Evaluating the Cancer Therapeutic Potential of Cardiac Glycosides

Abstract

Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na(+)/K(+)-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

Full text

Review Article
Evaluating the Cancer Therapeutic Potential of
Cardiac Glycosides
José Manuel Calderón-Montaño,1Estefanía Burgos-Morón,1Manuel Luis Orta,2
Dolores Maldonado-Navas,1Irene García-Domínguez,1and Miguel López-Lázaro1
1Department of Pharmacology, Faculty of Pharmacy, University of Seville, 41012 Seville, Spain
2DepartmentofCellBiology,FacultyofBiology,UniversityofSeville,Spain
Correspondence should be addressed to Miguel L´
opez-L´
azaro; mlopezlazaro@us.es
Received  February ; Revised  April ; Accepted  April ; Published  May 
Academic Editor: Gautam Sethi
Copyright ©  Jos´
e Manuel Calder´
on-Monta˜
no et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an
unsaturated lactone ring and the ability to induce cardiotonic eects mediated by a selective inhibition of the Na+/K+-ATPase.
Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias.
Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. ese compounds typically inhibit cancer
cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identied
cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models,
which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides
may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in
mice xenograed with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells
versus rodent cells. is paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer
therapeutic potential of cardiac glycosides in preclinical studies.
1. Introduction
Cardiac glycosides, also known as cardiotonic steroids, are
natural products with a steroid-like structure and an unsat-
urated lactone ring. ey usually contain sugar moieties
in their structure and have cardiotonic activity. Cardiac
glycosides containing the lactone -furanone are known as
cardenolides and those containing the lactone -pyrone are
known as bufadienolides (Figure ). Most cardiac glycosides
(e.g., digitoxin, digoxin, ouabain, and oleandrin) have been
isolated from plants, including Digitalis purpurea, Digitalis
lanata,Strophanthus gratus,andNerium oleander.Some
cardiac glycosides have also been found in amphibians and
mammals, including digoxin, ouabain, bufalin, marinobufa-
genin, and telecinobufagin. Several cardiac glycosides are
used in cardiology for the treatment of cardiac congestion
andsometypesofcardiacarrhythmias.emechanismby
which these drugs aect cardiac contractility is thought to
be mediated by a highly specic inhibition of the Na+/K+-
ATPase pump [–].
Over the years, several reports have suggested that cardiac
glycosides may have an anticancer utilization (reviewed in
[–]). In vitro and ex vivo experiments have revealed that
some cardiac glycosides (e.g., digitoxin) induce potent and
selective anticancer eects [,,], which may occur at con-
centrations commonly found in the plasma of patients treated
with these drugs []. Recent high-throughput screenings
of drug libraries have identied several cardiac glycosides
(e.g., digoxin, ouabain, and bufalin) as potent inhibitors of
cancer cell growth [–]. ese cardiac glycosides were also
able to block tumor growth in mice xenotransplanted with
human cancer cells, further supporting the idea that these
compounds should be evaluated in cancer patients [–].
e cardiac drugs digitoxin and digoxin, the semisynthetic
cardiac glycoside UNBS, and two extracts from the plant
Nerium oleander have entered clinical trials for the treatment
Hindawi Publishing Corporation
BioMed Research International
Volume 2014, Article ID 794930, 9 pages
http://dx.doi.org/10.1155/2014/794930

BioMed Research International
RO
OH
Cardenolide Bufadienolide
Digitoxin: R = digitoxose-digitoxose-digitoxose Bufalin: R = H
RO
OH
OO
O
O
F : Chemical structure of cardiac glycosides. e basic skeletons of cardenolides and bufadienolides and the structures of the
cardenolide digitoxin and the bufadienolide bufalin are shown.
of cancer (see http://clinicaltrials.gov/ and ref. [,,,,
]).
Research results also suggest, however, that cardiac gly-
cosides may not inhibit cancer cell proliferation selectively
in particular types of cancer [–]andthepotentinhi-
bition of tumor growth induced by cardiac glycosides in
mice xenograed with human cancer cells is probably an
experimental artifact caused by their ability to selectively
kill human cells versus rodent cells rather than by their
ability to selectively kill human cancer cells versus human
normal cells [–]. Aer reviewing such evidence, this
paper discusses experimental approaches that can be used to
reveal the cancer therapeutic potential of cardiac glycosides
in preclinical studies.
