Akt (a protein serine–threonine kinase) and
P 20 related critical components of the phosphoinositide-3 kinase
(PI3K) pathway; initiation of death receptor–mediated apoptosis;
0 sustained ROS production with consequent mitochondrial injury;
0 100 200 300 400 500 600
and inhibition of topoisomerases and reduction in expression of
Concentration (nM)
anti-apoptotic proteins, such as Bcl-xL and Bcl-2. Although the
B Panc-02 BxPC3 MiaPaca PANC-1 kaalrnle o orwfe lntah tpee rdrie mtpoao rirynt ehtdaib rmgiteieotc noh fao ncfa itsrhmdiissa ciom fg playoncrtotipasinrdote lesifn eizsr yaNtmiave+e., KaTc+h-tAiaoTt nPi sa(,T sceaa,b rnldeoi a2tc )
(cid:65) 1 glycosides such as bufalin and oleandrin should be thought of as
pleiotropic or multi-mechanistic anticancer agents. Few, if any, of
(cid:65) 3 these published reports on mechanism of action of cardiac glyco-
sides, however, address reasons for the well-established differential
(cid:66)-Actin in cardiac glycoside-mediated effects on human- vs rodent-derived
malignant cell lines.
Figure 3. Relative human and rodent tumor cell sensitivity to olean-
drin correlates with Na+,K+-ATPase subunit composition. A. Mouse
pancreatic cancer cells that lack expression of the α-3 subunit (Panc-02) are Species-Dependent Sensitivity and Selective
non-responsive to oleandrin whereas human tumor cell lines MiaPaca and Human Tumor Cell Response to Cardiac
Panc-1 (that contain high expression of α-3 relative to α-1) are extremely
Glycosides: Important Mechanistic Clues
responsive. The human pancreatic tumor cell line BXPC3 that contains a
very low level of the α-3 subunit, as per immunoblot analysis (B), is some-
what resistant to cytotoxic effects of oleandrin. These data suggest that it is
An unusual attribute of compounds such as oleandrin, bufalin,
the lack of α3 in rodent tumor cell lines that explains their resistance to car-
diac glycosides. In addition, the data suggest that it is the relative α3:α1 ratio and digitoxin is that they are, on the one hand, almost completely
correlates with human tumor cell sensitivity to lipid soluble cardiac glycosides nontoxic to rodent (mouse, rat, and hamster)-derived tumor cell
such as oleandrin.
lines but potently inhibit proliferation of monkey and human
signaling cascade that is initiated by interacting with neighboring tumor cell lines at nanomolar concentrations (33– 36). These
membrane proteins and organized cytosolic cascades of signaling results have been confirmed across a wide spectrum of human
molecules. These signaling complexes send messages to intracellu- and rodent tumor cell lines, including those of hematologic and
lar organelles via the activation of the protein tyrosine kinase Src, solid tumor derivation. This species-dependent disparity in tumor
transactivation of epidermal growth factor receptor (EGFR) by Src, cell sensitivity to a proposed antitumor agent is unusual. It may, in
activation of Ras and the p42/44 mitogen-activated protein kinases fact, have contributed to the conclusion in the 1970s that cardiac
[MAPKs, also termed extracellular-regulated protein kinases 1 and glycosides were without efficacy because murine P388 and L1210
2 (ERK1/2)], and increased generation of reactive oxygen species lymphocytic leukemia cell lines were, at the time, the only cell
(ROS) by mitochondria. Activation of these cellular pathways is lines available for anticancer drug development. The magnitude
also linked with translocation of Na+,K+-ATPase, through endo- of difference in response of murine as compared to human tumor
cytosis, to the nucleus (17). In fact, Xie and colleagues (17, 30) cell lines suggests that a fundamental difference in drug targeting
have referred to this as the Na+,K+-ATPase-Src-caveolin “signalo- exists and, thus, serves as a probe or model for re-examination of
some” complex (Figure 2). The view of Na+,K+-ATPase as a simple the pharmacologic role of this class of compounds in treatment of
ion-exchange pump situated solely at the cell membrane is thus human cancers.
