Biochemical Pharmacology 78 (2009) 1–10

Contents lists available at ScienceDirect

Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Commentary

UNBS1450 from Calotropis procera as a regulator of signaling pathways involved in

proliferation and cell death
Tom Juncker, Marc Schumacher, Mario Dicato, Marc Diederich *
Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Hôpital Kirchberg, 9 rue Edward Steichen, L-2540 Luxembourg, Luxembourg

A R T I C L E I N F O A B S T R A C T

Article history: Despite significant progress in oncology therapeutics in the last decades, the urge to discover and to
Received 31 October 2008 develop new, alternative or synergistic anti-cancer agents still remains. For centuries it has been known
Accepted 29 January 2009 that the coarse shrub Calotropis procera is a very promising source of ascaricidal, schizonticidal, anti-
bacterial, anthelmintic, insecticidal, anti-inflammatory, anti-diarrhoeal, larvicidal and cytotoxic
Keywords: chemicals. Different compounds like norditerpenic esters, organic carbonates, the cysteine protease
Calotropis procera procerain, alkaloids, flavonoids, sterols as well as numerous types of cardenolides have provided this
Cardenolide
plant for centuries with scientists’ interest. The chemical class of cardenolides and their related
Na+/K+ ATPase
bioactivity evaluation and structure–activity relationship (SAR) studies pointed out their therapeutic
UNBS1450
Nuclear factor kB (NF-kB) utility and led to the discovery of promising drug candidates. Recently the cardiotonic steroid UNBS1450
Apoptosis 01 (derived from 2-oxovoruscharin 02) from C. procera was shown to additionally exert an anti-cancer
Autophagy activity. UNBS1450 01 has been proven to be a potent sodium pump inhibitor, showing anti-proliferative
and cell death-inducing activities. This anti-cancer potential of UNBS1450 01 is achieved by
disorganization of the actin cytoskeleton after binding to the sodium pump at the cellular membrane,
by inducing autophagy-related cell death, by repressing NF-kB activation as well as by down-regulating
c-Myc in cancer cells. We aim to review pharmacologically important chemical extracts from C. procera
and focus more specifically on the anti-cancer activities of UNBS1450 01.
ß 2009 Elsevier Inc. All rights reserved.

1. Calotropis procera as a source of natural drugs C. procera, a member of the Asclepiadaceae, is a woody,
broadleaf evergreen coarse shrub, 3–5 m tall, widely growing in
1.1. C. procera the tropics. It is distributed in arid to semi-arid regions of the
Caribbean, Central America, South America, Africa, India and Israel,
According to literature, the origin of synthetic drugs develop- mainly appearing on plains and in the uplands. For decades, C.
ment lies within the upcoming of The Industrial Revolution. The procera, also referred to as ‘‘The Apple of Sodom’’ has been used
potentially more active synthetic products and the increasing especially in traditional folk medicine because of its pharmaco-
economic power of pharmaceutical companies were the main logically active compounds found in its roots, bark, leaves and
reasons for a preference for these new products compared to the mainly in its latex which exudates from damaged leaves. Mean-
natural compounds from mineral, plant and animal sources used while, chemical extracts from C. procera have been shown to have
for ages. ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insectici-
However, a significant number of these synthetic drugs are dal, anti-inflammatory, anti-diarrhoeal, larvicidal, cytotoxic and
obtained from natural precursors; it is even estimated that 11% of analgesic effects, thus explaining a growing demand in today’s
the drugs considered as basic and essential by the World Health medical research for the different parts of the plant [2–5].
Organisation (WHO) are strictly of plant origin. Digoxin, quinine,
quinidine, vincristine, vinblastine, atropine, morphine and 1.2. The latex of C. procera
codeine might be the most prominent. Furthermore, according
to Yue-Zhong Shu, 60% of current or future anti-tumor and More than 12,000 plant species contain latex, a milky fluid in
anti-infectious drugs are of natural origin [1]. which occurs a wide range of proteins, and for some 1000 species
also cis-polyisoprene that is the polymeric hydrocarbon of rubber
[6]. As the latex is abundant in the green parts of the plant, it is
* Corresponding author. Tel.: +352 2468 4040; fax: +352 2468 4060. thought to be produced and accumulated as a defense strategy
E-mail address: marc.diederich@lbmcc.lu (M. Diederich). against viruses, fungi and insects. Indeed, besides proteins

0006-2952/$ – see front matter ß 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2009.01.018
2 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10

