kfu invited seminar
TRANSCRIPT
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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
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Introduction
Jemal et al., 2010
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What are cardiac glycosides?
A B
C D
C3
C17
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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.
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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
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What are potential candidates from cardiac glycosides (CGs)?
Langenhan et al., 2005, Iyer et al., 2010, Wang et al., 2010
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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
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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
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Digitoxin monosaccharide analogs are more potent anticancer agents than their
disaccharide and trisaccharide counterparts
Iyer et al., 2010
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Cell cycle regulation in cancer therapeutics
Schwartz, 2005
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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.
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Digitoxin and D6-MA inhibit NCI-H460 cell viability and Na+/K+ATPase enzyme activity
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Digitoxin and D6-MA induces apoptosis in NCI-H460 cells
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Digitoxin and D6-MA exhibits selective cytotoxicity to NSCLC cells
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Feldstein and Gores, 2005
Apoptotic pathways
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Digitoxin and D6-MA induces extensive caspase-9 cleavage
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Digitoxin and D6-MA induces extensive caspase-9 cleavage
Iyer et al., 2010
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Digitoxin and D6-MA induces expression of cytochrome c
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Digitoxin and D6-MA induce G2/M phase arrest
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Digitoxin and D6-MA induced down-regulation of cyclin B, cdc2, and survivin
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Regulation of G2/M phase transition and cyclin B1/cdc2 complex
Wang et al., 2009
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Digitoxin and D6-MA-mediated G2/M phase arrest does not correlate with up-regulation of p53-related
signaling
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Digitoxin and D6-MA-mediated G2/M phase arrest does not correlate with up-regulation of Chk1/2
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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
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Digitoxin reduces cancer specific cell cycle regulatory proteins by modulating the Na+/K+ ATPase
signalosome
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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.
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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.
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Questions ?
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Synthesis of digitoxin-RON-neoglycosides
R-O-NH2*HCl : methoxyl HCLPry: pyridineT-Bu-NH2*BH3: borane tertiary butylamine
Langenhan et al., 2005
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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
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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.
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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