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Molecular characterization of apoptosis induced by PDT: implication of tumor
suppressor genes as prospective markers
A. Fekih, A. Major, F. Ludicke, A. Campana, andI. Irminger-Finger
Department of Obstetrics and Gynaecology, andBiology of Aging Laboratory, Department of Geriatrics, University of Geneva, Switzerland
What Is PDT?
Photodynamic therapy (PDT) is a revolutionary medical technology that uses lasers to activate light-sensitive pharmaceuticals to treat cancer and other diseases in a non-surgical, minimally invasive way.
How does it work ?
• Porphyrin absorption maximum at a wavelength corresponding to incident laser light.
• The excited singlet porphyrin can decay back to the ground state with release of energy in the form of fluorescence.
• Singlet can be converted into the triplet excited state (3P*) which is able to transfer energy to another triplet.
p0
1px
E
Fluo
resc
ence
INTERSYSTEN CROSSING
3px
Phos
phor
esce
nce
1O2
3O2
OXYGEN
Jablonski diagram
What are Photosensitisers
Photosensitisers are substances that absorb light energy and transform this energy in chemical reactions. Many different photosensitisers are currently under development for potential drug use, 8 of which in clinical trials.
Laser wavelengths for activation of photosensitive drugs used in PDT?
Each PDT drug, because of its unique chemical composition, requires a different activating wavelength of light. All current PDT drugs generally fall in the 630nm to 750nm range, the visible red to infrared range. 5-ALA, for example, is activated at 410nm to 630nm.
What is apoptosis
• programmed cell death characterized by– membrane changes– protein degradation– nuclear shrinkage– DNA fragmentation
What is necrosis
A pathological process caused by the progressive degradative action of enzymes generally associated with severe cellular trauma. It is characterized by mitochondrial swelling, nuclear flocculation, uncontrolled cell lysis, and ultimately CELL DEATH.
Morphological difference between apoptosis and necrosis
• Physiological process• The integrity of plasma membrane is
preserved for a long time• Condensation of the chromatin• Apoptotic bodies• Absence of inflammatory reaction• Phagocytosis by adjacent cells• One cell is affected• Intra-cellular organelles are intact
initially, then condensed• Loss of contact with the surrounding
cells and the extra-cellular matrix.
• Pathological process• Swelling then lysis of the cell• Rapid loss of the integrity of the plasma
membrane• Light compaction of the chromatin• No apoptotic bodies• Important inflammatory reaction
affecting neighboring cells• Destruction of mitochondria and
lysosomes• Exocytosis.
APOPTOSIS NECROSIS
Biochemical difference between apoptosis and necrosis
• Induced by physiological stimuli• Active and regulated process• Needs energy• Transcriptional activity associated• Translational activity associated• DNA fragmentation.
• Induced by non-physiological stimuli• Passive and non regulated process• Does not need energy • No transcriptional activity• No translational activity• Random DNA fragmentation.
APOPTOSIS NECROSIS
Genes reported to be implicated in PDT
• Bax, bik, bak• Bcl-X, Bad, P53• C-jun, hrk
• The apoptotic machinery can be broadly divided into two classes: sensors (FAS, p53, myc…) and effectors (bcl-2, bax, caspases…) 2
• Caspases are the ultimate effectors of apoptosis.
Pro-apoptotic genes1
Anti-apoptotic genes1
(1Novartis apoptosis pathway)
(2D. Hanahan, 2000)
•Bcl-2, Bcl-X, Rb•Mcl-1, A1, Brag-1, Bfl-1
Bax
p53pro-apoptotic genes anti-apoptotic genes
bax, bik,bcl-X, badp53, c-jun
hrk
bcl-2, bcl-X,Rb, Mcl-1
A1, brag-1,bfl
altered gene expression
bcl-2
bcl-2
bcl-X p53
Apoptotic pathways are complex
Activates
inhibited DNArepair
NUCLEUS
Possible pathway for ROS induced apoptosis5 AlA Light+
Emission of photons
NECROSIS APOPTOSIS
Loss of tumor suppressor genes
ROSROSReactive Oxigen Species Tumor suppressor
genes functioning
Tumor suppression pathways
Tumor suppressor genes are found to be implicated in
• DNA repair • Cell cycle arrest• Senescence• Apoptosis
Loss of tumor suppressor gene functions in tumor cells
• p53 loss in more than 50% of tumors• RB loss in many tumors• BRCA1 loss in breast and ovarian cancers
Breast cancer susceptibility gene products BRCA1 and BARD1
BARD1 interaction
Nuclear localizationsignals
Rad51 interaction
Granin motif
BRCTdomain
BRCA1interaction
Ankyrin repeats
BRCT domain
PCNA
p53 RNA-Pol II?
RHA
GOAL
• Defining genes that are implicated in PDT induced apoptosis
• Defining parameters controlling the extends of apoptosis and necrosis
• Fine-tuning of PDT
46 k D
66 k D
5 ALA - + - + + -UV - - + + + -Doxorubicin - - - - - +
p53 expression after PDT
53 kD
53 kD46 k D
66 k D
p53 -/- cell
TAC 2 cells
53 kD66 k D
46 k D
HeLa cell
Relative amount of p53 expression
Exp 1 Exp 3 Exp 5
p53TAC 2
HeLa
0
50
100
Exp 1 Exp 3 Exp 5
p53TAC 2
HeLa
Exp 1: 0 Exp 2: 5 ALA Exp 3: 1’ UV
Exp 3: 5 ALA+1’UV Exp 4: 5 ALA+2’UV Exp 6: Doxorubicin
BARD1 expression after PDT
TAC 2 cells
p53 -/- cell
HeLa cell
5 ALA - + - + + -UV - - + + + -Doxorubicin - - - - - +
97 kD
97 kD
97 kD
Relative amount of BARD1 expression
Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6
p53TAC 2
HeLa
0
50
100
150
Exp 1 Exp 2 Exp 3 Exp 4 Exp 5 Exp 6
p53TAC 2
HeLa
Exp 1: 0 Exp 2: 5 ALA Exp 3: 1’ UV
Exp 3: 5 ALA+1’UV Exp 4: 5 ALA+2’UV Exp 6: Doxorubicin
Quantification of apoptosis by AnnexinV staining in HeLa cells
HeLa + Doxorubicin HeLa + 1' UV
HeLa + 1' UV + 5 ALA HeLa + 2' UV + 5 ALA
annexin v
DN
A st
ain
Quantification of apoptosis by AnnexinV staining in embryonic stem cells
RD + 1'UVRD + Doxo
RD + 1' UV + 5 ALA RD + 2' UV + 5 ALA
annexin v
DN
A st
ain
Quantification of apoptosis by AnnexinV staining in TAC 2 cells
TAC 2 + Doxorubicin TAC 2 + 1' UV
TAC 2 + 1' UV + 5 ALA TAC 2 + 2' UV + 5 ALA
annexin v
DN
A st
ain
repair
eliminationapoptosis
ROS
Possible apoptotic pathway in PDT
ROSROS
necrosisdeath by damage
p53p53
BARD1BARD1BARD1
accumulation of damage
Conclusion• PDT induced apoptosis and necrosis are cell type
specific • Extend of apoptosis and necrosis depends on
– dose of ALA– time of light application
• Apoptotic potential depends on functional tumor suppressor genes
• Clinical application: PDT fine tuning should be the goal
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