involved in rubber biosynthesis, latex fluids have been shown to [14]. Contradictory to these findings, there is some evidence that
contain proteins implicated in plant defense and oxidative DL is also capable of inducing pro-inflammatory effects. In 2003,
metabolism [7,8]. Today, latex is considered as a promising source Shivkar et al. proved DL to be able to produce dose-dependent
of pharmacologically active molecules that might be chemically inflammatory responses resulting in rat paw edema similar to
modified to improve their effectiveness. carrageenan. Further studies suggested biogenic amines, especially
With regards to C. procera, although the pharmacological histamine, to be involved in DL driven inflammation [15].
potential of its latex has well been proven, only few active latex Similarly, by extracting DL sequentially with petroleum ether
molecules have been identified until today; most often latex is and methanol followed by a silica gel chromatography fractiona-
either used as an entity or is only poorly processed, mainly in order tion, Choedon et al. applied a DL treatment on X15-myc
to eliminate rubber compounds. Usually, the crude latex from C. transgenic, hepatocellular carcinoma developing mice [16].
procera is collected from non-cultivated plants by small incisions Not only did they discover an in vivo chemopreventive effect
near the younger leafs and is mixed 1:1 (v/v) into distilled water. of DL, they also noticed that DL has cytotoxic effects on Huh-7
The material is then kept under constant, gentle, agitation in order and COS-1 cancer cell lines. By chromatography separation, they
to prevent it from precipitating. It is maintained at room isolated a polar fraction responsible for the observed cytotoxic
temperature until being centrifuged at 5000  g, 10 min at 4 8C. potential. Apoptosis analysis revealed induced DNA fragmenta-
The precipitated material is pooled apart while the supernatant is tion as a mechanism.
further dialysed using membranes of 8000 Da cut-off, thus keeping
the protein fraction, before being lyophilized. Generally, authors 1.3. Ethanolic, methanolic and aqueous extracts
use both, the dialysis liquid containing low molecular weight
compounds, called the dialysis latex (DL), and the non-dialyzable Phytochemically, the plant has been investigated since the
latex (NDL) obtained after exhaustive dialysis and containing 1960s, especially for cardenolides [17,18], triterpenoids [19,20],
concentrated proteins. anthocyanins [21] and hydrocarbons [22]. Besides the use of plant
Soares et al. were among the first to show benefic, anti- latex, aqueous, methanolic, ethanolic and other organic plant
nociceptive effects of fractionated latex (NDL) [9]. After acetic acid extracts from different parts of the plant, namely flowers, buds,
induced writhes in mice, the latex was capable of inhibiting up to roots, stems and leaves, were among the very first approaches to
100% the abdominal constrictions in a dose-dependant manner develop natural drugs for classical and alternative medicine. In
(12.5–50 mg/kg). Morphine was used as a positive control (54.5% 1979, Malik and Chughtai described antimicrobial activity against
of inhibition). Similar results were obtained for formalin-induced pathogenic bacteria [23]. Moreover, organic extracts of C. procera
paw licking for a hot plate test. In contrast to morphine, they found were also shown to possess nematocidal [24,25], larvicidal [26],
that the opioid antagonist nalaxone did not alter the latex-induced anti-fertility [27] and even anti-cancer potentials [28,29]. In 1987,
antinociceptive effect. Mascolo et al. further elucidated the biological properties of C.
In 2006, Freitas et al., went a bit further and discovered pro- and procera and, using ruminant animal models, discovered anti-
anti-inflammatory potentials of dialysed and non-dialysable latex inflammatory, analgesic, antimicrobial and antipyretic activities in
[10]; dialysed latex of C. procera was shown to increase induced extracts from C. procera flowers. In the late 1980s, Fenado et al.
leukocyte, especially neutrophil, migration whereas anti-inflam- established a standard method to get crude aqueous extracts (CAE)
matory and anti-nociceptive activities were associated to non- from powdered C. procera flowers [30]. Similarly, in 1994 Asuzu
dialysable latex. and Onu managed to get crude methanolic extracts (CME) from C.
Since C. procera has been known to be effective against several procera using a Soxhlet apparatus [31]. Others used filtered
human diseases, the aim of the work of Ramos et al., was to ethanolic extracts to elucidate the plant’s pharmacological
elucidate larvicidal activities of different fractions of the plant’s potential [32,33].
latex [11]. Their evaluation was based on egg hatching and larval Meanwhile, the ethanolic extracts of flowers, buds, roots, stems
development of Aedes aegypti. A 10 mg/mL concentration of NDL and leaves were discovered to have a schizontocidal potential in
was able to completely block egg hatching; the inhibiting potential vitro. The different fractions of C. procera showed dose-dependant
of the DL fraction was significantly lower as a 5 times greater inhibitory effects on chloroquine sensitive and resistant Plasmo-
concentration (50 mg/mL) was needed in order to achieve 0% of egg dium falciparum strains. Antimalarial screening had been per-
hatching. Both, NDL as well as DL, also triggered severe larvae formed according to the method of Ang et al. [34]; parasitaemia
mortality in a dose and exposure time-dependent manner. had been adjusted to 1–1.5% before different concentrations of C.
Chromatography analysis revealed different protein and amino procera fractions were added. After an incubation time of 36 h, each
acids of NDL and DL latex, thus delivering promising outlooks for sample was analysed under the microscope.
future preparations of C. procera latex as a larvicidal compound. Furthermore, both, aqueous and methanolic extracts of
In contrast to latex fractionation, some authors used air dried powdered C. procera flowers, were shown to possess time-
latex; in most examples, latex was extracted from the aerial parts dependent anthelmintic activities in vitro [35]. Based on nematode
of the plant before being dried under shade (DL, dried latex). egg counting in sheep feces, the authors confirmed their observed
After being triturated 1:1 with gum acacia, DL administrated results in vitro: a maximum of 88.4% egg reduction (ECR, egg count
orally to albino rats and guinea pigs inhibited carrageenan and percent reduction) had been recorded in sheep treated with CAE
Freund’s adjuvant-induced edema as well as granuloma formation. and 77.8% in sheep treated with crude powder. CME had been
Further anti-inflammatory activities of DL resulted in fluid proven to be less effective.
exudation inhibition and in reduced UV-induced erythema onset Besides further insecticidal properties [36], organic C. procera
[12]. Similar results were obtained by Arya and Kumar. Shade dried extracts also possess interesting cytotoxic effects. Compared to the
DL, soxhlated to get methanolic extracts and triturated with gum reference compound cisplatin (IC50 0.9 mg/mL), Smit et al.
acacia as seen before, showed anti-inflammatory activity against demonstrated that ethanolic flos extracts (IC50 1.4 mg/mL) have
histamine, serotonin, compound 48/80, bradykinin and prosta- potent growth inhibition capacities when used on COLO 320 tumor
glandin mediated inflammatory reactions in induced rat paw cells.
edema [13]. The same research team also demonstrated that DL, More specific spectral data analysis of organic extracts of the
triturated this time in normal saline, is able to play an antioxidant plant revealed the presence of a norditerpenyl ester, calotropter-
and anti-hyperglycemic role in alloxan-induced diabetes in rats penyl, two pentacyclic triterpenoids, calotropursenyl acetate and
 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10 3

Fig. 1. Illustrative representation of the different pharmacologically important potentials of Calotropis procera extracts.

calotropfriedelenyl acetate as well as an organic carbonate, namely to temperature, denaturants and organic solvents, this makes it a
2-propenyl-20 -hydroxyethyl carbonate [37,38] (Fig. 1). very useful tool for food and pharmaceutical industry.

1.4. Procerain 1.5. Cardenolides

By further method standardizations and working condition Since the 1960s it has been known that the principal toxic
optimizations, the next step in characterizing the plant’s pharma- components in C. procera, capable of causing death in mammals
ceutical potentials consisted in the isolation of new, specific and when reaching high concentrations, are cardenolides [47]. Chemi-
pharmacologically important proteins and compounds, especially cally, cardenolides, cardiac glycosides named after their capacity to
with an enzymatic activity. influence heart beat, are 5bH,14b-hydroxy a,b-unsaturated g-
By default, plants are usually rich sources of various kinds of lactones widely distributed in plants. In 1983, Erdman was among
proteases, enabling protection, development of the plant, ripening the first to measure individual nutrient and cardenolide concentra-
of its fruits [39], nutritional reserve, degradation of seed storage tions in unextracted as well as in extracted C. procera [48]. Several
proteins thus allowing germination [40], activation of proenzymes cardenolides were positively identified: ascleposide, calactin,
as well as elimination of defective proteins [41]. Proteolytic calotoxin, calotropin, calotropagenin, coroglaucigenin, proceroside,
enzymes from plant sources, are of interest for the pharmaceutical usharidin and uzarigenin. Recently, with the aim to use cardenolides
research as the have been proven to be active over wide ranges of as potential anti-cancer agents, the list of cardiotonic glycosides
temperature and pH [42]. Consequently, an important number of found in C. procera has been enlarged by 2-oxovoruscharin 02, a
proteases have been isolated from lattices, fruits and seeds. Among derivative of voruscharin [49]. However, due to its high toxicity
these, most belong to the cysteine super family, one of five catalytic observed in mice, chemical modifications were necessary in order to
types of proteases [43]. Concerning the different Calotropis species, improve tolerance in vivo. The outcome, named UNBS1450 01, not
since 1979 it has been known that the latex of Calotropis gigantea only showed reduced toxicity, but also a severely increased
contains four cysteine proteases, namely Calotropin FI, FII, DI and therapeutic anti-cancer activity [50].
DII [44]. More recently, Dubey et al. purified a new cysteine Fig. 2 gives a short overview of how C. procera can be used in
protease from C. procera, named procerain. pharmaceutical science, either by applying rather simple methods,
Procerain was isolated by applying a 50% ammonium fractiona- to extract for example latex, or by more sophisticated approaches
tion of crude, gumless latex followed by CM-sepharose and SP- seeking to isolate specific compounds like cardenolides.
sepharose cation exchange chromatographies. Different physical
and chemical properties were then analysed: an SDS-PAGE 2. Bioactivity of cardenolides
electrophoresis revealed an average Mr of 28.8 kDa for procerain,
putting it in the common molecular mass range of 20–35 kDa for 2.1. Effect of cardenolides on the sodium pump
plant cysteine proteases. No carbohydrate could have been
determined by the phenol sulfuric acid method [45] thus differing Frequently named sodium pump, the Na,K-ATPase is an
procerain from glycoproteins. An isoelectric point of 9.32, defined integral-membrane protein with both an enzymatic and a
by isoelectric focusing on polyacrylamide gel, indicates a transporter function: in the conformational state E1, the phos-
predominance of basic amino acid residues. Total amino acid phorylated protein binds three intracellular Na+. These Na+ ions are
analysis revealed the presence of seven cysteines, one free and six then released into the extracellular fluid, in exchange to 2 external
forming disulfides [46], eight tryptophan and twenty tyrosine K+ ions and phosphate formation (state E2). With the Na+/K+
residues. In addition to proteolytic activity against different gradient being implicated in the regulation of animal cell volume,
natural substrates such as casein, azocasein, azoalbumin and in lyses prevention, in membrane potential control, in pH
haemoglobin, procerain also showed hydrolytic activity against homeostasis and in energy delivery for ion transport, the sodium
synthetic substrates. The enzyme’s activity was best over a broad pump is essential for cell viability and is thus ubiquitously
pH 7.0–9.0 range and together with its high stability with respect expressed throughout the different tissues [51,52].
4 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10

