Radiotherapy & Resistance
description
Transcript of Radiotherapy & Resistance
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2E Conférence Québécoise Sur La Résistance Thérapeutique du CancerQ-CROC
Montréal, November 5-6, 2010
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Potential Conflict of Interest
• Dr. Thierry Muanza– None
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Radiotherapy
Along with surgery and chemotherapy, radiotherapy is a mainstay of cancer treatment
75% cancer patients in the course of their disease will undergo radiotherapy
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Radiobiology IR local release of large amount of energy
~ 33eV dissipated / ionizing event, enough to break strong chemical bond
energy associated C=C bond is 4.9 eV
Types: Electromagnetic particulate
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IR: mechanism of action.
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Radiation Induced DNA Damage
http://www.radiation-scott.org/radsource
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Chromosomal Damage
Apoptosis Reproductive death
Necrosis
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Radiation Induced DNA Damage
ATM
G1 arrest
RepairG
2 ar
rest G1 arrest
Apoptosis
G2 arrest
pH2XA
P53
BAX
Cytochrome c
Apaf1 Caspase 9
Caspase 6Caspase 3
Apoptosis
P53
Cycline B
N Cycline-B-p34 cdc2
p21 WAF1 / CIPIcdK /Cycline D
Cycline E
pRb
E2F
pRb
E2F
Smac/ Diablo
Survivin
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DNA Repair Mechanisms
ATM DSB
Rad51 /Rad52
Rad52/MRE11/NBSI
BRAC1/BRAC2
Completion of DSB repair
DNA-PKcs / Ku80/70
MRE11/Rad51/NBSI
DNA ligase IV, XRCC4
RNA Pol II or XPC
XPD / XPB
ERCC1 / XPG
Completion of repair
DNA Pol / ligase
DNA Pol / DNA ligase III
XRCC1
BER NHEJ
HR
pH2aXNER
SSB
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Cell Survival Curves
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CA: RT sensitivity
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Cell cycle: RT sensitivity
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Tumor Oxygenation
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Re-oxygenation
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Effect of Oxygen
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RT resistance
Ma et al. JCO 21, 2003
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Radiosensitizers
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Radiosensitizers Nonhypoxic
Halogenated pyrimidines DNA Cycling tissues
Hypoxic Free radical process (fixed) Misonidazole (toxicity) Overgaard metaanalyisis: 4.6% LC, 2.8% OS
Hypoxic cytotoxins Bioreductive Rx
Tirapazamine (nitroxide) Mitomycin C
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Radiosensitizers
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Radiosensitizers
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Cell Proliferation Assay (MTT)
MDA-MB-468 cells
0
10
20
30
40
50
60
70
80
90
100
110
0 10 20 30 40 50 60 70 80 90 100 110
Drug Concentration (uM)
Per
cent
Con
trol
Cel
l Via
bilit
y
ZRBA1 with Radiation
Iressa with Radiation
ZRBA1
Iressa
Heravi, Muanza et al. ACD 20, 2009
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Cell Proliferation Assay (MTT)
0
20
40
60
80
100
120
140
No XRT RX then XRT RX+XRT XRT then RX
Per
cent
cel
l via
bilit
y
Control
ZRBA1
**
**
p<0.0049
**: P<0.01
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Colony Forming Assay
0 2 4 6 8 100.0001
0.001
0.01
0.1
1ZRBA1
Iressa
Control
Radiation Dose (Gy)
Sur
viva
l Fra
ctio
n
Radiation Dose (Gy) DER
2 4 6
1.62.22.9
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γH2AX Immunofluorescent Staining
Control
1 Gy
0.5 Gy
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γH2AX Immunofluorescent Staining
Control
ZRBA1 18uM
ZRBA1 9uM
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γH2AX Immunofluorescent Staining
Control 0.5 Gy
ZRBA1 9uM 0.5 Gy and ZRBA1 9uM
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γH2AX Immunofluorescent Staining
Control 1 Gy
ZRBA1 9uM 1 Gy and ZRBA1 9uM
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γH2AX Immunofluorescent Staining
0 Gy
0.5
Gy1
Gy0
5
10
15
20
25
Control
9 uMZRBA1
18 uM ZRBA1
*** *
* : P<0.01**: P<0.004
p<0.013
Fo
ci p
er
cell
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FACS Analysis
1 hrs post treatment
24 hrs post treatment
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Comet Assay
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Comet Assay
Control ZRBA1
Radiation
ZRBA1 and radiation
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EGFR Inhibitory Activity (ZRBA1)
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EGFR Signaling Pathway
TK
TGF
RAS
RAF
ERK1/2
BAD
ApoptosisCaspase 9
Caspase 3
Survival
AKT
EGFR
PSerine136
Serine112
PPI3K
P
Over-expression of EGFR associates
with activation of the AKT pathway.
P
HER2
MEK
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Conclusions
Significant cell killing in cells exposed to combination of ionizing
radiation with ZRBA1.
Higher G2M arrest in cells exposed to the combined treatment.
The most effective schedule for this combination is administration of
drug before and / or concurrent with radiation.
ZRBA1 potentiates the effect of radiation in MDA-MB-468 cells.
The combination of IR and ZRBA1 induces the formation of γH2AX foci.
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Future Plans
Better understanding of the molecular mechanism induced by this novel therapeutic approach.
Evaluate the status of DNA repair proteins.
Evaluate the expression levels of antiapoptotic and proapoptotic proteins.
Investigation for other possible modes of cell death. In-vivo correlative studies.
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Cancer Stem Cells Self-renewing cell
Pluripotent cell: recapitulate its tumor in SCID mice
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CSC: mechanisms of resistance
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CSC: RT resistance CSC enrichment Chk1/Chk2 inhibitor
S. Bao Nature 444, 2006
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CSC: RT resistance apoptosis Cell cycle arrest
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CSC: DNA damage repair kinetics
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Future directions Clinical models for identification of RT resistance
Rectal cancer CNS tumors Zevalin resistant lymphoma
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Acknowledgments
Dr. Thierry Muanza
- Mitra Heravi
- Lillian Lee
- Azusa Maeda
- Ava Schlisser
Dr. Danuta Radzioch
Dr. Bertrand Jean-Claude (Cancer Drug Research Laboratory)
- Dr. Zakaria Rachid
- Margarita Todorova
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Thank You!