Transcript amit-roy-national-institute-of-pharmaceutical-education

Decreased Camptothecin Sensitivity of Cancer Stem-Like cell
Population Correlates with Phosphorylation state of
DNA Topoisomerase I
6th World Congress on Biotechnology
New Delhi
05.10.2015
Dr. Amit Roy
Assistant Professor
Department of Biotechnology,
National Institute of Pharmaceutical
Education and Research (NIPER),
Hajipur, India
DNA Topoisomerase I
 DNA Topoisomerase I is an essential and ubiquitous enzyme that alters the
topological changes of DNA by breaking and rejoining of DNA stands.
 This ubiquitous enzyme play a pivotal role in vital cellular processes, like
DNA replication, transcription, recombination, and chromosomal
segregation during mitotic cell division.
 Topoisomerase I induces a transient single-stranded break of DNA duplex
and results in a reversible topoisomerase I-DNA covalent complex.
 Type IA (Prokaryotic): Enzyme links with 5' phosphate of the DNA strand
 Type IB (Eukaryotic): Enzyme links with 3' phosphate of the DNA strand
Human Topoisomerase IB
 Most eukaryotic type IB topoisomerases are monomeric enzymes,
including human Topo I, which comprises 765 amino acids (91 kDa).
Catalytic Mechanism of Topoisomerase IB
Cleavage
Stand
Rotation Religation
Agarose Gel
Elctrophoresis
Relaxed DNA
Supercoiled DNA
Electron Micrograph
Camptothecin (CPT)
 Camptothecin (CPT) is a plant alkaloid.
 CPT is most established inhibitor of DNA topoisomerase I.
Camptothecin is pharmacologically unique :
 Topoisomerase I is the only target of this drug.
 CPT binds neither to Top1 alone nor to DNA alone, but
only to the TopI-DNA cleavable complex.
Rolling-circle Enhanced Enzyme Activity Detection
(REEAD) System
Rolling Circle Amplification
Biosensor Microarray
Circularization
Hybridization of
detection probe
Hybridization and
Rolling circle
Rolling-circle Enhanced Enzyme Activity Detection
(REEAD) System
Control circle – Green
hTopI - Red
Models: HT-29 and Caco2
 Colorectal cancer is one of the most common causes of cancer
death worldwide.
 Human epithelial colon carcinoma cell lines have been widely
studied for their ability to differentiate into stem-like cells.
 Topo I-DNA cleavable complex formation induced by CPT is 4
to 7-fold reduced in fully differentiated cells compared with
proliferative cells in human intestinal cells.
 Cleavable complex formation and cytotoxicity induced by CPT
correlate with Topo I level and activity in cells at different stages
in their differentiation.
 Thus, high target levels correspond closely with drug sensitivity,
since proliferating cells contain larger amounts of Topo I.
REEAD assay is highly sensitive to detect drug responsiveness
in cancer cells
15 30 60 120 uM CPT
15 30 60 120
1
2
3
4
5
6
1
7
2
3
4
5
6
7
HT29
Caco2
HT29 Caco2
hTopI
Loading
Control
NE of HT29
NE of Caco2
0.8
hTopoI activty
HT29
Caco2
1
0.8
0.6
0.4
0.2
Relative TopoI Activity
Relative TopoI Activity
1.4
1.2
RT-PCR
CPT Sensitivity
0.7
HT29
Caco2
0.6
0.5
0.4
0.3
0.2
0.1
0
1X
10X
100X
1000X
Dilution of NE
10000X
0
DMSO
15 uM
30 uM
[CPT]
60 uM
120 uM
Cancer stem cells
 Cancer stem cells (CSCs) are a small sub-population of cancer
cells that have stem cell-like properties such as self-renewal and
the ability to differentiate into multiple cell types.
 CSCs are referred to as tumor-initiating cells (TICs) having the
ability to maintain the malignant population.
 Recent research suggested that CSCs are particularly resistant to
conventional chemoradiotherapy compared with the non-CSCs.
 CD44 and CD133 are cell-surface transmembrane glycoprotein,
which has been used in the identification of putative CSCs from
several solid tumors.
 The CD44 and CD133 markers may be the best to identify tumor
initiating cells of human colon cancer.
Differentiation of Cells
Survival (% DMSO)
NaBt-treated Caco2 cells are hypersensitive to CPT
90
80
70
60
50
40
30
20
10
0
Caco2
Caco2+ NaBt
0.1 uM 0.2 uM 0.5 uM
[CPT]
1 uM
5 uM
10 uM
hTopI Activity and CPT Sensitivity of NE
from NaBt-Treated Caco2 cells
Relaxation assay of endogenous Ni-NTA column
purified hTopI from NaBt+/- Caco2 cells
Control hTopI
1
2
3 4
5
6
7 8 9 10 11 12
NaBt-Treated hTopI
1
2
3 4
5
6 7
8
9 10 11 12
Lane1: DNA control, Lanes 2-11: Eluted fractions (1 to 10) respectively, Lane 12: Rest of the eluted collection.
CPT Sensitivity of Ni-NTA Column purified
endogenous hTopI from NaBt+/- Caco2 cells
Dephosphorylation of Ni-NTA Column purified
endogenous hTopI and CPT Sensitivity
Isolation of CD44+ and CD44- cells by Cells Sorting
hTopI Activity and CPT Sensitivity of
Caco2 cells (CD44+/-) from Cells Extract
1.40
Control
1.40
CD44+
1.20
CD44-
1.00
0.80
0.60
0.40
0.20
0.00
Relative hTopI Activity
Relative hTopI Activity
1.60
Control
1.20
CD44+
1.00
CD44-
0.80
0.60
0.40
0.20
0.00
1X
10X
100X
Dilution of Cell Extract
1000X
DMSO
15
30
[CPT, uM]
60
Phosphorylation of hTopI and CPT Sensitivity
Phosphorylation and Dephosphorylation
of cells extract and CPT Sensitivity
CONCLUSIONS
CD44 may be a good stem cell marker for colon cancer cell lines
like Caco2.
Phosphorylation of TopI increases the enzyme activity and drug
sensitivity.
Sodium butyrate (NaBt) treated cells are more drug sensitive
because of overexpression and hyper-phosphorylation of TopI.
CD44+ cells are supposed to be cancer stem cells and are chemoresistant. Because CD44+ cells having low TopI activity due to
hypo-phosphorylation of enzyme.
These studies are expected to provide important insights into the
cellular mechanisms behind TopI-targeted chemotherapy and
actual reason of chemo-resistant of cancer stem-like cells.
It will provide the better means for optimizing current clinical
use for the individual cancer patient.
PLoS ONE, 2014; 9(6): e99628
ACKNOWLEDGEMENTS
Dr. Hemanta K. Majumder,
IICB, Kolkata
Dr. Birgitta R. Knudsen,
Aarhus University, Denmark
Félicie, Rikke, Magnus, Jørn,
Sissel, David, Christine, Pia
Gerda, Noriko
Funding Agency:
Council of Scientific and Industrial Research (CSIR), Govt. of India
Danish Cancer Society, Denmark
Carlsberg Foundation, Denmark