2. Possible Misinterpretation of
Data from Preclinical Studies
Inhibition of cancer cell proliferation at low concentrations
and inhibition of tumor growth in animal models are the
most common parameters used by researchers to assess the
therapeutic potential of drug candidates in preclinical studies.
Based on this approach, researchers have proposed cardiac
glycosides as candidates for evaluation in clinical trials. is
section of the paper reviews evidence indicating that this
approach may be inadequate to reveal the cancer therapeutic
potential of cardiac glycosides.
2.1. Inhibition of Cancer Cell Proliferation at Low Concen-
trations Does Not Reliably Predict erapeutic Potential. e
key feature of an ecient anticancer drug candidate is its
ability to kill (or to inhibit the proliferation of) human cancer
cells at concentrations that do not signicantly aect human
nonmalignant cells. If the anticancer drug candidate does not
have this feature, it does not really matter whether or not it
can kill cancer cells at low concentrations. e reason is that
the drug concentrations required to kill the tumor cells of
cancer patients would also cause the death of their normal
cells and, therefore, would be lethal to these patients. It is
important to note that the therapeutic potential of a drug able
to kill cancer cells at a concentration of  millimolar without
signicantly aecting nonmalignant cells at a concentration
of  millimolar is probably higher than that of a drug that
kills both cancer and nonmalignant cells at a concentration
of  nanomolar.
Cancer researchers do not commonly use human non-
malignant cells to assess the therapeutic potential drug
candidates. Possible reasons are that they may consider
that the inhibition of human cancer cell proliferation at
low concentrations is an adequate parameter to predict
therapeutic potential or they prefer using animal models
instead. Researchers typically use mice xenotransplanted
with human cancer cells to reveal whether their drug can-
didates inhibit cancer cell growth selectively. If their drugs
inhibit tumor growth in these models without killing or
signicantly aecting the animals, they assume that their
drugs also inhibit the proliferation of human cancer cells
without signicantly aecting that of human nonmalignant
cells. Following this approach, researchers have proposed
several cardiac glycosides as candidates for clinical testing in
cancer patients [–,,].
Several research groups have evaluated the cancer thera-
peutic potential of cardiac glycosides by using human cancer
cells and human nonmalignant cells. For instance, we recently
observed that the cytotoxicity of digitoxin, digoxin, and
ouabain in breast cancer cells (MCF-) and melanoma cells
(UACC-) was similar than that in nonmalignant breast cells
(MCF-) and nonmalignantskin cells (VH-) []. Cliord
and Kaplan [] have recently reported that human breast
cancer cells were even more resistant to ouabain, digitoxin,
and bufalin toxicity than human nonmalignant breast cells.
Evidence has also shown, however, that digitoxin, digoxin,
and ouabain were approximately  times more cytotoxic
against human A lung cancer cells than against human
MRC- nonmalignant lung cells (– nM versus – nM)
[]. Ex vivo experiments, using cells from adult patients
with B-precursor or T-acute lymphoblastic leukemia (ALL),
acute myeloid leukemia (AML), and chronic lymphocytic
leukemia (CLL), as well as peripheral blood mononuclear
cells from healthy donors, have also shown that digitoxin
(but not ouabain) induced selective cytotoxicity (approxi-
mately-fold)incellsfrompatientswithT-andB-precursor
ALL []. In brief, although cardiac glycosides can inhibit

BioMed Research International 
the proliferation of cancer cells at very low concentrations
(nM), they usually inhibit the proliferation of human nonma-
lignant cells at similar concentrations; this strongly suggests
that their potential for cancer therapy is low. In contrast,
specic cardiac glycosides (e.g., digitoxin) can inhibit the
proliferation of particular types of cancer cells (e.g., lung can-
cer and acute lymphoblastic leukemia) at concentrations that
do not signicantly aect human nonmalignant cells; these
cardiac glycosides may have cancer therapeutic potential.
2.2. e Anticancer Activity of Cardiac Glycosides in Mice
Xenograed with Human Cancer Cells Is Probably an Exper-
imental Artifact. Several cardiac glycosides that are equally
toxic to human cancer cells and human nonmalignant cells
haveshownpotentanticancereectsinanimalmodels.
For instance, Cliord and Kaplan observed that human
nonmalignant breast cells were more sensitive than human
breast cancer cells (e.g., MDA-MB-) to the cytotoxic
eects of bufalin [], and it has recently been reported
that bufalin reduces tumor growth in mice xenotransplanted
with human MDA-MB- breast cancer cells []. ese
apparent controversies can be explained by the ability of
cardiac glycosides to kill human cells at concentrations much
lower (approximately – fold) than those required to
kill rodent cells [,].
