Effects of Oxygen on Radiation Therapy

February 19, 2009

Zhong Yun, PhD

Department of Therapeutic Radiology, HRT-313Yale University School of MedicineTel: 737-2183; zhong.yun@yale.edu

Outline

1. A mechanism of hypoxia signal transduction

2. Oxygen effects on Radiotherapy---OER and factors affecting OER---Reoxygenation---Overcoming tumor hypoxia

---Oxygenation enhancement---Hypoxia sensitizers---Hypoxia toxins---Hypoxia-activated gene therapy

The Hypoxia-Inducible Factor Family

Denko, N.C., Nature Reviews Cancer 8, 705-713 (September 2008)

Mechanisms of hypoxia-inducible factor 1 (HIF1) stabilization

pOpO22

PHD1,2,3

Anaerobic Metabolism

degradation

Angiogenesis

VE

GF

Glut-1

ROS

mt

Metastasis LOX

; E-cadherin

Muscle tissue survival

Cul2

Elong.

HIF1,2

Notch

Embryogastrulation

Oct

-4

Lung Morphogenesis

EG

FR

Ten

asci

n-C

Erythropoesis

EPO

FIH

VHL

DNA Repair

Stem Cell Differentiation

Tu

mor

igen

esis

Tu

mor

igen

esis

Dif

fere

nti

atio

n-D

evel

opm

ent

Dif

fere

nti

atio

n-D

evel

opm

ent

msh2/msh6

Mitoch. O2 consumption PDK1 Proteasome

OH

OH

HIF

O2

Maxwell & Coumenis, 2006

Semenza GL, Drug Discovery Today, 12:853-9, 2007

Semenza GL, Drug Discovery Today, 12:853-9, 2007

Biology of Tumor Hypoxia

HypoxicRegion

Blood Vessel

O2 / DrugConcentration

Gene/Protein Regulation

Increased Glycolysis

Increased Angiogenesis

Increased Genomic Instability

Selection of Apoptosis Resistance

Chemo/Radio-therapy Resistance

Evans and Koch, 01

Hypoxia (EF5, Red) and Proliferation (Ki67, Green)

Fei P. et al. Cancer Cell, 6:597, 2004

Proapoptotic BNIP3L is Induced by p53 under Hypoxia

C.V. Dang, et al. Nature Reviews Cancer 8, 51-56, 2008

The HIF1, HIF2 and MYC protein–protein interaction transcriptional network.

Kinzler KW and Vogelstein B Nature, 379, 1996Graeber TG et al. Nature 379, 1996

Hypoxia Selects for Apoptosis Resistant Clones

Graeber TG, et al. Nature, 379:88, 1996

Hypoxia Selects for Apoptosis Resistant Clones

• Point mutations• Oxidative base damage• DSBs/SSBs• Gene amplification• DNA over-replication

Hypoxia Increases Genome Instability

Genetic instability induced by the tumor microenvironment. Reynolds, T.Y., S. Rockwell, and P.M. Glazer. Cancer Res, 1996. 56(24): p. 5754-7.

Diminished DNA repair and elevated mutagenesis in mammalian cells exposed to hypoxia and low pH. Yuan, J., Narayanan, L., Rockwell, S. and P.M Glazer. Cancer Res, 2000. 60(16): p. 4372-6.

Decreased expression of the DNA mismatch repair gene Mlh1 under hypoxic stress in mammalian cells. Mihaylova, V.T., Bindra, R.S., Yuan, J., Campisi, D, Narayanan, L., Jensen, R., Giordano, F., Johnson, R.S., Rockwell S, and P.M. Glazer. Mol Cell Biol, 2003. 23(9): p. 3265-73.

Mihaylova VT, et al. Mol. Cell. Biol. 24:8504, 2004

Hypoxia Suppresses Mismatch Repair (MMR)

Bindra RS, et al. Mol. Cell. Biol. 24:8504, 2004

Hypoxia/Reoxygenation Represses Homologous Recombination (HR)

Hypoxia/Reoxygenation Represses Homologous Recombination (HR)

Bindra RS, et al. Mol. Cell. Biol. 24:8504, 2004

Oxygen Influences Biological Effects of Radiation

1. Less damage to tightly pressed skin, Swartz 1912,

2. Radiation inhibited germination of vegetable seeds only in the presence of O2, Petry 1923

3. Oxygen effect on tumor radiosensitivity championed by Mottram 1930s

4. 1st quantitative study on O2 effects on radiation-inducedgrowth inhibition of broad bean, Gray 1953

5. O2 levels decrease in respiring tumor cells located away from blood vessels, Tomlinson and Gray 1955

Brown JM & Wilson WR, 2004

Ability of ionizing radiation to kill cells is highly dependent on pO2

---Tomlinson and Gray , 1950s

Oxygen Enhancement Ratio (OER)

The ratio of HYPOXIC to AEROBIC IR doses needed to achieve the SAME biological effects.