Fig. 2. Calotropis procera cartoon showing the three main approaches for using the plant’s intrinsic pharmaceutical potential.

The Na,K-ATPase is a heterodimer formed of an a and b Antiaris toxicaria, exhibited cell proliferation inhibition in
subunit. The a subunit, with a molecular mass of 106 kDa, different cancer cell lines together with 10 other cardenolides
contains a ten segment transmembrane domain (M) and three purified from the same source. Concerning structure–activity
different cytosolic domains, namely a nucleotide binding relationship, a reduction of 19-CHO and mono-glycosidation of
domain (N) binding ATP, a phosphorylation domain (P) and the 3-hydroxy group resulted in a higher cytotoxicity to different
an actuator domain (A). The abundantly glycosylated b subunit cancer cell lines by factor 2 and over 100 respectively. In contrast,
of 40 kDa has a single transmembrane segment, a short a b-hydroxylation of carbon 12 reduced the induction of
intracellular N-terminal and a larger extracellular site. In apoptosis [56]. However, an additional acetoxy group did not
contrast to the catalyzing a subunit, the b subunit is required modify the cytotoxicity [57,58]. These observations had been
for the biogenesis and activity of the holoenzyme. A third confirmed in a previous publication [57]. Structural modification
subunit FXYD has been reported to be associated to the a and b of the core unit as an additional alcohol group or an epoxy group
subunits in some tissues. at C8 or a double bond between C16 and C17 led to a decrease of
Endogenous and exogenous cardiac glycosides, for example cytotoxicity [58]. Additionally, Ueda et al. isolated 15 cardeno-
cardenolides, act by binding to extracellular H1–H2, H3–H4 and H5–H6 lides from the Vietnamese medicinal plant Streptocaulon
loops of the catalytic a subunits of sodium pumps. It had been shown juventas. They proved that di-glycosidation did not alter or even
that these cardenolide compounds are tightly fixed to the E2 decrease the activity. Hence, the hydroxyl groups at C1 and C5
conformational enzyme state, leading to inactivation [53]. E2- were important for the anti-proliferative property. These authors
phosphoenzyme-cardiotonic compound complexes were found to be concluded that bioactivity of the isolated cardenolides-set
formed irreversibly. With varying Kd values, the inhibition complexes depends on the hydrophilicity of the sugar unit linked to C3-
are probably internalized and degraded. Inhibitors of the Na+,K+ OH because a free hydroxyl C20 and acetylating of C30 OH reduced
transporter lead to an increase of the cytosolic Na+ concentration and the cytotoxicity [57].
are thus influencing the thermodynamics of the Na+,Ca2+ exchange The research group of O’Doherty developed a novel stereo-
pump finally resulting in an increased Ca2+ concentration in selective synthetic method to glycosylate the aglycone core units
sarcoplasmic reticulum [54] (Na+-Lag hypothesis). of cardenolides. The comparative study of the synthesized mono-,
However, recent studies suggest that this Na+ pump inhibition di- or tri-saccharide derivatives 05–07 of digitoxigenin 04 showed
is not necessary to obtain an inotropic effect on myocytes [55]. By that the linkage of a monosaccharide increased the bioactivity [59],
interacting with cardiac glycosides, the Na+–K+ pump complex has which confirmed the previously described studies by Yao and
been shown to act as a signalosome. Various intracellular signaling Kadota [56,57]. The higher anti-proliferative properties of the tri-
pathways are responsive to cardiac glycoside binding; Ca2+ could saccharide derivatives of digoxin and gitoxin underlined this result
play the role of a second messenger, the phosphatidylinositide [60]. In detail, the stereocenter at C20 of the sugar unit plays a
30 -kinase and protein kinase B can be activated as well as the SRC- crucial role in cytotoxicity, as the unnatural epimers of the
EGFR-RAS-RAF-ERK cascade. In this way, cardiac glycosides may cardenolides loose almost their complete apoptotic activity against
influence cell proliferation, cell differentiation and finally cell cancer cells [61].
death by apoptosis (Fig. 3). The anti-inflammatory bioactivity of cardenolides proved to be
sensible on chemical modifications of the core unit. Although, an
2.2. Cardenolide derivatives and their structure–activity relationship additional acetoxy function at C16 does not alter the observed
properties, an additional alcohol group at C8, modification of the
Recently, Toxicaroside D 03, isolated via bioassay-guided C14 hydroxyl group or a double bond between C16 and C17
fractionation from a ethanolic stem extract of the rainforest tree resulted in a lower anti-inflammatory activity [58].
 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10 5

