Download - KFU Invited seminar
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