Gupta and colleagues [] evaluated some time ago the
cytotoxicity of numerous cardiac glycosides (i.e., ouabain,
digitoxin, digoxin, convallatoxin, SC, bufalin, gitaloxin,
digoxigenin, actodigin, oleandrin, digitoxigenin, gitoxin,
strophanthidin, gitoxigenin, lanatosides A, B, and C, alpha-
and beta-acetyl digoxin, and alpha- and beta-methyl digoxin)
against a number of independent cell lines established
from human, monkey, mouse, Syrian hamster, and Chinese
hamster. e authors observed that all cardiac glycosides
exhibited greater than -fold higher toxicity towards the
human and monkey cells in comparison to the rodent cells
(mouse, Syrian hamster, and Chinese hamster). ey also
provided strong evidence that the species-related dierences
in sensitivity to cardiac glycosides were mediated by the
Na+/K+-ATPase enzyme. ey obser ve d that th e Na+/K+-
ATPase enzyme of rodent cells was inhibited at much higher
concentrations of cardiac glycosides than the Na+/K+-ATPase
of human cells. ey also observed a good correlation
between these concentrations and those reported for inhibi-
tion of the Na+/K+-ATPasefromisolatedheartmusclesof
thesamespecies[]. More recent evidence suggests that
the expression and cellular location of Na+/K+-ATPase alpha
subunits in dierent types of cells may explain why they are
more or less susceptible to the cytotoxic activity of cardiac
glycosides [–].
Several years ago, a PNAS paper reported that digoxin
blocked tumor growth in mice xenotransplanted with several
types of human cancer cells []. e authors observed
that digoxin prolonged tumor latency and inhibited tumor
xenogra growth in mice when treatment was initiated
before the implantation of P-Myc, P-Myc-Luc, PC,
and HepB cells. Digoxin also arrested tumor growth when
treatment was initiated aer the establishment of PC and
P-Myc tumor xenogras []. Based on the observations
of Gupta and colleagues []andontheplasmalevels
of digoxin in cardiac patients, we discussed the fact that
the potent anticancer eects induced by digoxin in mice
harboring human cancer cells []werenotrelevanttothe
treatment of human cancer and these anticancer eects were
probably due to interspecies dierences in sensitivity []. In
other words, the marked reduction in tumor growth induced
by digoxin in mice xenograed with human cancer cells
was probably caused by the ability of cardiac glycosides to
selectively kill human cells versus rodent cells rather than
by their ability to selectively kill cancer cells versus normal
cells. Perne et al. [] later reported experimental data that
further supported this idea. Despite these and other reports
[,], numerous publications containing this probable
experimental artifact continue to appear in the scientic
literature.
is section of the paper now reviews reports that have
used mice xenotransplanted with human cancer cells to
evaluate anticancer eects of cardiac glycosides (Tab le ).
e results of the following reports should probably be
reinterpreted.
Digoxin. Svensson et al. [] carried out in vitro and in vivo
studies to evaluate the anticancer activity of the cardenolide
digoxin. ey studied the eect of digoxin on the growth of
tumor cell lines and primary endothelial cells from dierent
species. e most sensitive cell lines in vitro were the human
SH-SYY and SK-N-AS neuroblastoma cell lines; the IC50
values were  and  ng/mL, respectively. ey also reported
that digoxin signicantly reduced the growth of human SH-
SYY neuroblastoma cells xenotransplanted in immunode-
cient mice. e authors concluded that digoxin might be
a specic neuroblastoma growth inhibitor. e authors also
reported that the in vitro and in vivo anticancer eects of
digoxin were dramatically reduced when the murine Neuro-
a neuroblastoma cell line was used instead of the human
neuroblastoma cell lines []. Zavareh et al. []reported
data suggesting that cardiac glycosides were inhibitors of
N-glycan biosynthesis. Since aberrant N-linked glycans are
known to contribute to cancer progression and metastasis,
the authors studied whether digoxin could inhibit cellular
migration and invasion. ey used two mouse models of
metastatic cancer in which human PPC- prostate cancer
cells were injected into immunodecient mice. ey found
that digoxin reduced distant tumor formation in both models
and concluded that this cardiac glycoside could be a lead
for the development of antimetastasis therapies. As discussed
before,Zhangetal.[] found that digoxin inhibited hypoxia-
inducible factor  (HIF-), a transcription factor highly
involved in cancer development, and suggested that this
eect might be observed in patients taking this drug. ey
also reported that digoxin blocked tumor growth in mice
xenotransplanted with several types of human cancer cells.
ese data suggested that digoxin had anticancer potential
[]. Wong et al. [] reported data suggesting that digoxin
was a potential antimetastasis compound. ey investigated
whether digoxin could reduce metastases in human MDA-
MB- tumor-bearing mice. Digoxin blocked metastatic

BioMed Research International
T : e antitumor activity of cardiac glycosides in mice xenograed with human cancer cells is probably caused by their ability to
selectively kill human cells versus rodent cells rather than by their ability to selectively kill human cancer cells versus human nonmalignant
cells.