outmoded. Further research on this enzyme, including its role in Whereas the differences between human and murine
regulation of cell proliferation and its inhibition through cardiac responses to cardiac glycosides are interesting and should be stud-
glycosides, is clearly warranted. ied further, the differential effects of cardiac glycosieds on human
The diverse mechanisms reported to specifically be involved tumor vs normal cells are essential to their usefulness as a therapy.
in cardiac glycoside-mediated control of malignant cell prolifera- Several published studies have confirmed the observation that car-
42
Cardiac Glycosides as Novel Cancer Therapeutic Agents
diac glycosides have a selective effect on malignant but not normal some prognostic value if that patient is to be subsequently treated
cell proliferation. For example, oleandrin suppresses the activation with a cardiac glycoside.
of certain transcription factors and potentiates ceramide-induced Mijatovic et al., on the other hand, suggest that, rather than
apoptosis in human tumor cells but not in normal, primary the α3 subunit, it is the α1 subunit of Na+,K+-ATPase that could
human cells (19). In vitro observations that leukemia cells under- represent a novel anticancer target (47). They have shown that
go apoptosis in the presence of oleandrin and bufalin, but that human lung cancer cell lines overexpressing the α1 subunit were
normal leukocytes do not, are also consistent with the hypothesis sensitive to a few select cardenolides. They noted that the cardiac
of a potentially therapeutic, selective therapeutic effect of cardiac glycosides produced a marked change in the actin cytoskeleton,
glycosides on tumor growth (37–39). Not only do cardiac gly- suggesting this abets tumor cell death. Whether it is altered
cosides appear to be more effective at inhibiting proliferation of expression of α1, as suggested by Mijatovic et al., or an elevation
malignant cells than normal cells, but they also are more effective of α3, as indicated by our own work, or perhaps even a specific
at sensitizing tumor cells to irradiation, which would appear to ratio of α3:α1 that is most important as a predictor of cell sensi-
increase their potential utility in the clinic. Research reported by tivity, remains to be determined. More research, using human tis-
several investigators (40–42) indicates that cardiac glycosides sen- sues and not just cell lines, will no doubt shed light on the poten-
sitize human tumor but not normal cells to subsequent radiation tial importance and perhaps even prognostic value of the enzyme
treatment. These data suggest that it may be possible to exploit subunit composition within individual types of tumors.
differences in the Na+,K+-ATPase pumps of normal as opposed to It is significant that the relative composition of Na+,K+-ATPase
tumor cells to improve the therapeutic index of radiation therapy. subunits may not be static within human tissues. The relative ratio
Modern drug development seeks specific biochemical differ- of α subunits within the enzyme may shift when tissues are trans-
ences between malignant and normal cells that may be critical to formed from a benign to a malignant state. Sakai et al. (46) recent-
survival of cancer cells. One then attempts to develop selective ly showed, for example, that a decrease in the α1 isoform and an
inhibitors to disrupt these pathways. Na+,K+-ATPase, however, is increase in the α3 subunit occurs in colon tissue when a normal
a ubiquitous enzyme present in every mammalian cell. At first phenotype changes to a malignant one. If, as our data suggest, it
appearance, therefore, it would appear to make Na+,K+-ATPase is the relative expression of α3 that is important for determining
an anticancer target of dubious value unless, of course, the target sensitivity of a tissue to inhibition by cardiac glycosides then, in
were found to be fundamentally different in normal versus malig- essence, the report by Sakai et al. suggests that the tumor becomes
nant human cells or between rodent and mammalian cancer cells. a more sensitive target than normal tissue to cardiac glycoside
Our recent data suggest that, in fact, there is a difference in the therapy (46). Given the current as well as proposed clinical trials
basic subunit composition of Na+,K+-ATPase that might explain of cardiac glycosides for treatment of cancer, specific determina-
the differential species-dependent sensitivity to cardiac glycosides. tion of enzyme subunit composition in specific tissue types as
Although human tumor cells and tissues commonly express both well as pathologic characterization may prove to be a timely tool
α1 and α3 subunits, all rodent tumor cell lines we have examined to help optimize the effectiveness of this class of potential cancer
to date only express the α1 subunit (Figure 3). Early reviews of therapeutic agents.