nephrine, drives both, cardiac and arterial myocytes into

molecular remodeling for example by internalizing b-adrenergic
receptors, by altering G protein expressions, by lowering K+
channel expressions and by increasing Na+/Ca2+ pumps, thus
favoring severely reduced intrasarcoplasmic Ca2+ concentrations
[65].
Exogenous as wells as endogenous cardiac glycosides act
according to the Na+-Lag hypothesis: as seen before, partial
inhibition of the Na+–K+ pump by these cardiac glycosides results
in higher Na+ concentrations leading to increased Ca2+ concentra-
tions thus increasing cardiac output. Certain vertebrates endo-
genously produce cardiac glycosides with a steroid core, namely
the cardenolides Ouabain and Digoxin as well as the Bufadieno-
lides Marinobufagenin, Telocinobufagin and 19-Norbufalin. More-
over, since the 1990s there is increasing interest in the possible role
of the sodium pumps in myocytic hypertrophy [66]. On in vitro
myocyte cultures, inhibition of Na+,K+-ATPases by cardiotonic
compounds like ouabain resulted in increased contractility,
growth, protein synthesis, early proto-oncogene responses, AP-1
and NF-kB transcription factor activations thus explaining the
beneficial effect of digitalis, another cardiotonic steroid, on heart
failure. Despite being the oldest drug in cardiovascular medicine,
digitalis is still being used in contemporary heart failure
treatments.
Due to cardiotonic steroid-induced signaling pathways, the
various endogenously synthesized cardiac glycosides constitute a
new class of steroid hormones, regulating blood pressure, arterial
tension and insulin release.
Because of the evidence that cardiac glycosides might
influence cell proliferation and differentiation and that cancer
cells have enhanced Na+,K+ activity, further studies trying to
Fig. 3. Schematic representation of Na+/K+ ATPase inhibition by cardiotonic steroids.
elucidate the effects of cardiac glycosides on tumor growth came
Two conformationally different states are known for the Na+/K+ ATPase: in E1, up. In 2001, Stenkvist discovered a reduced death rate (6% versus
enzyme phosphorylation on the phosphorylation domain P enables intracellular 34%) of breast cancer patients when treated for a long period (22
binding of 3 Na+ in exchange to 2 K+, thus leading to state E2. The 3 Na+ are released years) with digitalis glycosides [67]. In the same context,
in the extracellular fluid whereas 2 new K+ are imported. Through hydrolysis, the
leukemia has been shown to be less frequent in a group of
enzymatic complex returns to state E1 with Pi release. Cardiotonic steroids (CS) like
cardenolides become tightly bound to the different extracellular loops (H1–H2, H3– patients treated with cardiac glycosides for heart failure. Also
H4, H5–H6) of the enzymatic a subunit. The holoenzyme is blocked in state E2, no bufalin, a bufadienolide cardiac glycoside, has been used for ages
further ion exchange is possible. in Chinese medicine to treat hepatocarcinoma and leukemia.
Meanwhile, numerous studies indicated that cardiac glycosides
A low anti-proliferative activity is not necessary for the are able to block several cancer cell types in the G2/M cell cycle
reversal of multi-drug resistance (MDR) as one cardenolide, even phase [68,69]. In order to further evaluate the cytotoxic effects of
exhibiting a cytotoxicity at a concentration range up to 77 mM, cardiac glycosides, it is of great importance to develop non-
might serve as potential future lead MDR-cancer reversal agent cardiotonic cardiac glycosides. UNBS1450 01, a semi-synthetic
due to the calcein accumulation in MDR human ovarian cancer derivative of 2-oxovoruscharin 02, one of the first non-cardiotonic
2780AD cells [58]. This compound had been isolated first by compounds, first described in 2005, just entered phase I of clinical
Neumann and its structure determination had been carried out by trials (Table 1).
the research teams of Reichstein and Yamauchi [62–64] (Figs. 4
and 5). 3. UNBS1450 and its anti-cancer activity
Interestingly, in the case of the hemi-synthetic derivative 01 of
200 -oxovoruscharin 02, the reduction of the carboxyl function into a 3.1. The impact of UNBS1450 on the Na+K+-ATPase isozymes
primary alcohol did not result in a higher cytotoxicity but the in
vivo toxicity was reduced by factor 10 [49] (Fig. 4). Several isozyme forms are known for Na+,K+-ATPases, depend-
The design of novel anti-cancer drugs derived from natural ing on tissue and cell type: 4a and 3b isoforms have been
products is still a matter in the development of potent cancer identified so far. Until today, severely increased expression of the
therapeutics. There is still a need and potential in further a1 subunit has been associated to the clinical patterns of non-
investigation of natural sodium pump inhibitors. small cell lung cancers (NSCLC) and glioblastoma [70,71].
For both studies, sodium pump inhibition assays were
2.3. Pharmaceutical activity of cardenolides performed using a heterologous protein expression system. Given
the advantage of producing high amounts of recombinant Na+/K+-
With chronic heart failure being a widespread health problem ATPases, baculovirus-transfected Sf-9 insect cells expressing
in modern society, there is an urge for new therapy drugs. a1b1, a2b1 and a3b1 isozymes were used to analyse the
According to today’s knowledge, arterial hypertension and inhibitory capacities of several cardenolides, among which
coronary artery disease are among the main causes for the UNBS1450 01. When compared to ouabain and digoxin, UNBS1450
development of heart failure diseases. Constant exposure to high 01 showed a greater Na+/K+-ATPase isozyme inhibitory potential.
concentrations of hypertension-producing hormones like epi- The a3b1 dimer was mostly affected by an UNBS1450 inhibition,
6 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10

Fig. 4. Chemical structure of a small selection of natural sodium pump inhibitors and its analogues.

with a determined Ki of 3.2 nM. The a2b1 and a1b1 isozymes Mijatovic et al. were among the very first to investigate a
were also inhibited by UNBS1450 01, but to a lesser extent, with Ki possible repressive potential of cardiotonic steroids and especially
values of 15 and 160  70 nM respectively. Compared to classic cardenolides on constitutive NF-kB activation [73]. In their work
cardenolides, the double-linked sugar moiety as well as the trans- they used a new hemi-synthetic derivative of 2-oxovoruscharin 02,
conformational steroid structure seem to further potentiate the a compound recently identified by themselves in C. procera and
inhibitory activity of UNBS1450 01, rendering this cardenolide isolated by liquid chromatography from methanolic extracts from
especially promising for additional anti-cancer studies based on the root barks of C. procera. In contrast to stereotype cardenolides,
modified cell proliferation, differentiation and cell death by inhibited the chemically modified 2-oxovoruscharin named UNBS1450 01
sodium pump driven signaling. shows a double-linked sugar moiety and a trans-conformational
According to recent findings, sodium pump signaling through steroid core.
UNBS1450 01 binding leads to disorganization of the actin As a first result, they managed to show that UNBS1450 01 is able
cytoskeleton, increased non-apoptotic cell death by autophagy, to influence NF-kB activity in human A549 cells. In fact, a 24-h
down-regulation of c-Myc in cancer cells, NF-kB deactivation and treatment with UNBS1450 01 with a concentration of 10 nM was
general modifications of the nucleolar morphology. sufficient to point out induced accumulation of I-kBb, however not
of I-kBa. For the latest, phosphorylation levels were decreased
3.2. The impact on the NF-kB signaling pathway after 4–8 h post-treatment. The expression of cdc34, an enzyme
implicated in I-kBa ubiquitination, was also decreased. Further
As many typical cancer cell lines, A549, non-small cell lung NF-kB binding (Trans-AM) as well as NF-kB reporter gene
cancer cells, show constitutive NF-kB pathway activation, and thus (luciferase reporter plasmid construct pNF-kB-Luc) assays con-
become able to escape cell death by apoptosis, necrosis, autophagy, firmed the in vitro inhibitory potential of UNBS1450 01 on A549
senescence, mitotic catastrophe and paraptosis [72]. NF-kB tumor cells. Secondly, preliminary in vivo studies revealed
activation favors multi-drug resistant cancer development. increased survival of A549 orthotopic xenograft-bearing nude

Fig. 5. SAR studies on analogues of natural sodium pump inhibitors. Summary of structure–activity relationship (SAR) results of natural sodium pump inhibitors and derivatives
concerning the observed anti-cancer cytotoxicity and anti-oxidant properties. The pharmacophores identified during the studies are highlighted in colour [49,56–64].
 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10 7