Cardiac
glycoside
Antitumor activity in mice
xenograed with human cancer
cells
Selective cytotoxicity
against human cells
versus rodent cells
Selective cytotoxicity against human cancer cells versus
human nonmalignant cells
Arenobufagin Liver HepG/ADM [] N.D. N.D.
Bufalin
Breast MDA-MB- [],
osteosarcoma UOS/MTX
[], and pancreatic Mia Paca-
[]
>-fold []
NO: breast cancer versus breast nonmalignant [];
-fold: ovarian cancer versus endometrial
nonmalignant []
Bufotalin Liver R-HepG []>-fold []N.D.
Digitoxin N.D.
>-fold [];
>-fold []
-fold: lung cancer versus lung nonmalignant [];
-fold: lung cancer versus lung nonmalignant [];
-fold: ALL versus PBMCs nonmalignant []; -fold:
AML versus PBMCs nonmalignant []; -fold: CLL
versus PBMCs nonmalignant []; -fold: breast cancer
versus breast nonmalignant []; NO: breast cancer
versus breast nonmalignant []; NO: skin cancer
versus skin nonmalignant []
Digoxin
Brain SH-SYY [], brain
SK-N-AS [], breast
MDA-MB- [,], breast
MDA-MB- [], liver HepB
[], prostate PC [], prostate
PPC- [], and transformed
human B-lymphocytes
P-Myc []
>-fold [,]
NO: breast cancer versus breast nonmalignant []; -
fold: lung cancer versus lung nonmalignant []; NO:
skin cancer versus skin nonmalignant []; -fold: brain
cancer versus umbilical vein endothelial nonmalignant
[]; NO: breast cancer versus umbilical vein
endothelial nonmalignant []; NO: colorectal cancer
versus umbilical vein endothelial nonmalignant []
Lanatoside C Brain U []>-fold []N.D.
Ouabain
Brain SH-SYY [], ocular
YLUC [], pancreatic BON
[], promyelocytic leukemia
HL- [], and prostate PPC-
[]
>-fold [,,]
NO: breast cancer versus breast nonmalignant [,];
- fold: lung cancer versus lung nonmalignant [];
NO: skin cancer versus skin nonmalignant []; -fold:
ALL versus PBMCs nonmalignant []; -fold: AML
versus PBMCs nonmalignant []; NO: CLL versus
PBMCs nonmalignant []
Periplocin Liver Huh- []andlungA
[]
>-fold []∗;NO:
[]
>-fold: liver cancer versus PBMCs nonmalignant
[]∗
UNBS
Brain U-MG [], lung A
[,], lung NCI-H [,],
prostate PC- [], and skin
VM- []
>-fold []
-fold: brain cancer versus lung and skin
nonmalignant []; -fold: prostate cancer versus
lung and skin nonmalignant []
N.D.: not determined; NO: no selective cytotoxicity; ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; CLL: chronic lymphocytic leukemia;
PBMCs: peripheral blood mononuclear cells; ∗not specied if the PBMCs were human cells or rodent cells (we contacted the authors without success).
niche formation and breast cancer metastasis in the lungs,
andtheauthorsdiscussedthefactthatthiseectwasprobably
due to inhibition of HIF-. e most relevant conclusion of
this work was that digoxin might be useful to treat patients
with HIF--overexpressing breast cancers []. Zhang et al.
[] observed that digoxin reduced tumor growth and inhib-
ited the metastasis of human MDA-MB- breast cancer
cellstothelungsinmicexenograedwiththesecells,without
causing any sign of toxicity in the animals. ey concluded
that clinical trials were warranted to investigate whether the
concentrations of digoxin achievable in patients are sucient
to inhibit tumor growth and metastases []. Schito et al. []
reported that HIF- promoted lymphatic metastases of breast
cancer and the use of the HIF- inhibitor digoxin strongly
decreased tumor growth and blocked lymphangiogenesis and
lymphaticmetastasisinmicebearinghumanbreastcancer
cells. e authors suggested that digoxin might be useful to
treat patients with high risk of lymphatic metastases [].
