BBB seminar, February 2, 2006
MITOCHONDRIA AND
CELL PROLIFERATION
Mitochondrium
Karl Johan Tronstad
[email protected]
Mitochondria - history
1890
Altman: Intracellullar granules, similar to bacteria, ”bioblasts”
1932
Bensley og Hoerr: Isolated mitochondria from guinea pigs
1949
Lehninger: Metabolic pathways localized to the mitochondria
(fatty acid oxidation and TCA-cycle)
1952-53
Palade/Sjostrand: Mitochondrial membrane structure (TEM)
1955-62
Chance/ Hafeti/Beinert/Crane: The respiratory chain
1961
Mitchell: The Chemiosmotic theory
1964/65
Schatz/Nass: Mitochondrial DNA
1996
Liu et al: Cytokrom c release during apoptosis
Mitochondrial structure
Outer Membrane
Inner membrane
Matrix
Intermembrane space
From: Fawcett, A Textbook of Histology,
Chapman and Hall, 12th edition, 1994
Mitochondrial network in a fibroblast (COS-7).
Mitochondria: Green, Microtubuli: Red
From: Yaffe M.P. 1999, Science 283: 1493-97
Scorrano et. al. 2002,
Dev Cell, 2: 55-67
Mitochondrial facts
• Organelles i Eukaryotic cells
(endosymbiotic theory)
• Have their own DNA (mtDNA)
(“The 47th chromosome”, multiple copies in each cell)
• Contributes in - energy metabolism
- regulation of cell death
• Hepatocytes:
1000-2000 mitochondria constituting about 1/5
of the cell volume
1
Mitochondrial interference
in cell proliferation
• Energy metabolism
- Energy (ATP) supply
- Reactive oxygen species (ROS)
- Nutritional environment
• Signalling
- Ca2+ uptake and efflux
- Cardiolipin
- Cell cycle
• Apoptosis
- Permebility transition
- Release of soluble molecules
• Differentiation
-?
Cell fates - malignancy
Differentiation
Conditions
and signals
Mitosis
Rest
Malignant
transformation
Anticancer
treatment
Tumor
Cell death (apoptosis)
Cancers have high aerobic glycolysis
Modified fatty acids
H
OH
C
Substituents:
• F, Br, S, Se, OH
• Hydrocarbon branches
• Amino/amide-groups
• Aromatic rings
Cancer cell target mechanisms:
Other modifications:
• Double/triple bounds
• Isomerisation (cis, trans)
Source:Gatenby & Gillies, Nat Rev Cancer, 2004
TTA (tetradecylthioacetic acid)
O
•
•
•
•
•
Histon deacetylase (HDAC)
Eicosanoid metabolism (COX-II)
Nuclear receptors (RXR/RAR, PPAR)
Protein prenylation (Ras)
Mitochondria
Tronstad et al, Expert Opin. Ther. Targets, 2003
Mitochondria as target organelles
Drugs
Transporters
?
Cardiolipin
β-oxidation
TCA-cycle
on
dr
io
n
Respiration
M
ito
ch
• Is not β-oxidized
• Stimulates oxidation of other fatty acids
• Mitochondrial proliferation
• Ligand and activator or PPARs
• Hypolipidemic effects
• Immunomodulating effects (anti-inflammatory)
• Low toxicity
Berge RK et al, Curr Opin Lipidol, 2002
2
Reduced tumor growth,
prolonged survival
Inhibition of glioma cell growth
BT4Cn rat gliomas
120
3-Thd incorporation
(% of control)
Intracranial
BT4Cn
100
D54Mg
80
GaMg
60
40
Subcutaneous
20
0
0
50
100
150
200
TTA (µM)
Tronstad et al, Biochem Pharmacol, 2001
Proliferation of AML blasts
from patients
incorporation 10 4 cpm
3 H-Thd
Patient 2
2.0
10 Patient 3
Patient 4
2.0
105
Patient 5
2.0
2.0
1.6
8
1.6
1.6
PA
1.5
1.2
6
1.2
1.2
TTA
1.0
0.8
4
0.8
0.8
0.5
0.4
2
0.4
0.4
0.0
0.0
0
0.0
0.0
Patient 6
12
8
6
4
12 Patient 9
Patient 8
0.4
10
0.3
9
0.2
6
0.1
2
Patient 13
6
4
0.3
0
Patient 15
0.6
2
0.0
Patient 16
0.6
2
0.4.