Table 1 chronically administrated (12 times) with 10 mg/kg of

Main endogenous and exogenous cardenolides and their pharmaceutical activity.
UNBS1450 01 showed best results, with a population down to
Digoxin Partial inhibition of the Na+–K+ pump Endogenous 45% only after 40 days. Higher concentrations (20 mg/kg)
Marinobufagenin leading to increased intracellular Ca2+
seemed to be less encouraging since the outcome was
19-Norbufalin concentrations; increased contractility
Ouabain and metabolism in myocytes in vitro less promising than for 10 mg/kg, probably due to slight toxic
Telocinobufagin effects.
Comparable results were obtained for per os administrations of
Bufalin Beneficial effect in hepatocarcinoma Exogenous
and leukemia treatment UNBS1450 01, nevertheless requiring higher dosages. Oral uptake
Digitalis Still in use for heart failure treatments of UNBS1450 01 at 80 mg/kg significantly increased mouse
UNBS1450 One of the first non-cardiotonic survival. Whereas the 50% survival barrier was broken shortly
cardenolides; promising anti-cancer
after 20 days post-tumor graft in control mice, with 80 mg/kg of
potentials
UNBS1450 01 this point was delayed to day 40.
A principal characteristic of many cancer cells is their high
invasiveness; glioblastoma cells for instance are active, ‘‘self-
mice when treated with different concentrations of the maximum propelled’’ brain cancer cells, highly capable of invading brain
tolerated dose of UNBS1450 01. tissue by shape and volume modifications [74]. Today it is believed
that ion transport plays an important role in enabling cancer cell
3.3. The effect of UNBS1450 on cell proliferation and cancer migration [75]. Until very recently, the impact of the Na+,K+-
ATPase in cancer cell migration remained unexplored. In early
Starting with in vitro anti-proliferation assays in 2005, Van 2008, Lefranc et al. tried to establish a link between glioblastoma
Quaquebeke et al. analysed cytotoxicity levels of several cell migratory invasion, sodium pump and cardenolide adminis-
cardenolides, among which UNBS1450 01, on 57 human cancer tration [71]. At first, they demonstrated that in human glioblas-
cell lines; for each compound average IC50 values were toma cells the a1 subunit of the Na+,K+-ATPase is overexpressed
determined by MTT assays after 72 h of incubation. As reference when compared to normal human brain tissue. Using immuno-
drugs, a tubulin inhibitor (taxol) and a topoisomerase I inhibitor fluorescent staining, they were able to show that a1 subunits co-
(SN-38) were chosen. Concerning the individual sensitivity of localize with caveolin-1 in the cytoplasmic protrusions of U373-
each human cancer cell line, significant differences were observed MG glioblastoma cells.
between UNBS1450 01, taxol and SN-38 suggesting a different Additionally, UNBS1450 01 was able to exert a cytotoxic effect
antitumor mechanism for UNBS1450 01. In terms of average on human U373-MG cells; the same was true for rat C6 cells, but to
growth inhibition however, UNBS1450 01 was proven to be as a lesser extent. For U373-MG, concentrations of around 100 nM of
potent as taxol, with a mean IC50 of 2.7 nM for UNBS1450 01 and both UNBS1450 01 as well as ouabain rendered cell survival nearly
2.5 nM for taxol. SN-38 was significantly less potent. Furthermore, impossible. For rat C9 cells, ouabain was practically ineffective
when applicated on multi-drug resistant tumor bladder cells, whereas high concentrations reaching 10 mM were required to
UNBS1450 01 showed rather astonishing capacities in inhibiting completely abolish cell viability. An impact of UNBS1450 01 on
cell growth: vincristine and adriamycin resistant tumor cells intracellular ATP concentrations in human glioblastoma cells had
forced taxol to reach concentrations of 10,000 nM to become then been suggested. Next, computer-assisted phase-contrast
cytotoxic whereas UNBS1450 01 became effective at 45 nM. microscopy analysis permitted to gain further insights in cellular
Complementary to these data, UNBS1450 01 also showed proliferation, morphology and motility as well as on ion home-
cytotoxic effects on rat glioma, mouse mammary carcinoma ostasis. They discovered that in the presence of 10 nM UNBS1450
and melanoma cells but at a far less extent when compared to 01, U373 cells are well capable of undergoing mitosis, but fail to
UNBS1450 01 anti-tumor effects in human cancer cells. This divide into daughter cells and to form lamellipodia. Nevertheless,
feature could relate to the double mutation observed in the a1 the observed, round-shaped U373 cells remained alive for several
subunit of rodent sodium pump. days (mitochondria staining). However, contrary to a possible
More recently, the observed findings were confirmed: hypothesis, the administration of 10 nM of UNBS1450 01 did not
UNBS1450 01 is also capable of repressing in vitro growth of result in either an increased Ca2+ nor an increased Na+ concentra-
A549 tumor cells as well as growth and migration of glioblastoma tion, which at the same time constitutes one of the major
cell lines. advantages of UNBS1450 when compared to other cardiac
UNBS1450’s in vivo anti-cancer activity has been investigated glycosides like digitalis. Increasing intracellular Ca2+ favors
so far by tumor growth analysis in xenograft models. Generally, arrhythmias in otherwise healthy people. The observed changes
by administering UNBS1450 01 chronically to nude mice bearing were probably due to irreversibly modified actin cytoskeleton,
orthotopic xenografts of NSCLC and glioblastoma cell lines, thus explaining reduced migratory capacities of these human
cytotoxicity of this hemi-synthetic cardenolide has been glioblastoma cells (Fig. 6).
determined by intraperitoneal or per os injections. In both
cases, the maximum tolerated dose (MTD) had been previously 3.4. UNBS1450-induced cell death
elucidated acutely in healthy mice. When compared to classical
cardenolides such as ouabain and digoxin as well as to its 3.4.1. By apoptosis
precursor, 2-oxovoruscharin 02, UNBS1450 01 showed signifi- It has been reported in literature that cardiac steroid glycosides
cantly increased MTD values: for ouabain and digoxin, MTD are a priori potentially able to induce cell death by apoptosis. For
values were 5 and 10 mg/kg respectively, whereas for UNBS1450 example it has been observed in vitro that different leukemia cell
01 the MTD value raised up to 120 mg/kg. As a result, chronic lines such as THP-1 [76], K562 [77], HL-60, U937 and ML1 [78]
administration of UNBS1450 01 was realized with different undergo apoptosis when treated with different concentrations of
concentrations, from a 32- to an 8-fold reduced MTD [73]. Bufalin or Digoxin. However, when treated with rather low
Interestingly, UNBS1450 01 at concentrations from 5 to 20 mg/ nanomolar doses of these cardiac glycosides, it might preferen-
kg was proven to be able to increase grafted mice survival. In tially stimulate differentiation and proliferation. It is believed that
control mice, about 20 days were sufficient to bring the mouse nanomolar concentrations lead to low-frequency oscillations of
population down to 45%. In contrast, mice which had been Ca2+ concentrations favoring NF-kB activation and protection
8 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10

Fig. 6. In vitro and in vivo effects of UNBS1450.