Gayed et al. [] observed that specic concentrations of
digoxin inhibited blood vessel formation but not tumor
growth in mice injected with the human C- prostate cancer
cell line.
Ouabain. Several cardiac glycosides were identied by
Antczak et al. [] as potent antiretinoblastoma agents in
vitro. One of them, the cardenolide ouabain, induced a drastic
tumor regression in immunodecient mice injected with
human YLUC retinoblastoma cells, without inducing any

BioMed Research International 
signicant toxicity on the host. In light of the results of their
study, the authors proposed that digoxin, which is widely
used in patients with cardiac disease, could be repositioned
for the treatment of retinoblastoma []. Simpson et al.
[] identied the cardiac glycosides ouabain, peruvoside,
digoxin, digitoxin, and strophanthidin as anoikis sensitizers.
Because resistance to anoikis permits cancer cells to survive
in the circulation and improves their metastatic potential,
the authors evaluated in mouse models of metastasis whether
ouabain could block distant tumor formation. ey observed
that ouabain reduced the number of tumors in human PPC-
prostate cancer cells bearing mice. ey also reported that
systemic administration of ouabain decreased the survival
and growth of human PPC- prostate cancer cells and human
BON pancreatic cancer cells xenograed into nude mice
[]. Hiyoshi et al. [] reported that ouabain induced quies-
cence in neuroblastoma cells in vitro and a marked reduction
in tumor growth when human neuroblastoma cells were
xenograed into immune-decient mice. Based on these
ndings, the authors concluded that ouabain could be used in
chemotherapies to suppress tumor growth and/or arrest cells
to increase the therapeutic index in combination therapies.
Tail ler e t al. [ ] identied the cardiac glycoside ouabain
as a potential antileukemic compound. ey observed that
ouabain was highly ecient in inhibiting the growth of
human acute myeloid leukemia cells xenotransplanted in
immunodecient mice, without exerting signicant toxic-
ity on the host. e authors concluded that ouabain was
a promising antileukemic agent whose activity should be
evaluated in prospective clinical studies [].
UNBS1450. Mijatovic et al. []investigatedthein vitro and
in vivo anticancer activity of UNBS, a semisynthetic
derivative of the natural cardenolide UNBS (isolated
from the African plant Calotropis procera). ey observed
that UNBS was able to inhibit cell growth of four
dierentnon-smallcelllungcancercells(A,NCI-H,
A, and CAL-T) at nanomolar concentrations. is
cardenolide also signicantly decreased tumor growth in
nude mice xenograed with human NCI-H cancer cells
andincreasedthesurvivalratesinmicexenograedwith
human A cancer cells. e authors observed in another
study [] that the cytotoxic potency of UNBS in A
lungcancercellswassimilarthanthatoftheanticancerdrugs
paclitaxel and SN (the active metabolite of irinotecan)
and much higher than that of cisplatin, carboplatin, and
oxaliplatin. UNBS also decreased tumor growth in mice
xenotransplanted with A lung cancer cells and human
NCI-H lung cancer cells []. Another study revealed
that UNBS inhibited the proliferation of human prostate
cancer cells (LNCaP, PC-, and DU) and increased the
survival of mice transplanted with human PC- prostate can-
cer cells []. Lefranc et al. [] reported that UNBS was
more cytotoxic on human glioblastoma cells (U-MG and
TG)thanonhumannormalbroblasts(WI-andWSI)
at nanomolar concentrations. is compound also inhibited
the proliferation of rat C glioblastoma cells at micromolar
concentrations. UNBS increased the survival of mice
graed with human U-MG glioblastomas cells, without
observabletoxiceectsontheanimals.Mathieuetal.[]
reported that UNBS blocked cell proliferation in several
human melanoma cell lines in vitro (IC50 values between 
and  nM) and improved the survival of immunodecient
mice graed with human VM- melanoma brain metastasis
cells.
Periplocin. Lu et al. [] reported that the natural cardenolide
periplocin induced similar cytotoxicity against a panel of
human lung cancer cell lines than against a rodent lung cancer
cell line (LL/). ey also observed antitumor activity in mice
transplanted with both the human A lung cancer cell
line and the murine LL/ Lewis lung cancer cell line. Cheng
et al. [] have recently reported that periplocin displayed a
potent cancer cell growth inhibitory activity in vitro and in
vivo. Periplocin inhibited cell growth of human HAT/VGH
hepatocellular carcinoma with an IC50 of  nM and was less
toxic to normal peripheral blood mononucleated cells. e
authors also observed that periplocin showed an inhibition
of tumor growth when human Huh- hepatoma cells were
injected into immunodecient mice, without observing clear
side eects on the host.
