Digitoxin, a cardiac glycoside with the potential to provide a new hope for

cancer therapy

Hosam A. Elbaz, Ph.D.Center for Molecular Medicine and Genetics

Department of Radiation OncologyWayne State University

Introduction

Jemal et al., 2010

What are cardiac glycosides?

A B

C D

C3

C17

Evidence for anticancer effect for cardiac glycosides (CGs)

• Historical records indicated CGs potential anticancer activity.

• Stenkvist et al. (1979-2001), and Goldin et al., (1984) showed that women on digitalis therapy

– developed more benign forms of breast tumors, and

– 9.6-times lower cancer recurrence rate when compared to control patients.

What are potential candidates from cardiac glycosides (CGs)?

• Digitoxin is a promising candidate for future CGs anticancer research because:

– Anticancer effect at therapeutic doses (15 – 40 nM)

– Long half life (≈7 days)• 97% bound to plasma proteins• Large Vd

– Complete clinical profile

What are potential candidates from cardiac glycosides (CGs)?

Langenhan et al., 2005, Iyer et al., 2010, Wang et al., 2010

Manipulation of the glycosidic linkage and saccharide moiety of digitoxin

n=1; Digitoxin mono-O-sacchariden=2; Digitoxin di-O-sacchariden=3; Digitoxinn= α-L-rhamnose; D6-MA

(B) O’Doherty’s digitoxin O-saccharides

n=1; Digitoxin mono-MeON-sacchariden=2; Digitoxin di-MeON-sacchariden=3; Digitoxin tri-MeON-saccharide

(A) Langenhan’s digitoxin RON-saccharides

Digitoxin O-saccharides are more potent than digitoxin MeON-saccharides against NSCLC cells

(B) O’Doherty’s digitoxin O-saccharides

(A) Langenhan’s digitoxin RON-saccharides

Iyer et al., 2010

Digitoxin monosaccharide analogs are more potent anticancer agents than their

disaccharide and trisaccharide counterparts

Iyer et al., 2010

Cell cycle regulation in cancer therapeutics

Schwartz, 2005

Objective and Hypothesis

• Objective: – compare digitoxin with D6-MA with respect to their

cytotoxic mechanisms

• Hypothesis: – therapeutically relevant doses of digitoxin and D6-MA

would decrease cell viability due to G2/M arrest and apoptosis in NCI-H460 cells.

– D6-MA is mechanistically more potent than digitoxin.

Digitoxin and D6-MA inhibit NCI-H460 cell viability and Na+/K+ATPase enzyme activity

Digitoxin and D6-MA induces apoptosis in NCI-H460 cells

Digitoxin and D6-MA exhibits selective cytotoxicity to NSCLC cells

Feldstein and Gores, 2005

Apoptotic pathways

Digitoxin and D6-MA induces extensive caspase-9 cleavage

Digitoxin and D6-MA induces extensive caspase-9 cleavage

Iyer et al., 2010

Digitoxin and D6-MA induces expression of cytochrome c

Digitoxin and D6-MA induce G2/M phase arrest

Digitoxin and D6-MA induced down-regulation of cyclin B, cdc2, and survivin

Regulation of G2/M phase transition and cyclin B1/cdc2 complex

Wang et al., 2009

Digitoxin and D6-MA-mediated G2/M phase arrest does not correlate with up-regulation of p53-related

signaling

Digitoxin and D6-MA-mediated G2/M phase arrest does not correlate with up-regulation of Chk1/2

Digitoxin affects Na+/K+ATPase differently depending on its concentrationOld theory

Digitoxin (0.5-5 μM)New theory

Digitoxin (0.01-0.1 μM)

Na+/K+ ATPase inhibition

Intracellular Na+

Na+/Ca2+ exchanger activation

Intracellular Ca2+

Cardiac contractility Apoptosis

Na+/K+ ATPase signalosome(MAPK, SRC, Akt, and PLC signaling)

Intracellular eventsManipulated gene expression

Anticancer effects

Digitoxin reduces cancer specific cell cycle regulatory proteins by modulating the Na+/K+ ATPase

signalosome

Discussion and Conclusions

• Na+/K+ ATPase inhibition by either digitoxin or D6-MA does not account for drug or analog cytotoxic effects.

• Na+/K+ ATPase signalosome activation is a viable possibility.

• Digitoxin and D6-MA are selective to NSCLC cells.

• Digitoxin and D6-MA induced caspase-9 cleavage, but not caspase-8.

• Induced cytochrome c expression contrasts previous claims of general inhibition of protein synthesis.

Discussion and Conclusions

• Digitoxin and D6-MA induce G2/M phase arrest and cyclinB1 and cdc2 down-regulation.

• Inhibited expression of cyclin B1, cdc2, survivin, and Chk1/2 explains the potent and selective cytotoxic effect of digitoxin and D6-MA.

• G2/M phase arrest and down regulation of cyclinB1 and cdc2 are not directly controlled by up-regulation of p53 signaling or checkpoint kinase signaling.

Questions ?

Synthesis of digitoxin-RON-neoglycosides

R-O-NH2*HCl : methoxyl HCLPry: pyridineT-Bu-NH2*BH3: borane tertiary butylamine

Langenhan et al., 2005

Why D6-MA exhibits greater cytotoxic potency than digitoxin?

• Prof. Karlish tested digitoxin and D6-MA on human Na+/K+ATPase isoforms.

– D6-MA is non- selective in binding to α1β1 and α2 / α3β1.

– Digitoxin and other cardiac glycosides tend to selectively bind to α2 / α3 subunit over α1

Lipinski’s rule of five

• Adequate absorption or permeation are more likely when:

– There are less than 5 H-bond donors.– The molecular weight is under 500.– The LogP is under 5.– There are less than 10 H-bond acceptors.

D6-MA shows a better profile with respect to Lipinski’s rule of five

Digitoxin D6-MA

Molecular weight

764.98 520.65

Partition coefficient

4 (2.8 – 4.54) 3.346 (2 – 4.32)

H-bond donors 5 4

H-bond acceptors

13 8