1. 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 Oncology
Wayne State University

2. Introduction
Jemal et al., 2010

3. What are cardiac glycosides?
A B
C D
C3
C17

4. 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.

5. 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

6. What are potential candidates from cardiac
glycosides (CGs)?
Langenhan et al., 2005, Iyer et al., 2010, Wang et al., 2010

7. Manipulation of the glycosidic linkage and
saccharide moiety of digitoxin
n=1; Digitoxin mono-O-saccharide
n=2; Digitoxin di-O-saccharide
n=3; Digitoxin
n= α-L-rhamnose; D6-MA
(B) O’Doherty’s digitoxin O-
saccharides
n=1; Digitoxin mono-MeON-
saccharide
n=2; Digitoxin di-MeON-saccharide
n=3; Digitoxin tri-MeON-saccharide
(A) Langenhan’s digitoxin RON-
saccharides

8. 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

9. Digitoxin monosaccharide analogs are more
potent anticancer agents than their
disaccharide and trisaccharide counterparts
Iyer et al., 2010

10. Cell cycle regulation in cancer therapeutics
Schwartz, 2005

11. 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.

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

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

14. Digitoxin and D6-MA exhibits selective
cytotoxicity to NSCLC cells

15. Feldstein and Gores, 2005
Apoptotic pathways

16. Digitoxin and D6-MA induces extensive
caspase-9 cleavage

17. Digitoxin and D6-MA induces extensive
caspase-9 cleavage
Iyer et al., 2010

18. Digitoxin and D6-MA induces expression of
cytochrome c

19. Digitoxin and D6-MA induce G2/M phase
arrest

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

21. Regulation of G2/M phase transition and cyclin
B1/cdc2 complex
Wang et al., 2009

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

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

24. Digitoxin affects Na+/K+ATPase differently
depending on its concentration
Old 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 events
Manipulated gene expression
Anticancer effects

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

26. 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.

27. 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.

28. Questions ?

29. Synthesis of digitoxin-RON-neoglycosides
R-O-NH2*HCl : methoxyl HCL
Pry: pyridine
T-Bu-NH2*BH3: borane tertiary butylamine
Langenhan et al., 2005

30. 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

31. 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.

32. 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