Lanatoside C. Badr et al. [] identied the cardenolide
lanatoside C as a sensitizer of glioblastoma cells to tumor
necrosis factor-related apoptosis-inducing ligand (TRAIL)-
induced cell death. ey observed that lanatoside C, alone or
in combination with TRAIL, reduced tumor growth in nude
mice harboring human U glioblastoma cells.
Bufalin. Chen et al. [] identied the bufadienolide bufalin
as a potential agent for the treatment of pancreatic cancer in
combination with the standard anticancer drug gemcitabine.
ey found that bufalin inhibited the growth on three pancre-
atic cancer cell lines (Bxpc-, Mia PaCa-, and Panc-) and
it synergistically increased gemcitabine-induced cancer cell
growth inhibition and apoptosis. e combination of bufalin
with gemcitabine was also found to signicantly reduce
tumor growth in mice bearing human Mia Paca- pancreatic
cancer cells. Xie et al. []investigatedthein vitro and in
vivo antiosteosarcoma activity of bufalin. ey observed that
bufalin strongly inhibited the cell growth of dierent human
osteosarcoma cell lines, including the methotrexate-resistant
UOS/MTX cell line. ey also found that the treatment
with bufalin induced signicant tumor growth inhibition
in mice xenotransplanted with the human UOS/MTX
osteosarcoma cell line, without decreasing the body weight
oftheanimals.eauthorsconcludedthatbufalinmightbe
an alternative chemotherapeutic agent to treat osteosarcoma,
particularly in methotrexate-resistant cancers []. Wang
et al. [] have recently reported that bufalin was a potent
inhibitor of the steroid receptor coactivators SRC- and SRC-
. Because these coactivators have been implicated in cancer
progression, the authors investigated whether bufalin could
also block cancer cell growth in cell culture and animal
models. ey observed that bufalin inhibited the growth of
human MCF- breast cancer cells and human A lung
cancer cells at nanomolar concentrations (–nM); these
concentrations also resulted in inhibition of the steroid

BioMed Research International
receptor coactivator SRC- and were below those reported
to be tolerated by humans (. nM). ey also found that
bufalin inhibited tumor growth in mice xenotransplanted
with human MDA-MB- breast cancer cells.
Arenobufagin. Zhang et al. [] recently observed that the
bufadienolide arenobufagin induced a potent cell growth
inhibitory activity on cancer cells both in vitro and in
vivo. ey tested its anticancer activity on several human
cancer cell lines (hepatoma, breast adenocarcinoma, cervix
adenocarcinoma, lung cancer, colon cancer, leukemia, and
gastric adenocarcinoma). Arenobufagin inhibited the growth
of all cancer cell lines at nanomolar concentrations, includ-
ing multidrug-resistant cancer cell lines. Arenobufagin also
inhibited the growth of human HepG/ADM hepatocellular
carcinoma cells xenograed into immunodecient mice,
without causing side eects on the hosts. e authors con-
cluded that their results may provide a rationale for future
clinical application using arenobufagin as a chemotherapeu-
tic agent for the treatment of patients with hepatocarcinoma
[].
Bufotalin. Zhang et al. [] observed that four bufadienolides
from Venenum Bufonis, a traditional Chinese medicine,
displayed inhibitory eects on the growth of human HepG
hepatocarcinoma cells and human R-HepG multidrug hep-
atocarcinoma cells. One of them, bufotalin, was also able
to signicantly inhibit the growth of human R-HepG cells
xenograed into immunodecient mice, without observing
any life-threatening toxicity in the animals. e authors
discussed the fact that their study supports the possible
development of bufotalin as a potential agent in the treatment
of multidrug resistant hepatocellular carcinoma [].
Data from preclinical studies reporting antitumor eects
in rodent xenogras of plant extracts containing cardiac
glycosides may also need reinterpretation. For instance,
Han et al. [] reported that an extract from the plant
Streptocaulon juventas induced a strong inhibitory eect
on the proliferation of human lung A adenocarcinoma
cells. A bioassay-guided fractionation revealed that the most
cytotoxic fraction in vitro also induced antitumor eects in
athymicnudemicetransplantedwithhumanAcancer
cells without exerting side eects on the mice. Following
HPLC and NMR spectrometry, the main components of
this active fraction were identied as the cardiac glycosides
digitoxigenin, periplogenin, and periplogenin glucoside [].