Dose (Gy)

0 10 20 30

Su

rviv

ing

Fra

ctio

n

0.01

0.1

1

AerobicHypoxic

1/e1/e

OER =D0 (hypoxic)

D0 (aerobic)= 3.22

D0D0

OER =D0 (hypoxic)

D0 (aerobic)

Radiosensitivity and pO2

1.0

2.0

3.0

0 20 40 60 80155 760 2280

Air 100%O2

HBO(3 ATA)

VenousBlood

Rel

ati

ve

Rad

iose

nsi

tivi

ty

Oxygen tension (mmHg)

(Rockwell et al.)

Half maximum sensitivity = 3 mmHg or 0.5% O2

Kirkpatrick JP, et al. Intl. J. Rad. Oncol. Biol. Phys. 59:822, 2004

CHO Cells

Re

lati

ve

Ra

dio

se

ns

itiv

ity

The Oxygen Fixation Hypothesis

X-rays + H2O H + H3O+ + OH

HO2

H2O2

O2-

H2O+ + e-H2O OH

R

RHRO2

O2

H+

O2

O2

t1/2: 10-10 sec for ion pairs; 10-5 sec for free radicals

Non-repairable

Parameters Affecting OER

1. Nature of cellular sensitivity

2. Nature of radiation (x-ray, neutron, alpha particle, or LET)

3. pO2

5. Cell Cycle ( cells in S-phase are more resistant.)

4. Time of oxygen presence

1. CellularSensitivity

Resistant Cells

High OER

Sensitive Cells

Low OER

2. Nature of Radiation X-rays

neutrons -particles

LET = 2.0

LET and OER

60

4. Oxygen effect is time-dependent

1. O2 needs not to be present DURING radiation

2. O2 has to be present WITHIN milliseconds after radiation

Radiation Ion pairs Free Radicals

T = 10-10 sec T = 10-5 sec (0.01 msec)

Time delay is determined by lifetime of free radicals

Sinclair W and Morton R, Radiat. Res. 29:450, 1966Sinclair W, Radiat. Res. 33:620, 1968

5. Cell Cycle and Radiation Sensitivity

Most Sensitive Phase:G2/M

Most Resistant Phase:Late SEarly G1

OER varies slightly during cell cycle

S phase(OER = 2.8-2.9)

G2/M phase(OER = 2.3-2.4)

G1 phase >>

OER remains unchanged for ALL dose levels in a given phase of cell cycle.

ReoxygenationNormoxic

Hypoxic

Necrotic

X-ray

Recovery

X-ray

X-ray

X-ray

Hypoxic Fraction

No change

Decreased

Increased

Factors Affecting Reoxygenation

2. Clearance of dead cells by macrophages, etc.

1. Rate of angiogenesis.

3. Inflammatory response

4. Tissue remodeling.

Mouse mammary Carcinoma

Rat sarcoma

Mouse fibrosarcoma

Mouse osteosarcoma

Mouse fibrosarcoma

Variable Rates of Reoxygenation

Strategies to Overcome Tumor Hypoxia

1. Radiation Dose Fractionation- Limiting damage to normal tissue- Reoxygenation of hypoxic tumor cells

2. Improve Tumor Oxygenation- Hyperbaric oxygen breathing- Compounds to improve oxygenation (Motexafin Gadolinium or MGd)- Allosteric modifiers of hemoglobin-oxygen binding such as RSR13

(to reduce O2 affinity for efficient O2 discharge)

3. Hypoxic Cell Sensitizers (Nitroimidazoles with high electron affinity)- Misonidazole: more active, but toxic (peripheral neuropathy)- Etanidazole: less toxic, but no benefit- Nimorazole: less active, much less toxic (benefit in H&N cancer--Danish study)

4. Hypoxia Cytotoxins (Bioreductive agents that selectively kill hypoxic cells)- Tirapazamine: high Hypoxic toxicity ratio- Mitomycin C: low Hypoxic toxicity ratio

MGd Improves Tumor Oxygenation

0 10 20 30 40 50

Rel

ativ

e F

req

uen

cy (

%)

0

10

20

30

40

50

60

70

0 10 20 30 40 500

10

20

30

40

50

60

70

Oxygen Partial Pressure (mmHg)

0 10 20 30 40 50

Rel

ativ

e F

req

uen

cy (

%)

0

10

20

30

40

50

60

70

Oxygen Partial Pressure (mmHg)

0 10 20 30 40 500

10

20

30

40

50

60

70

(A) Control

(C) 40 mol/kg MGd - 6 Hours

(B) Mannitol Control - 6 Hours

(D) 40 mol/kg MGd - 8 Hours *

*

* p < 0.01

**

**

E. Donnelly & S. Rockwell, 2005

MGd: Clinical Tumor Oxygenation Observations

• Effects of MGd on tumor oxygenation were measured during Phase I head and neck cancer clinical trials– Median Tumor pO2

• Before treatment: 6.8 mmHg• 1 mg/kg MGd, one week: 27.0 mmHg

– Improved Tumor Response• 8 of 9 patients experienced complete tumor response• 7 patients still in remission after 9 months

D.M. Brizel, Duke University

Brown JM & Wilson WR, 2004

Activation of Hypoxia-Selective Prodrugs

JM Brown, Stanford Univ.

Topo II

TPZ Preferentially Kills Hypoxic Cells

Brown JM & Wilson WR, 2004

A Strategy for Hypoxia-Activated Gene Therapy

Semenza GL, Drug Discovery Today, 12:853-9, 2007