against apoptosis whereas higher concentrations would lead to situation, autophagy sometimes constitutes an alternative path-
apoptosis due to sustained, high levels of Na+ and Ca2+[79]. way to apoptosis, sometimes a possibility to escape apoptosis.
In contrast to many other cardiac steroids, UNBS1450 01 does In 2006, first studies indicating UNBS1450-induced autophagy
not seem to induce cell death by apoptosis in vitro, at least when in cancer cells appeared [80]. Working on NSCLC cell lines, cell
working with adherent cell lines (evidence provided by flow death was indeed relevant after treatments with UNBS1450 01: a
cytometry analyses after propidium iodide and Annexin V 24 h treatment with 100 nM of UNBS1450 reduced cell viability to
staining). To our knowledge, the entire studies on UNBS1450’s about 70%, when prolonged to 72 h, 10 nM were sufficient to
anti-proliferative and anti-cancer activity has been carried out on obtain similar results. Consequently, PARP cleavage analysis
solid tumor cell lines. UNBS1450 01 stimulation has been shown to permitted to exclude cell death through apoptosis, as PARP
induce non-apoptotic cell death [71,80]. remained intact. Complementary performed Tunel assays ulti-
mately confirmed the absence of apoptotic cell death. Cell cycle
3.4.2. By autophagy analysis revealed increased percentages of cells in S and G2 phases
Recent studies on UNBS1450-induced cell death claim pro- whereas microscopy images clearly showed severe cytoplasmic
autophagic effects to be responsible for the observed cytotoxic vacuole formation. Further Mijatovic et al. made clear that
potential [71]. It has been reported that during autophagy, besides UNBS1450 01 induces Hsp70 downregulation thus rendering
apoptosis, one of the two self-destructive processes, intracellular possible lysosomal membrane permeabilization, typically inhib-
organelles are enclosed in characteristic double- or multi- ited in lung cancer cells by heightened levels of Hsp70 which
membraned vacuoles which finally fusion with lysosomes thus constitute a prosurvival function. Lysosomal permeabilization is a
leading to degradation [81]. Together with apoptosis, cellular ‘‘self- known indicator of cell death through autophagy; these findings of
eating’’ has the aim to eliminate superfluous, damaged or aged 2006 then strengthened the hypothesis of non-apoptotic cell death
cells or organelles like mitochondria and endoplasmic reticulum. by ‘‘self-eating’’ in UNBS1450 treated cancer cells.
Moreover, being implicated in macromolecule catabolism, autop- In the same context UNBS1450 01 has been assigned to
hagy helps cells in overcoming stress situations, thus avoiding induced cell death through autophagy in different human
probable cell death by apoptosis for instance. Depending on the glioblastoma cell lines [71]. In a first step, acridine orange

Fig. 7. Apoptosis and autophagy-related cell death induced by UNBS1450.

 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10 9

staining was used in order to quantify acidic vesicular organelles [2] Sharma P, Sharma JD. Evaluation of in vitro schizontocidal activity of plant
parts of Calotropis procera—an ethnobotanical approach. J Ethnopharmacol
whose abundance is tightly associated to autophagy. Indeed, the 1999;68:83–95.
presence of nanomolar UNBS1450 01 significantly increased [3] Dewan S, Sangraula H, Kumar VL. Preliminary studies on the analgesic activity
staining in U373-MG and U87-MG cells (from 3 to 42 and from 4 of latex of Calotropris procera. J Ethnopharmacol 2000;73:307–11.
[4] Kumar S, Dewan S, Sangraula H, Kumar VL. Anti-diarrhoeal activity of the latex
to 65%). At the same time, the expressions of LC3 and beclin-1, of Calotropis procera. J Ethnopharmacol 2001;76:115–8.
two specific autophagy markers, were found to be stimulated by [5] Kumar VL, Shivkar YM. In vivo and in vitro effect of latex of Calotropis procera
UNBS1450 01. on gastrointestinal smooth muscles. J Ethnopharmacol 2004;93:377–9.
[6] Yeang HY, Arif SA, Yusof F, Sunderasan E. Allergenic proteins of natural rubber
Besides lung cancer and glioblastoma cell lines, UNBS1450 01 latex. Methods 2002;27:32–45.
has also been found to induce probably autophagy-related cell [7] Azarkan M, Amrani A, Nijs M, Vandermeers A, Zerhouni S, Smolders N, et al.
death in human prostate cancer cell lines by severe nucleolar Carica papaya latex is a rich source of a class II chitinase. Phytochemistry
1997;46:1319–25.
targeting [82]. In fact, UNBS1450 01 efficiently inhibited apopto-
[8] Nallamsetty S, Kundu S, Jagannadham MV. Purification and biochemical
sis-resistant PC-3 prostate cancer cell growth at 100 nM, without characterization of a highly active cysteine protease ervatamin A from the
caspase-3 and caspase-9 activation and PARP cleavage. Intracel- latex of Ervatamia coronaria. J Protein Chem 2003;22:1–13.
lular Ca2+ concentrations were not modified, in contrast to cellular [9] Soares PM, Lima SR, Matos SG, Andrade MM, Patrocinio MC, de Freitas CD, et al.
Antinociceptive activity of Calotropis procera latex in mice. J Ethnopharmacol
ROS load. Not only did UNBS1450 01 (10 and 100 nM) severely 2005;99:125–9.
increase ROS production, computer-assisted phase-contrast [10] Freitas CD, Oliveira JS, Miranda MR, Macedo NM, Sales MP, Villas-Boas LA, et al.
microscopy revealed that it again completely disorganized the Enzymatic activities and protein profile of latex from Calotropis procera. Plant
Physiol Biochem 2007;45:781–9.
actin cytoskeleton as seen before and disturbs nucleolar morphol- [11] Ramos MV, Bandeira Gde P, de Freitas CD, Nogueira NA, Alencar NM, de Sousa
ogy. The nucleolus domain constitutes the site of ribosome PA, et al. Latex constituents from Calotropis procera (R. Br.) display toxicity
biogenesis and is thus indispensable to cell survival. It has been upon egg hatching and larvae of Aedes aegypti (Linn.). Mem Inst Oswaldo Cruz
2006;101:503–10.