0.4
1
0.2
0.2
3
2
4
2
3
4
5
0
0
1
2
3
4
0.0
5
Tronstad et al, Leukemia, 2003
0
1
2
3
4
5
0.0
0
1
2
3
4
0
5
n = 23
1
2
1
103
Patient 18
4
6
0.1
0
0
1
2
3
4
0
5
0
1
2
3
4
102
5
Control
TTA
8CPT-cAMP
Induction
IPC-81 Bcl-2
IPC-81 Bcl-2
Capase-8
Oxidative stress
Cellular damage
Second messengers
Specific pathways
*
40
0
*
*
0
3
6 9 12 15 18 21 24
TTA exposure (hrs)
80
*
Decision
*
*
20
*
*
60
DNA
damage
Bax
*
*
20
0
Protease
activation
cyt c,
Smac/DIABLO
Δψ↓
Caspase-9
(Caspase-3)
40
0
1
2
3
4
5
cAMP exposure (hrs)
6
Degradation
60
*
% apoptosis
% apoptosis
80
p53
Bcl-2
100
IPC-81
IPC-81 Bcl-2
TTA
p<0.005
Growth factor
Chemicals
deprivation
Radiation
ROS
Bid
100
Control
Apoptotic pathways
Receptor-mediated
signals
IPC-81
IPC-81
Time
PA
p<0.05
Fatty acid concentration (10 2 µM)
Induction of apoptosis in leukemia cells
Time
104
0
Patient 17
10
8
0.2
0.0
8
0.6
0.4
0.3
Reduced
growth of AML
blasts from
patients
Patient 12
10
0.9
1
0
Patient 14
3
Patient 11
1.2
2
3
0.0
0
0.5
Patient 10
3
incorporation (cpm)
Patient 1
106
3 H-Thd
Figure 1
2.5
Berge K et al, Carcinogenesis, 2001
ROS
Membrane
alterations
Nuclear
apoptosis
Cytoplasmic
alterations
Tronstad et al,Chemistry & Biology, 2003
3
control
TTA-induced apoptosis
TTA
Apoptosis
cyt c (ng/µl protein)
Cytochrome c
Glutathione
*
3
6
0.4
0.0
0.0
0
3
*
*
0.3
0.2
0.1
0.1
6
9
2
ΔΨm Ø
TTA
0 1
2 3 4(h)
12
15
18 hrs
execution
1
Cyt C
Activation of
effector caspases
GSSG
( e
GSH Ø
Generation of
oxidative stress
f
9
12
15
18 h
0
6
12
18 h
procaspase-3
PARP
*
0.2
commitment
mitochondrion
*
TTA
cAMP
*
0
0.3
nmol/mg protein
14
12
10
8
6
4
2
0
ΔΨ
Caspase-3
red fluorescence
fold increase
TTA
inititation
0.4
IPC-81
1.2
0.8
*
0.4
0.0
ctr
cAMP
20
*
15
10
0
3
Nuclear condensation
and fragmentation
*
*
*
6
*
*
h
Transcriptional
regulation
g
*
5
Cytosolic glutathione
depletion (TTA)
i
GSH
0
*
uncleaved
cleaved
GSSG + GSH
25
cAMP
Cell fragmentation
(blebbing)
9 12 15 18 hrs
TTA
nucleus
Tronstad et al, Chemistry & Biology, 2003
Respirometry
The respiratory chain
-measuring oxygen consumption
Intermembrane Space
rotenone
Antimycin A
2H+
CN-
4H+
2H+
3H+
cyt
c
UQ
I
II
FADH2
NADH
FAD
NAD+
H+
III
½O2
FAD
Acyl-CoA
dehdrogenase
Succinate
Pyruvate
Fo
IV
V
ETFP
2H+
F1
H2O
2H+
ADP + Pi
Ascorbate/TMPD
3H+
Matrix
Uncoupling effects of fatty acids,
HL-60 cells
Oxygen consumption in HL-60 cells
Complex IV
Cytochrome c oxidase
(150µM, 5.5h)
KCN
30
25
20
15
ADP
Oxygen flux
Oxygen flux
pmol oxygen/(s*mill cells)
antimycin A
succinate
ascorbate
Cells
+ digitonin
+ rotenon
TMPD
10
pmol oxygen /(s*mill cells)
cytochrome c
35
Control (DMSO)
TTA
10
30
50
100 µM
15
rotenone
5
0
18
16
TTA
DMSO
12
10
0
0
20
14
-5
5
Palmitic acid
22
AntiA
20
10
24
Succinate
25
Oxygen flux
Complex II
Succinate:CoQ reductase
control
TTA
500
1000
1500
time (s)
2000
2500
0
20
40
60
80
FA concentration (µM)
-5
500
1000
1500
2000
2500
3000
time (s)
4
Involvement of a cyclosporin A
sensitive pore?
