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Na,K-ATPase isoform-selective cardiac glycosides-a potential anti-cancer drug ?
Adriana Katz,
Cherniavsky-Lev M., Ainbinder E., Tal D. and Karlish SJDWeizmann Institute of Science, Israel
Ion channels and pumps as cancer targets!
Over the years, several reports have suggested that cardiac
glycosides may have an anticancer utilization. In vitro and ex vivo
experiments have revealed that some cardiac glycosides induce potent and
selective anticancer effects, which may occur at concentrations commonly
found in the plasma of patients treated with these drugs.
Na+ /K+ -ATPase could be targeted to combat chemoresistant cancers.
Na,K ATPase is a vital protein in all mammalian cells.
b Subunit targets into the membrane, 3 isoforms: 1b 2b 3b
a subunit
FXYD
Catalytic moiety, 4 isoforms:a1, ubiqutous , all tissuesa2, skeletal muscle, heart, eyesa3, neurons braina4, sperms
membrane
Cytoplasm
Extracellular space
accessory protein, regulator
DigoxinSpecific inhibitor
. . Na,K -ATPase is an oligomeric transmembrane protein, localized to the
basolateral plasma membrane in most epithelial cells.
Na,/K-ATPase pumps Na+ and K+ against their physiological gradients.
Na,K pump is essential for many physiological processes
Renal function, and regulation of hypertension .
Cardiac contraction
Regulation of intra ocular pressure, IOP.
And many more….
All of the isoforms are expressed in a tissue and functional specific manner.
1/1
ab 1 1a b
2/ 2a b
3 1a b3/ 1a b
1/ 1a b 2/ 1a b
Expression of isoforms in different tissues.
2/ 3a b
1/ 1a b
OOCH3
HOHO
CH2OH
HOHO
OO
OHHO
HOH3C
The cardiac glycosides are an important class of naturally occurring
drugs whose actions include both beneficial and toxic effects on the heart.
Cardiac glycosides (CG)
Lactone ring
Steroid
Glycone
Digoxin
Withering W (1785). “An account of the foxglove and some of its medical uses: with practical remarks on dropsy and other diseases.”
Digitalis purpurea
O
O
CH3
OH
CH3
H14
17 20
3
21
22
OO
OH
H3CO
O
OH
H3C
HO
OO
OH
H3C
HO
Bufo bufo Bufalin
O
OCH3
OH
CH3
H14
3OH
HOStrophanthus gratus
Ouabain
OOCH3
HOHO
CH2OH
HOHO
OO
OHHO
HOH3C
Cardiac glycosides, naturally occurring in plants and animals.
Cardiac glycosides have a long history of therapeutic application.
Plants containing cardiac steroids have been used as poisons and heart drugs
at least since 1500 B.C.
The early understanding of their positive inotropic effects facilitated their use as effective
drugs for the treatment of heart-related pathologies, yet their toxicity remains a serious problem.
More recently, considerable in vitro, in vivo and epidemiological data support novel roles
for CG’s such as inducing apoptosis and inhibit the growth of cancer cell lines.
Control +10uM CG +20uM CG
Liang Fei Ye,et al.2013 Oncology letters‑
Slingerland M. et al
Na+,K+ ATPase
Na+/Ca+2
exchang.Na+/H+ exchang.
Proposed mode of action of cardiac glycosides, CG
The decrease in intracellular K+ and increase in intracellular Na+ and Ca2+ following inhibition of the Na+/K+-ATPase may induce apoptosis
Na,K-ATPase as a versatile signal transducer ?
Na+/K+-ATPase may
also act as a signal transducer.
When intact cells are exposed
to digitalis drugs (e.g., ouabain
and digoxin) specific inhibitors
of this enzyme various cell
signaling pathways are
activated leading to highly cell-
specific down-stream
consequences.
Proliferation of CG-treated cells.
Breast cancer cells are not more sensitive to CG’s cytotoxicity
than are normal cells.
Normal Cancer cells
Clifford RJ, and Kaplan JH 2013 PLOS ONE 8,
Purification and stabilization of isoforms of human Na,K-ATPase expressed in Pichia pastoris
Isoforms of human Na,K-ATPase expressed in Pichia pastoris
FX
YD
1
Detergent , C12E8Phosphatidyl serine,SOPSPhosphatidyl choline, PCCholesterol
Functional, stable, detergent-soluble FXYD complex
Enables different combinations of isoforms
a1a23MW
Human isomers/1 purified enzymes
Isoform selectivity is determined by the sugar component!
Digoxin, is partially 2-selective CG, while Ouabain shows very low
selectivity
Bo
un
d 3 H
-Ou
ab
ain
, fr
ac
tio
n o
f c
on
tro
l
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1
0
0.2
0.4
0.6
0.8
1
1.2
0.001 0.01 0.1 1 10
Digoxigenin
Digoxin
Digoxigenin, μM
Digoxin, μM
Cardiac glycoside affinity and selectivity for the isoforms.