3. Possible Approaches to Reveal the Cancer
Therapeutic Potential of Cardiac Glycosides
in Preclinical Studies
As discussed before, the key feature of an ecient anticancer
drug candidate is its ability to kill (or to inhibit the pro-
liferation of ) human cancer cells at concentrations that do
not signicantly aect human nonmalignant cells. Ideally, the
drugcandidateshouldkillallthecancercellsofthepatients
without signicantly aecting their normal cells. Because this
is dicult to achieve, one can settle for less. A drug that
improves the ability of our current anticancer drugs to kill
cancer cells at concentrations that do not signicantly aect
nonmalignant cells could be therapeutically useful.
In vitro, one can evaluate whether the drug candidate
improves the selective cytotoxicity of the standard anticancer
drugs towards cancer cells by using the following approach.
e rst step in this approach is the selection of a panel of
human cancer cell lines and human nonmalignant cell lines
(or primary cells). Because the cytotoxicity of some drugs
dependsonthenatureofthetissuefromwhichtheyoriginate,
one should select nonmalignant cell lines of the same tissue
origin than that of the selected cancer cell lines. A small
number of cancer cell lines may be sucient to reveal the
therapeutic potential of a drug for a particular type of cancer.
However, the selection of a low number of nonmalignant cell
lines reduces the chances of nding toxicity on a specic
tissue that would limit the possible therapeutic use of the
drug. e next step is to treat the selected cell lines with
several concentrations of the drug candidate and of the
anticancer drugs most commonly used in the treatment of the
selected cancers. en, cell viability or cell death is estimated
with a cytotoxicity test (e.g., SRB assay and MTT assay), and
cytotoxic parameters (e.g., IC50 values) are calculated. e
following step is to calculate one or several selectivity indexes
for the drug candidate and for the anticancer drug. ese
selectivity indexes can be calculated by dividing the IC50
values in the nonmalignant cell lines by the IC50values in the
cancer cell lines. For instance, if the mean IC50 value of a drug
in a variety of nonmalignant cells originated from several
tissues is  𝜇MandthemeanIC
50 value of the drug in
several cell lines derived from a specic cancer is  𝜇M, the
selectivity index for this particular cancer would be . Finally,
the following question must be answered: is the selectivity
index of the drug candidate higher (or at least similar) than
that of the standard anticancer drug? If the answer is no,
the drug candidate does not have therapeutic potential and
shouldnotbetestedinanimalmodels.Iftheanswerisyes,
the drug candidate has chemotherapeutic potential, which
should be conrmed by using in vivo experiments.
Rodent xenogra models are the most common animal
models used by researchers to evaluate the therapeutic
potential of anticancer drug candidates in vivo.However,as
discussed before, these models may be inadequate to evaluate
the therapeutic potential of cardiac glycosides. To the authors’
knowledge, all cardiac glycosides tested in human cells and
rodent nonmalignant cells have shown greater than -
fold higher toxicity towards the human cells in comparison
to the rodent cells. is does not mean, however, that all
compounds having the basic chemical structure of cardiac
glycosides (a steroid skeleton with an unsaturated lactone
ring)willbemoretoxicagainsthumancellsthanagainst
rodent cells. One can test the suitability of using tumor
xenogras to evaluate the in vivo therapeutic potential of a
particular cardiac glycoside by testing if the cytotoxicity of
the cardiac glycoside against a panel of human nonmalignant
cells is similar than that against a panel of rodent nonmalig-
nant cells. If the compound behaves similarly in both types
of cell lines, its in vivo anticancer activity can be evaluated
in mice xenograed with human cancer cells. If the rodent

BioMed Research International 
cell lines are more resistant than the human cell lines to
the cytotoxicity of the cardiac glycoside, these models are
probably inadequate to evaluate its anticancer eects in vivo.