Palmitic acid
TTA
35
Cyclosporin A
ctr
Fatty acid oxidation
TTA
5
Fatty acid (50µM)
30
25
Ketogenesis
1.6
*
*
20
15
succinate
10
Cells
+ digitonin
+ rotenon
5
0
(µmols/h/2 mil cells)
4
(nmols/h/2 mill cells)
pmol oxygen/(s*mill cells)
TTA increases fatty acid oxidation
and ketogenesis in primary hepatocytes
*
3
2
*
1
0
-5
0
400
800
1200
1600
2000
Substrate:
2400
1.2
*
0.8
0.4
0
PA
EPA
DHA
PA
EPA
DHA
time (s)
Grav et al, JBC, 2003
TTA stimulates respiration in hepatocytes
Lowered membrane potential
140
Oxygen uptake
(ngatoms oxygen/h/2 mill cells)
1800
1600
120
*
*
1400
80
60
40
0
1000
800
600
400
200
0
Substrate:
*
*
100
20
1200
59 ΔpH (mV)
TTA
Δψ (mV)
ctr
UCP-2 mRNA, relative to control
Increased oxygen consumption
Induced UCP-2 expression in rat hepatocytes
-20
-40
-60
Liver
6.0
4.0
*
3.0
2.0
1.0
0.0
control TTA 150 TTA 300
-80
EPA
ctr
Rat
treatment:
Respiration
substrate:
control
TTA
ctr
TTA
Succinate
ctr
Fish-oil TTA
UCP-2 protein
TTA
33 kD
Palmitoyl-L-carnitine
Grav et al, JBC, 2003
7.0
UCP-2 mRNA, relative to control
TTA
-120
PPARα dependent and independent
induction of UCP-2
6.0
Wild type
*
Biological activity of fatty acids
A
TT
PPARα deficient
Proliferation
5.0
PPARs
4.0
3.0
*
2.0
1.0
*
Metabolism
*
0.0
Control
Grav et al, JBC, 2003
control
-100
DHA
Grav et al, JBC, 2003
Purified
hepatocytes
*
-140
PA
Primary
hepatocytes
5.0
Fish-oil
TTA
Fibrate
Apoptosis
Energy
Redox
Death
(apoptosis)
5
Mitochondria as targets
for cancer therapy
Drugs
Changes in energy metabolism
Oxidative damage
Transporters
Respiration
Permeability transition
β-oxidation
Cardiolipin
Institute of Medicine
Haukeland University Hospital, Bergen
Rolf Kristian Berge
Kjetil Berge
Endre Dyrøy
University of Oslo
Therese H. Røst
Hans J. Grav
Oddrun A. Gudbrandsen
Per Ole Iversen
Hege V. Wergedahl
Christian Drevon
Ziad Muna
Pavol Bohov
Norwegian University of Science
and Technology, Trondheim
Bjørn Tore Gjertsen
Tom Chr. Martinsen
Øystein Bruserud
Helge Waldum
Bjarte Sko Erikstein
Emmet McCormack
Differentiation
Department of Biomedicine
Stein Ove Døskeland
Camilla Krakstad
Kari Fladmark
M
ito
ch
?
Apoptosis
on
dr
io
n
Proliferation
Coworkers
Reduced
tumor growth
The Norwegian Cancer
Society
The Research Council
of Norway
6