the biochemical properties of Na+,K+-ATPase suggested that cardi-
ac glycoside binding may be equal to all four α subunit isoforms; Cardiac Glycoside-Mediated Cancer Cell
however, more recent studies have shown a clear preferential Death: Autophagy and Apoptosis
binding of cardenolides to the α3 form over that of the α1 or α2
isoforms (43–45). For example, O’Brien et al. (45) cite a 1000-fold Although it is clear that lipid-soluble cardiac glycosides (i.e.,
difference in binding of ouabain to the α3 isoform over that of digitoxin, oleandrin, and bufalin) have a potent ability to pro-
α1. Given the fact that rodent tumor cells possess the α1 subunit, duce human tumor cell death, the mechanisms by which this is
lack expression of α3, and are unresponsive to inhibition of prolif- accomplished are still being defined. Apoptotic cell death medi-
eration with cardiac glycosides, we suggest that the α3 subunit is ated by cardenolides has been demonstrated in a number of cell
critical. The increased expression of α3 over α1 subunits has also lines. Sreenivasan et al., for example, have shown that oleandrin
been noted in human colon colorectal cancer and colon adeno- produced an increase in expression of Fas and Tumor Necrosis
carcinoma cell lines (e.g., KM12-L4, T-84, HT-29, and WiDr), Factor Receptor 1 (TNFR1), resulting in potentiation of apoptosis
whereas no significant expression of the α3 isoform protein was in tumor cells but not in normal primary cells, such as peripheral
noted in the normal kidney and renal tissues (46). Moreover, blood mononuclear cells or neutrophils (48). Fas–Fas ligand and
human tumor cell lines with a low ratio of α3:α1 are relatively TNF–TNFR1 death pathways are important mediators of apopto-
resistant to growth inhibition with cardiac glycosides but those sis (49). Another recent report has shown that oleandrin, bufalin,
tumor cell lines with high α3:α1 ratios are very sensitive (Figures digoxin, and digitoxin initiate apoptosis induced by Apo2L/TNF-
3 and 4). This finding, of course, also suggests that determination related apoptosis-inducing ligand (TRAIL) in non-small-cell lung
of the relative α3:α1 ratio in tumor biopsy specimens may have cancer cells by increasing the expression of death receptors 4 and
February 2008
Volume 8, Issue 1 43
Review
5 (18). Because Apo2L/TRAIL induces apoptosis in tumor cells Cardiac Glycosides and Cancer Prevention
with little if any toxicity to normal cells, this cytokine is of great
interest to cancer researchers. The selective cardenolide activation Recent investigations of potent cardiac glycosides have focused on
of death receptors may very well contribute to the observation their potential application to the treatment of established cancers;
that compounds such as oleandrin are relatively selective in their however, at least one report has suggested that there may also be
cytotoxic activity. a chemopreventive role for this class of agents. That is, Afaq et al.
Oleandrin elicits caspase-associated apoptosis in human have suggested that oleandrin might serve as an effective agent
prostate carcinoma cells (50). Interestingly, however, treatment of for the prevention or treatment of skin cancer (57). Their research
human PANC-1 pancreatic cancer cells produces clear hallmarks investigated the topical application of oleandrin to CD-1 mice to
of autophagy, including formation of autophagosome bodies with counteract the effects of TPA (12-0-tetradecanoylphorbol-13-ace-
damaged mitochondria and expression of light chain-1 protein, an tate), a widely used skin tumor promoter. The topical application
early indicator of autophagosome formation (51). Frese et al. have of TPA to mouse skin or its treatment in certain epidermal cells is
also suggested that the apoptotic potential of cardiac glycosides known to result in several biochemical alterations, changes in cel-
depends on the cell type treated (18). Our data on the differential lular functions, and histological changes leading to dermal tumor
effects of oleandrin on tumor cells, such as pancreatic vs prostate promotion. The data of Afaq et al. clearly show that application of
tumor cells, as compared to oleandrin-treated normal human cells oleandrin to skin prior to TPA administration affords significant
concurs with this. inhibition of TPA-induced skin edema, hyperplasia, epidermal
ornithine decarboxylase (ODC) activity, and protein expression of
Cardiac Glycosides and Estrogen ODC and cyclooxygenase-2 (COX-2), classical markers of inflam-
Receptor Interaction mation and tumor promotion. Their data also show that topical
application of oleandrin prior to TPA inhibits activation of PI3K
Selected cardiac glycosides may be of particular importance in the and phosphorylation of Akt, activation of NF-κB, and degradation
treatment of human breast cancer. Chen et al. (15) have recently and phosphorylation of the inhibitor of NF-κB α protein (IκBα).