reported that nucleolar regions are of great importance in cancer [12] Sangraula HDS, Kumar VL. Evaluation of anti-inflammatory activity of latex of
cells as it has been proven that tumor cells retain tumor Calotropis procera in different models of inflammation. Inflammopharmacol-
suppressors, like p53 and MYC for instance, inside the nucleoli. ogy 2001;9:257–64.
[13] Arya S, Kumar VL. Antiinflammatory efficacy of extracts of latex of Calotropis
The observed impairments of CDK and Myc protein expressions
procera against different mediators of inflammation. Mediators Inflamm
might thus be mechanisms of nucleolar disorganization and 2005;2005:228–32.
induced autophagy (Fig. 7). [14] Roy S, Sehgal R, Padhy BM, Kumar VL. Antioxidant and protective effect of latex
of Calotropis procera against alloxan-induced diabetes in rats. J Ethnopharma-
col 2005;102:470–3.
4. Conclusion [15] Shivkar YM, Kumar VL. Histamine mediates the pro-inflammatory effect of
latex of Calotropis procera in rats. Mediators Inflamm 2003;12:299–302.
[16] Choedon T, Mathan G, Arya S, Kumar VL, Kumar V. Anticancer and cytotoxic
Taken together, these results suggest that the novel hemi-
properties of the latex of Calotropis procera in a transgenic mouse model of
synthetic cardenolide UNBS1450 01 from C. procera is able to hepatocellular carcinoma. World J Gastroenterol 2006;12:2517–22.
repress cancer cell proliferation and even induce cell death. [17] Grout D, Curtis R, Hassall C, Jones T. Cardiac glycosides of Calotropis procera.
Tetrahedron 1962;26:1281–5.
Despite the fact that the plant’s beneficial effects on many different
[18] Seiber J, Nelson C, Lee S. Cardenolides in the latex and leaves of seven Asclepias
pathologies have been admitted many centuries ago, the real species and Calotropis procera. Phytochemistry 1982;21:2343–8.
breakthrough is still to come. Both, the entire latex as well as [19] Saber A, Hifney, Maharan G, Rizkallah M. Sterols and pentacyclic triterpenes of
organic extracts, might well have proven their usefulness in Calotropis procera. In: Bulletin of the Faculty of Pharmacy, vol. 7. Cairo
University; 1969. p. 91–104.
treating different clinical entities, only recent advances in research [20] Saxena V, Saxena Y. Isolation and study of triterpenoids from Calotropis
revealed the true potential lying within C. procera. By chemically procera. J Res Indian Med Yoga Homeopathy 1979;14:152–4.
modifying a novel cardenolide isolated from this tropical plant, [21] Tiwari K, Masood M, Rathore S, Minocha P. Study of anthocyanins from the
flowers of some medicinal plants. Vijnana Parishad Anusandhan Patrika 1978;
researchers discovered with UNBS1450 01 a new and very 21:177–8.
promising weapon in cancer therapy. [22] Carruthers I, Griffiths D, Home V, Williams L. Hydrocarbons from Calotropis
In contrast to many other anti-cancer agents, UNBS1450 01 procera in northern Australia. Biomass 1984;4:275–82.
[23] Malik N, Chughtai M. Antimicrobial activity of Calotropis procera. A prelimin-
does not induce apoptosis but autophagy, at least in solid tumor ary study. Pakistan J Sci 1979;31:127–9.
cells. Essentially by deactivating NF-kB, down-regulating Hsp70 [24] Nandal S, Bhatti D. Preliminary screening of some weeds and shrubs for their
and by modifying nucleolar structure, UNBS1450 01 leads to nematocidal activity against Meloidogyne javanica. Indian J Nematol 1983;13:
123–7.
vacuole formation, actin cytoskeleton impairment and finally cell
[25] Masood A, Haq S, Anjum S, Saxena S. Further studies on the effect of some plant
death. extracts on the mortality of Meloidogyne incognita. J Sci Res Plants Med
Further insights, particularly concerning its usefulness in 1980;1:18–22.
[26] Girdhar G, Deval K, Mittal P, Vasudevan P. Mosquito control by Calotropis latex.
leukemia treatment, still remain to be investigated.
Pesticides 1984;18:82–7.
[27] Prakash A, Gupta R, Mathur R. Effect of oral administration of forty two
Acknowledgements indigenous plants extracts on early and late pregnancy in albino rats. Probe
1978;27:315–23.
[28] Dhar M, Dhar M, Dhawan B, Mehrotra M, Roy C. Screening of Indian plants for
The authors thank C. Cerella and M.H. Teiten for critical reading. biological activity: part I. Indian J Exp Biol 1968;6:231–47.
T.J. is a recipient of an Aide à la Formation-Recherche (AFR) [29] Ayoub S, Kingston D. Screening of plants used in Sudan folk medicine for
fellowship and M.S. is a recipient of a Télévie fellowship. Dr. anticancer activity. Fitoterapia 1981;52:281–4.
[30] Fenado MRWS, Thabre MI, Karunayaka EH. J Ethnopharmacol 1989;7.
Diederich’s research at the Laboratoire de Biologie Moléculaire et [31] Asuzu IU, Onu OU. Anthelmintic activity of the ethanolic extract of Piliostigma
Cellulaire du Cancer (LBMCC) is financially supported by thonningii bark in Ascaridia galli infected chickens. Fitoterapia 1994;65:291–7.
‘‘Recherche Cancer et Sang’’ foundation, by the ‘‘Recherches [32] Mishra PPN, Guru PY, Katiyar JC, Tandon JS. Antimalarial activity of traditional
plants against erythrocytic stages of P. berghei. Int J Pharmacognosy 1991;29:
Scientifiques Luxembourg’’ association, by ‘‘Een Häerz fir kriib- 19–23.
skrank Kanner’’ association, by the Action Lions ‘‘Vaincre le Cancer’’ [33] Mascolo N, Sharma R, Jain SC, Capasso F. Ethnopharmacology of Calotropis
association and by Télévie Luxembourg. procera flowers. J Ethnopharmacol 1988;22:211–21.
[34] Ang HH, Chan KL, Mak JW. In vitro antimalarial activity of quassinoids from
Eurycoma longifolia against Malaysian chloroquine-resistant Plasmodium fal-
References ciparum isolates. Planta Med 1995;61:177–8.
[35] Iqbal Z, Lateef M, Jabbar A, Muhammad G, Khan MN. Anthelmintic activity of
[1] Shu YZ. Recent natural products based drug development: a pharmaceutical Calotropis procera (Ait.) Ait. F. flowers in sheep. J Ethnopharmacol 2005;102:
industry perspective. J Nat Prod 1998;61:1053–71. 256–61.
10 T. Juncker et al. / Biochemical Pharmacology 78 (2009) 1–10

