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.