suggested several reasons why cardenolides should be developed These authors, therefore, recommend the use of chemopreven-
as anti-breast cancer drugs. These include the facts that: 1) Na+,K+ tive agents (i.e., oleandrin) in formulations such as emollients or
-ATPase is a key player of cell adhesion and is involved in cancer patches for the prevention or treatment of skin cancer (57). This
progression; 2) the enzyme serves as a versatile signal transducer suggestion is all the more relevant when considered in light of our
involving a number of hormones, including estrogens; and 3) the own work which shows a potent ability of oleandrin to inhibit
aberrant expression and activity of this enzyme in breast cancer human melanoma proliferation (58).
implicates an etiologic or at least contributing role of Na+,K+-
ATPase in the development and progression of this malignant In Vivo Efficacy and Development of
disease. For example, there is now strong evidence that Na+,K+- Cardiac Glycosides for Clinical
ATPase plays an important role in the assembly of tight junctions Cancer Therapy
(TJs) and cell adhesion (52–55). Chen et al. convincingly argue
that altered expression and malfunction of Na+,K+-ATPase may Rodent tumor cells fail to respond to cardiac glycosides in vitro.
lead to abnormal TJ structure and, thus, to altered cell adhesion Similarly, it has been very difficult to demonstrate an in vivo
important in the progression of breast cancer. As mentioned pre- response of syngeneic rodent tumors to administration of this class
viously, there are also strong data supporting the role of Na+,K+- of compounds. However, as shown in Figure 4, there is no ques-
ATPase in a complex signalosome involved in transmitting mem- tion that human tumor cell lines are extremely sensitive to treat-
brane signals to the nucleus. ment with cardiac glycosides such as oleandrin and bufalin. Thus,
A series of reports suggests that estrogen receptor (ER) ligands it is possible that human tumor xenografts would respond. Indeed,
(e.g., 17β-estradiol and estrogen-like molecules) can also serve as this is exactly the case, as shown by several investigators. Han et
ligands of Na+,K+-ATPase. Use of 17β-estradiol enhances Na+,K+- al. (7), for example, explored the response of a human hepatocel-
ATPase activity (56) possibly through improvement of the interac- lular carcinoma cell line (BEL-7402) implanted orthotopically
tion of the enzyme with ATP as well as Na+ and K+ ions. Because (i.e., transplantation of cells or tissue into its normal anatomical
the interaction of 17β-estradiol with ERs serves as an important site) in liver tissue to intraperitoneal treatment with bufalin. They
determinant of breast cancer growth, and cardiac glycosides can found that this toad-derived cardiac glycoside produced significant
block this interaction, cardenolides could be considered effective reductions in tumor volumes and a prolongation in life-span of
modulators of estradiol-dependent breast cancer proliferation. the animals. Importantly, no adverse morphological changes were
noted in myocardial, hepatic, or renal tissues. Another interesting
report involved use of the semi-synthetic cardenolide UNBS-1450
against orthotopically implanted human non-small-cell lung cancer
44
Cardiac Glycosides as Novel Cancer Therapeutic Agents
BxPC3 PANC-1
shown, however, that single cardenolides, or those derived from
various extracts of plants and animals (Table 1), represent potent
3(cid:65) compounds with selective effects against human tumor cell lines
e-
a,K-TPas annodli dxee ndoegrirvaafttsiv. eF oorf ae xcaamrdpialec, gUlyNcBoSsi-d1e4 5o0ri,g ain saelmly iissyonlathteedti cfr coamrd aen-
NA
African plant (Calotropis procera), has shown promising activity
against human non-small cell lung carcinomas growing as xeno-
grafts in nude mice (59). UNBS-1450 can be safely administered
drin to mice in doses that are twenty-four times greater than that of
an ouabain and twelves times greater than that of the parent com-
e
Ol pound oxovoruscharin (64). With good activity against non-small
cell lung cancers in mice with metastases in the brain and liver,
UNBS-1450 is entering phase I clinical trials in Belgium (17).