[36] Moursy LE. Insecticidal activity of Calotropis procera extracts of the flesh fly. [60] Lopez-Lazaro M, Pastor N, Azrak SS, Ayuso MJ, Austin CA, Cortes F. Digitoxin
Sarcophaga haemorrhoidalis fallen. J Egypt Soc Parasitol 1997;27:505–14. inhibits the growth of cancer cell lines at concentrations commonly found in
[37] Ansari SH, Ali M. Norditerpenic ester and pentacyclic triterpenoids from root cardiac patients. J Nat Prod 2005;68:1642–5.
bark of Calotropis procera (Ait) R. Br Pharmazie 2001;56:175–7. [61] Langenhan JM, Peters NR, Guzei IA, Hoffmann FM, Thorson JS. Enhancing the
[38] Gallegos Olea RS, Oliveira AV, Silveira LM, Silveira ER. Organic carbonate from anticancer properties of cardiac glycosides by neoglycorandomization. Proc
Calotropis procera leaves. Fitoterapia 2002;73:263–5. Natl Acad Sci USA 2005;102:12305–10.
[39] Brady C. Fruit ripening. Annu Rev Plant Physiol 1985;38:155–78. [62] Neumann W. Über Glykoside des Oleanders. Berichte der deutschen che-
[40] Kembhavi AA, Buttle DJ, Knight CG, Barrett AJ. The two cysteine endopepti- mischen Gesellschaft 1937;70:1547–54.
dases of legume seeds: purification and characterization by use of specific [63] St Janiak P, Weiss E, Reichstein T. ‘‘Alpha’’-,‘‘beta’’- and ‘‘delta’’-anhydrodigi-
fluorometric assays. Arch Biochem Biophys 1993;303:208–13. toxigenin. Helv Chim Acta 1967;50:1249–66.
[41] Rudenskaya GN, Bogacheva AM, Preusser A, Kuznetsova AV, Dunaevsky Ya E, [64] Abe F, Yamauchi T, Shibuya H, Kitagawa I. Triterpenoid glycosides from bark of
Golovkin BN, et al. Taraxalisin—a serine proteinase from dandelion Taraxacum Meliosma lanceolata. Phytochemistry 1996;42:809–14.
officinale Webb s.l. FEBS Lett 1998;437:237–40. [65] Schoner W, Scheiner-Bobis G. Endogenous and exogenous cardiac glycosides:
[42] Dubey VK, Jagannadham MV. Procerain, a stable cysteine protease from the their roles in hypertension, salt metabolism, and cell growth. Am J Physiol Cell
latex of Calotropis procera. Phytochemistry 2003;62:1057–71. Physiol 2007;293:C509–36.
[43] Boller T. Roles of proteolytic enzymes in interaction of plant and other [66] Xie Z, Askari A. Na(+)/K(+)-ATPase as a signal transducer. Eur J Biochem
organisms. In: Dalling M, editor. Plant Proteolytic Enzymes. CRC Press; 2002;269:2434–9.
1986. p. 68–96. [67] Stenkvist B. Cardenolides and cancer. Anticancer Drugs 2001;12:635–8.
[44] Abraham KI, Joshi PN. Studies on proteinases from Calotropis gigantea latex. I. [68] McConkey DJ, Lin Y, Nutt LK, Ozel HZ, Newman RA. Cardiac glycosides
Purification and some properties of two proteinases containing carbohydrate. stimulate Ca2+ increases and apoptosis in androgen-independent, metastatic
Biochim Biophys Acta 1979;568:111–9. human prostate adenocarcinoma cells. Cancer Res 2000;60:3807–12.
[45] Hounsell E, Davies M, Smith K. Chemical methods of analysis of glycoproteins. [69] Haux J, Solheim O, Isaksen T, Angelsen A. Digitoxin, in non-toxic concentra-
In: Walker J, editor. The Protein Protocols Handbook. Humana Press; 2002. p. tions, inhibits proliferation and induces cell death in prostate cancer cell lines.
803–4. Zeitgenössische Onkologie 2000;32:11–6.
[46] Ellman L. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7. [70] Mijatovic T, Roland I, Van Quaquebeke E, Nilsson B, Mathieu A, Van Vynckt F,
[47] Watt J, Breyer-Brandwijk M. Medicinal and Poisonous Plants of Southern and et al. The alpha1 subunit of the sodium pump could represent a novel target to
Eastern Africa. E. & S. Livingstone; 1962. combat non-small cell lung cancers. J Pathol 2007;212:170–9.
[48] Erdman MD. Nutrient and cardenolide composition of unextracted and sol- [71] Lefranc F, Mijatovic T, Kondo Y, Sauvage S, Roland I, Debeir O, et al. Targeting
vent-extracted Calotropis procera. J Agric Food Chem 1983;31:509–13. the alpha 1 subunit of the sodium pump to combat glioblastoma cells.
[49] Van Quaquebeke E, Simon G, Andre A, Dewelle J, El Yazidi M, Bruyneel F, et al. Neurosurgery 2008;62:211–21 [discussion 21-2].
Identification of a novel cardenolide (20 0 -oxovoruscharin) from Calotropis [72] Okada H, Mak TW. Pathways of apoptotic and non-apoptotic death in tumour
procera and the hemisynthesis of novel derivatives displaying potent in vitro cells. Nat Rev Cancer 2004;4:592–603.
antitumor activities and high in vivo tolerance: structure–activity relationship [73] Mijatovic T, Op De Beeck A, Van Quaquebeke E, Dewelle J, Darro F, de Launoit Y,
analyses. J Med Chem 2005;48:849–56. et al. The cardenolide UNBS1450 is able to deactivate nuclear factor kappaB-
[50] Mijatovic T, Lefranc F, Van Quaquebeke E, Van Vynckt F, Darro F, Kiss R. mediated cytoprotective effects in human non-small cell lung cancer cells. Mol
UNBS1450: a new hemi-synthetic cardenolide with promising anti-cancer Cancer Ther 2006;5:391–9.
activity. Drug Dev Res 2007;68:164–73. [74] Sontheimer H. Malignant gliomas: perverting glutamate and ion homeostasis
[51] Horisberger JD. Recent insights into the structure and mechanism of the for selective advantage. Trends Neurosci 2003;26:543–9.
sodium pump. Physiology (Bethesda) 2004;19:377–87. [75] Ransom CB, O’Neal JT, Sontheimer H. Volume-activated chloride currents
[52] Faller LD. Mechanistic studies of sodium pump. Arch Biochem Biophys 2008; contribute to the resting conductance and invasive migration of human glioma
476:12–21. cells. J Neurosci 2001;21:7674–83.
[53] Erdmann E, Schoner W. Ouabain-receptor interactions in (Na+K+)-ATPase [76] Kurosawa M, Tani Y, Nishimura S, Numazawa S, Yoshida T. Distinct PKC
preparations from different tissues and species. Determination of kinetic isozymes regulate bufalin-induced differentiation and apoptosis in human
constants and dissociation constants. Biochim Biophys Acta 1973;307: monocytic cells. Am J Physiol Cell Physiol 2001;280:C459–64.
386–98. [77] Numazawa S, Honma Y, Yamamoto T, Yoshida T, Kuroiwa Y. A cardiotonic
[54] Keller M, Pignier C, Egger M, Niggli E. F90927: a new member in the class of steroid bufalin-like factor in human plasma induces leukemia cell differentia-
cardioactive steroids. Cardiovasc Drug Rev 2007;25:210–20. tion. Leuk Res 1995;19:945–53.
[55] Xie Z, Cai T. Na+–K+—ATPase-mediated signal transduction: from protein [78] Zhang S, Malmersjo S, Li J, Ando H, Aizman O, Uhlen P, et al. Distinct role of the
interaction to cellular function. Mol Interv 2003;3:157–68. N-terminal tail of the Na,K-ATPase catalytic subunit as a signal transducer. J
[56] Jiang MM, Dai Y, Gao H, Zhang X, Wang GH, He JY, et al. Cardenolides from Biol Chem 2006;281:21954–62.
Antiaris toxicaria as potent selective Nur77 modulators. Chem Pharm Bull [79] Aizman O, Uhlen P, Lal M, Brismar H, Aperia A. Ouabain, a steroid hormone that
(Tokyo) 2008;56:1005–8. signals with slow calcium oscillations. Proc Natl Acad Sci USA 2001;98:13420–4.
[57] Ueda JY, Tezuka Y, Banskota AH, Tran QL, Tran QK, Saiki I, et al. Antiprolifera- [80] Mijatovic T, Mathieu V, Gaussin JF, De Neve N, Ribaucour F, Van Quaquebeke E,
tive activity of cardenolides isolated from Streptocaulon juventas. Biol Pharm et al. Cardenolide-induced lysosomal membrane permeabilization demon-
Bull 2003;26:1431–5. strates therapeutic benefits in experimental human non-small cell lung
[58] Zhao M, Bai L, Wang L, Toki A, Hasegawa T, Kikuchi M, et al. Bioactive cancers. Neoplasia 2006;8:402–12.
cardenolides from the stems and twigs of Nerium oleander. J Nat Prod [81] Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: cross-
2007;70:1098–103. talk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007;8:741–52.
[59] Zhou M, O’Doherty G. The de novo synthesis of oligosaccharides: application to [82] Mijatovic T, De Neve N, Gailly P, Mathieu V, Haibe-Kains B, Bontempi G, et al.
the medicinal chemistry SAR-study of digitoxin. Curr Top Med Chem 2008;8: Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor
114–25. activity. Mol Cancer Ther 2008;7:1285–96.