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"Combination of HDACi and Nucleoside Analog in inducing Apoptosis in Leukemia Cell Lines ". - Devangi Shah. I Thank. Dr. Steven Grant Dr. Roberto Rosato Jorge Almenara Stefanie Coe Dr. Allison Johnson, HHMI Coordinator. APOPTOSIS : Programmed cell death - PowerPoint PPT Presentation
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Dr. Steven GrantDr. Steven Grant
Dr. Roberto RosatoDr. Roberto Rosato
Jorge AlmenaraJorge Almenara
Stefanie CoeStefanie Coe
Dr. Allison Johnson, HHMI CoordinatorDr. Allison Johnson, HHMI Coordinator
APOPTOSIS : Programmed cell death
HDAC : Histone deacetylases are a class of enzymes
HDACI : HDAC Inhibitors are involved in anticancer activity
LAQ824 : HDAC Inhibitor
FLUDARABINE :A nucleoside analog
U937 CELLS : Leukemia cell lines
• A class of enzymes that remove acetyl groups fromA class of enzymes that remove acetyl groups from
lysine amino acid on a histone.lysine amino acid on a histone.
•Altered expression and mutations of genes encoding Altered expression and mutations of genes encoding
HDACs have been linked to tumor developmentHDACs have been linked to tumor development
• Inhibits cell growth in a dose- and time–dependent mannerInhibits cell growth in a dose- and time–dependent manner
• Induces upregulation of p21, caspase cascade activation and Poly Induces upregulation of p21, caspase cascade activation and Poly (ADP) ribose polymerase (PARP) cleavage(ADP) ribose polymerase (PARP) cleavage
• Inhibits Inhibits DNA synthesis
• Active against both dividing and resting cells. Active against both dividing and resting cells.
• One of the newer chemotherapy drugs which has been around One of the newer chemotherapy drugs which has been around and in use for a few years. and in use for a few years.
Dose response of LAQ824 and Fludarabine to U937
cancer cells
Study of Proteins in U937 cells treated with LAQ824 and Study of Proteins in U937 cells treated with LAQ824 and FludarabineFludarabine
• Treatments :Treatments :
- Control- Control
- LAQ824 (40 nM)- LAQ824 (40 nM)
- FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM)- FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM)
- L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7- L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7
• All LAQ824 treatments for 48 hoursAll LAQ824 treatments for 48 hours
• All Fludarabine (FD) treatments for 24 hoursAll Fludarabine (FD) treatments for 24 hours
• Cells and Cell CultureCells and Cell Culture
• Drugs and ChemicalsDrugs and Chemicals
• Flow CytometryFlow Cytometry
Percentage of Apoptosis in U937 cells induced by LAQ824 and Fludarabine
using DIOC6 dye
0102030405060708090
100
Treatment
Pe
rce
nta
ge
of
Ap
op
tos
is
Percentage of Apoptosis in U937 Cells induced by LAQ824 and Fludarabine
using 7AAD dye
0102030405060708090
Treatment
Per
cen
tag
e o
f Ap
op
tosi
s
(p21, PARP, Caspase-3, Caspase-7 and Caspase-9)(p21, PARP, Caspase-3, Caspase-7 and Caspase-9)
• TreatmentsTreatments
- Control- Control
- LAQ824 40 nM- LAQ824 40 nM
- FD 0.4 µM- FD 0.4 µM
- L/F 0.4- L/F 0.4
• Cells and Cell CultureCells and Cell Culture
• Drugs and ChemicalsDrugs and Chemicals
• Flow CytometryFlow Cytometry
• Western Blot AnalysisWestern Blot Analysis
p21
Control LAQ824 FD 0.4 L/F 0.4
PARP
C L40 4h 8h 16h 24h 28h 32h 40h 48h
C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h
Cleavage
Fragment
C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h
C L40 4h 8h 16h 24h 28h 32h 40h 48h
C L40 4h 8h 16h 24h 28h 32h 40h 48h
C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h
Cleavage
Fragment
LAQ824 and Fludarabine interact synergistically LAQ824 and Fludarabine interact synergistically
in U937 cancer cells inducing cell death by in U937 cancer cells inducing cell death by
apoptotic events.apoptotic events.
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