Figure 4. Relationship of expression of Na+,K+-ATPase α3 subunit and
To date, there is only a single plant extract, Anvirzel™,
binding of oleandrin to human pancreatic cancer cell membranes.
Cells were stained using a mitochondrial dye, Mitotracker, (red color) and a derived from Nerium oleander, that has progressed through a
nuclear Herscht dye (blue). Top row: Cells were also exposed to an antibody Phase I trial in the United States. Anvirzel is a hot-water extract
to the α3 subunit of Na+,K+-ATPase (green color). Bottom row: Cells were
of oleander whose encouraging preclinical activity (65) led to a
incubated with a fluorescent analog of oleandrin (green color). The presence
of green staining (top row) indicates the relative presence of the α3 subunit successful investigational new drug application (IND) from the
which is more prevalent in the PANC-1 than the BXPC3 cells. There is a cor- FDA and to a Phase I clinical trial performed at the Cleveland
responding uptake of oleandrin (bottom row) only in those cells (PANC-1)
Clinic between 2000 and 2001. No objective responses were
that express the α3 subunit.
noted which might be because the limited time of exposure to
(59). Chronic oral administration of this compound produced a the product and intramuscular route of administration limited
beneficial therapeutic effect against these orthotopically implanted the total volume of extract that could be administered on a daily
A549 tumor cells. Finally, digoxin inhibits human neuroblastoma basis. A longer time of exposure and a different route of admin-
growth in vitro and significantly reduced the growth of human istration may impact response. Also, no dose-limiting toxicities
neuroblastoma tumor in vivo in mice (60), whereby the antitumor were found. The product known as PBI-05204 was produced in
effect arose, at least in part, from an antiangiogenic effect because response to the need for a formulation and route of administration
digoxin was also found to be effective in the chicken chorioallanto- suitable for adequately exploring the anticancer potential of an
ic membrane (CAM) assay. Thus, although limited, evidence exists oleander extract. PBI-05204 is a modified supercritical CO extract
2
for antitumor activity against human tumor xenografts. of organically grown Nerium oleander that has been especially for-
In this age of targeted therapeutics, however, where one mulated for oral administration to humans. An IND for evaluation
strives for selective effects within tumor cells so as to increase of PBI-05204 as a “botanical drug” was obtained from the FDA in
effectiveness and also minimize toxicity to normal tissues, one September, 2007, and a Phase I clinical trial in patients with solid
might reasonably question why cardiac glycosides with a known tumors has now been initiated at the University of Texas M.D.
narrow therapeutic index would ever be considered for clini- Anderson Cancer Center. Given the expanding knowledge of the
cal development as an anticancer therapy. There is little doubt multiple anticancer mechanisms for cardenolides, the outcome of
that the potential for serious cardiovascular toxicity exists with these early stage clinical trials of potent plant extracts, such as the
many, if not all cardiac glycosides, but the risks appear manage- proposed botanical drug PBI-05204 and simpler single chemical
able because the effective concentrations (i.e., in the nanomolar entities such as UNBS-1450, will be of great interest. The results
range) of these agents to control cancer-cell proliferation are well may hold the key as to whether this class of potent natural prod-
below concentrations that produce cardiac toxicity (61). Because ucts is worthy of further development as primary and/or adjuvant
the cardiovascular toxicities associated with this class of agents therapy for malignant diseases.
are well documented, careful monitoring of plasma concentra-
tions may allow for the continued safe use of digitalis and related Conclusions
compounds. Moreover, specific antibody-based treatments, such
as digoxin-specific F antibody fragments (Digibind; DigiTAb), as Our understanding of the spectrum of the pharmacologic activities
ab
well as an older treatment, activated charcoal, are available to res- of cardiac glycosides has increased significantly since the discov-
cue patients receiving accidental over-medication (62, 63). ery of their effectiveness for treatment of congestive heart failure.
The desirable goal of producing a synthetically derived car- It is now recognized that certain cardiac glycosides are involved
diac glycoside with potent ability to inhibit proliferation of human in complex cell signal transduction mechanisms that may have
tumors but without the potential to cause cardiac related toxicity, important consequences in their application to the prevention
unfortunately, has not yet been fully achieved. Investigators have and/or treatment of malignant diseases. Development of clinically
February 2008
Volume 8, Issue 1 45