Animal models using mice transplanted with mouse cancer
cells may also be inadequate when human cells are more
sensitive than rodent cells to the cytotoxicity of the cardiac
glycoside. e reason is that the therapeutic target responsible
for the death of the human cells may be dierent than that
responsible for the death of the rodent cells and, therefore,
resultsobtainedinmicemaynotbeextrapolatedtohumans.
e anticancer activity of cardiac glycosides displaying
a similar cytotoxic prole in nonmalignant cells originated
from both human and mouse tissues can be assessed by using
tumor xenogras or other rodent models. It is important
to remember that most cancer patients requiring therapy
with anticancer drugs have metastatic disease and patient
survival is the parameter used by oncologists as an endpoint
of clinical interventions designed to assess drug ecacy
in patients with cancer (other parameters used by many
preclinical researchers as an endpoint for their experiments,
such as measurements of tumor volumes, do not necessarily
predictsurvival).Itisessential,therefore,toselectanimal
models of metastasis and to assess animal survival as an
endpoint for the experiments. In our opinion, animals with
metastasis should be treated with equitoxic concentrations
of the cardiac glycoside and of the standard anticancer drug
usedinthetypeofcancerunderstudy.en,oneshould
evaluate whether the cardiac glycoside improves the survival
rates induced by the standard anticancer drug. If the cardiac
glycoside improves (or at least matches) the selectivity index
(in vitro)andthesurvivalrates(in vivo)ofthestandard
anticancer drugs, it should be considered for clinical trials
testing.
Rodent models are inappropriate for testing the anti-
cancer activity of cardiac glycosides that kill human non-
malignant cells at lower concentrations than those required
to kill rodent nonmalignant cells. ese models, however,
could provide information on the pharmacokinetics of the
cardiac glycoside. ese models may also help detect possible
toxicity not detected by using a panel of human nonmalignant
cell lines; they could help detect toxicity not mediated by
inhibition of the Na+/K+-ATPase (which seems to be the
main determinant for the species dierences in sensitivity
to cardiac glycosides). In our opinion, a cardiac glycoside
that kills human nonmalignant cells at lower concentrations
than rodent nonmalignant cells should pass the following
tests before being considered for evaluation in clinical trials.
First, it should match or improve the selectivity indexes of the
standard anticancer drugs when they are evaluated in a panel
of human cancer cell lines derived from a particular type
of cancer versus a variety of human nonmalignant cell lines
and primary cells derived from a variety of human tissues.
Second, in vivo experiments (e.g., rodent models) should
exclude pharmacokinetic and toxicological limitations that
may compromise the in vivo anticancer activity of the cardiac
glycoside. Finally, if the cardiac glycoside is in clinical use
for the management of other diseases or if clinical data
already exist on its plasma and tissue concentrations, one
should also consider whether the anticancer eects observed
in preclinical studies may occur at concentrations within or
below the concentration range tolerated by humans.
4. Conclusion
Preclinical research has shown that cardiac glycosides can
both inhibit cancer cell proliferation at very low concen-
trations and induce potent anticancer eects in mice trans-
planted with human cancer cells. Based on these observa-
tions, cardiac glycosides have been considered as potential
anticancer drug candidates that should be evaluated in
clinical studies. is paper has reviewed evidence indicating
that cardiac glycosides may not selectively inhibit the prolif-
eration of human cancer cells and these compounds have the
ability of killing human cells at concentrations much lower
than those required to kill rodent cells (approximately –
 fold). is strongly suggests that the potent anticancer
eects induced by cardiac glycosides in mice transplanted
with human cancer cells may be an experimental artifact
caused by their ability to selectively kill human cells versus
rodent cells rather than by their ability to kill human cancer
cells versus human nonmalignant cells. It has also been
discussed that inhibition of cancer cell proliferation at low
concentrations is not an adequate parameter to predict the
therapeutic potential of a drug candidate. e key feature of
an ecient anticancer drug is its ability to kill (or inhibit the
proliferation of ) human cancer cells at concentrations that
do not signicantly aect human nonmalignant cells. Based
on this principle, an approach to evaluate the therapeutic
potential of cardiac glycosides in preclinical in vitro studies
has been proposed. is approach is also based on the
idea that only drug candidates that match or improve the
ability of the approved anticancer drugs to kill human cancer
cells at concentrations that do not signicantly aect human
nonmalignant cells have a chance to be ultimately used in
cancer therapy. A test for revealing the suitability of using
rodent models for the evaluation of the anticancer activities
of cardiac glycosides in vivo has also been proposed. If the
cardiac glycoside passes this test, several recommendations
have been made for the evaluation of its cancer therapeutic
potential in these models. If the cardiac glycoside fails to pass
this test, an alternative approach for revealing its possible
therapeutic potential has been discussed. It is the hope of
the authors that this paper may help researchers evaluate
the therapeutic potential of cardiac glycosides in preclinical
studies.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
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