CGCalculated Kd±SEM, nM Ratio of Kd’s, ± SE
1a 2 3a 1a / 2a 1a / 3a
Ouabain 9.8±0.33 21.9±0.56P=0.0001
11.1±1.3 0.44±0.01 0.88±0.1
Digoxin 87±6.0 25.6±2.8P=0.001
25±2.4P=0.001
3.39±0.43 3.48±0.41
Digoxigenin 270±21 332±11 307±27 0.81±0.06 0.88±0.1
-Methyl digoxin
129±20 43±7P=0.018
31±4P=0.009
3.0±0.67 4.16±0.84
Digitoxin 38±3 18.3±4P=0.02
14±3.9P=0.008
2.07±0.48 2.70±0.78
Digitoxigenin 101±13.4 125±13.6 134±15.6 0.8±0.13 0.75±0.13
Bufalin
Marinobufagenin
42.5±6.5
2240±137
45±8
2470±45
40±11
2430±205
0.94±0.22
0.91±0.06
1.06±0.33
0.92 ±0.1
Isoform selectivity of Cardiac glycosides
Na,K-ATPase structure with bound ouabain
Ouabain
The residues common to 2a and 3a and different in a1 are all located on the extracellular loops at the entrance to the Ouabain binding site, close to the sugar moiety.
In vitro growth inhibitory concentrations at 50% (IC50) in human cancer cells after
three days of culture in the presence of the drug of interest
Inhibition of cancer cell proliferation at low concentrations is used to assess the
therapeutic potential of drug candidates in preclinical studies.
-9.00 -8.00 -7.00
-150
-100
-50
0
50
100
150
SF-268 (central nervous system)
Digoxin
DM
Ouabain
GBR
D.EA
Ol
Log of Sample Conc. (Molar)
Ce
ll G
row
th (
%)
2.
-9.00 -8.00 -7.00 -6.00 -5.00
-100
-50
0
50
100
150
A549 (non small cell lung carcinoma )
D.MAD.EAO.SCD.EOCF3FDGL
Log of Sample Conc. (Molar)
Cell
Gro
wth
(%)
-9.00 -8.00 -7.00 -6.00 -5.00
-100
-50
0
50
100
150 HFF (Human foreskin fibroblast)
D.MAD.EAO.SCD.EOCF3FDGL
Log of Sample Conc. (Molar)
Cell
Gro
wth
(%)
3.
0.3 3 30 300-120
-100
-80
-60
-40
-20
0
20
40
60
80
100
120
140
Gamabufotalin- rhamnoside conc.,nM
Cell
Gro
wth
, %
LLC-PKMCF-7HCT-116TK-10
Cell lines: LLC-PK pig kidney, MCF-7 breast cancer , HCT-116 colon cancer, TK-10 renal cancer.
In vitro growth inhibitory concentrations at 50% (IC50) in human cancer
cells after two days of culture in presence of GBR, the most effective CG.
1.
0 0.05 0.1 0.15 0.2 0.250
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.1
ACNHSF 268MCF 7MDA-MB-23Sk-Mel-5HCT 116A549TK 10ovcar 3
Ki of CG's on purified Na,K-ATPase
IC5
0 o
f C
G f
or
ca
nc
er
ce
ll lin
es
Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effects
DigoxinGBR OleandrinHellebrig. Ouabain
Hellebrin
20 40 60 80 100 120 140 160 180 200 2200
10
20
30
40
50
60
70
Ki, nM
IC
50
, n
m
1
2
3
45
6
1. Digoxin2.Hellebrin3.Ouabain4.Oleandrin5.Hellebrigenin6. Gamabuf. Rhamnoside
4 repetitions with 14 different cancer cells.
Correlation of Ki for inhibition of human α1β1 by cardiac
glycosides and the growth inhibition effects
Cell growth is linearly correleted to the affinity of individual CG
to the Na,K-ATPaes
0
20
40
60
80
100
0 20 40 60 80 100 120
Relative binding capacity (%of α1-GFP)
Via
bilit
y at
30
nM O
uaba
in (
% o
f N
T)
α1-GFP
Si-α1-8
Si-α1-5
Si-α1-3
Viability is linearly correlated with number of active pumps in partially silenced H1299 cells
0
20
40
60
80
100
120
0 0.5 1 1.5
0
20
40
60
80
100
120
0 200 400 600 800 1000 1200
Viab
ility
(%of
NT)
Viab
ility
(%of
NT)
Bmax (pmol oub*mg protein-1)Total α1 (a.u.)
hff h1299
ovcar3
hekpanc1
helaskmel5
mcf7lncap
a549llcpk1
mdamb231
hff
h1299
ovcar3hek
panc1
helaskmel5
mcf7lncap
a549llcpk1
mdamb231
h129
9hff
mda
-mb-
231
llcpk
1
a549
skm
el5
ovca
r3
lncap
mcf7 hela
hek
panc
1
anti α1
anti α2
anti α3
α1 α2 α350 ng Pur. Pr.A
B C
Viability is linearly correlated with number of active pumps in a panel of cells
Although cardiac glycosides can inhibit the proliferation of cancer cells at very
low concentrations (nM), they inhibit the proliferation of human nonmalignant
cells at similar concentrations; this strongly suggests that their potential for cancer
therapy is low.
More experimental data are needed to further decipher the structure-activity
relationship between CG’s and cancer cell cytotosicity.
Viability is linearly correlated with number of active pumps– CG affect cancer cell viability only through binding and inhibition of NaK ATPase transport
Steven Karlish
Marina Cherniavsky-Lev
Elena Ainbinder
Daniel Tal
Yotam Nadav
Michael Habeck
Acknowledgments
Thank you for your attention.