Workshop Summary
and
Clinical Implications
Sidney M. Hecht, Director
Center for BioEnergetics
Biodesign Institute
Arizona State University
Mitochondria, Metabolism and Cancer Workshop
March 23, 2012
Mitochondria in Health & Disease
• Mitochondria have about 1600 imported gene
• products
ppppppp
- about half have specialized functions and are
organ-specific
- functions include lipid metabolism, signal
transduction
• Clinical expression of mitochondrial disease
requires a high level of mutations
• Mitochondria are essential for nuclear DNA
synthesis, and hence for tumor cell growth
• Mitochondrial DNA fragments can be inserted
into nuclear genes
Cancer as a Metabolic Disease
• Cancer results from damage to cellular respiration
- many types of damage produce a similar response
• Tumor cells produce most of the ATP from Gln by a
compensatory fermentative process
• Caloric restriction is associated with reduced blood
glucose and increased ketone bodies
• Caloric restriction leads to reduced tumor growth, is
strongly antiangiogenic and pro-apoptotic and targets
NF-kB-mediated inflammation
• by
Mitochondrial Dynamics
• Primary mitochondrial disorders can be caused by
any of about 240 mutations
- ROS, mitochondrial membrane potential
• There are also secondary mitochondrial disorders
- [ATP], redox state, PTP, signaling
• Mitochondria within the same cell can have different
ATP levels and mitochondrial membrane potential
• Nature of mitochondrial network (fragmented,
dynamic, hyperfused) may control metabolic activity
Mitochondria, Oxygen Sensing and Cancer
• The mitochondrion is an oxygen sensor
- in different cell types there may be very different
responses to oxygen sensing by mitochondria
• HIF-1a may be a key player in altering cells even
without frank hypoxia
• Cancer cells have increased membrane potential,
and this is reversed by DCA, which induces apoptosis
(via pyruvate dehydrogenase)
• DCA decreases vascular perfusion in tumors
• Mitochondrion has symbiotic relationship with
endoplasmic reticulum
Silencing Mitochondrial Genes
• Mitochondrial DNMT1 leader peptides direct GFP to the
mitochondria
• Mitochondrial DNA contains both 5mC and 5hmC modifications
• Loss of p53 preferentially upregulates the mitochondrial
isoform of DNMT1
• Mitochondrial transcription patterns change in a gene specific
fashion with an increase in mtDNMT1
• mtDNMT1 expression is regulated by factors that respond to
oxidative stress; the mediators are NRF1 and PGC1a, which
interact with the DNMT1 promoter
• 2-HG is a competitive inhibitor of TET enzymes and
histone demethylases
Bioenergetics
• Cancer cells have altered glycolysis and respiration
• Cancer cell mitochondria support cell proliferation
rather than efficient ATP production
• Key factors for killing mitochondria
- slow glycolysis
- block lactate dehydrogenase/stimulate purvate
dehydrogenase to promote TCA cycle and OXPHOS
- target VDAC to form apoptosome
- block NADH to deplete ROS scavenging systems
- promote Ca2+ loading (block Na+/Ca2+ exchange)
Oxidative Stress and Cancer
• Animal models in which antioxidant systems have
been knocked out exhibit enhanced age-related
cancer development
• Malignant tumors often have increased levels of
DNA base oxidation
• p53 has 10 cysteine residues and has been observed
to be nitrated in human gliomas
p53 Protein Has Both Pro- and Antioxidant
Functions
Production of Reactive Oxygen Species
by Human Tumor Cell Lines
• Constitutive H2O2 (but not superoxide)
production has been observed for several
cancer cell lines
• Sustained increase in H2O2 shown to be
essential to maintain transformed state
• Inhibitors of mitochondrial respiration had
no effect on H2O2 production
• Cancer cells have enhanced resistance to
H2O2-induced cell killing, even in the
absence of catalase
Strategies for Antitumor Therapy Based on
Elevated Peroxide Levels in Cells
• Suppress H2O2, causing a reversion of
transformed phenotype
• Enhance ROS, potentially disadvantaging tumor
cells which already contain elevated H2O2
• Use elevated H2O2 to selectively activate toxins in
tumor cells
Activation of Prodrug
with the Generation
of a Quinone Methide
and Ferrocenium Ion
Mitochondrially Targeted Compounds
H
N
H
N
O
OCH3
H3CO
N+ +HN
NH+
ditercalinium
P+
CH3O
N+
CH3O
mitoQ10
O
Non-Peptidic Oligoguanidinium Vectors that
Selectively Internalize Into Mitochondria
Internalization of Oligomers 1 and 2 by HeLa
Cells
Cytotoxicity of Oligomers 1 and 2
Kinetics of Uptake of 0.5 mM Oligomer 1 Into
HeLa Cells
Distribution of Oligomer 1 and Colocalization
with MitoTracker Orange
Hydrogen Peroxide Sensing
• Involves proteins that react with H2O2 faster than
its reaction with glutathione
• Enzymes that process H2O2 (peroxidases,
catalase) have any of several reactive groups
(Cys-thiolate, SeCys, Fe heme, vanadate)
• Proteins modified by H2O2 almost all involve
acidic Cys-thiolates
• Protein modification occurs at 1-700 nM
intracellular [H2O2], below levels associated with
redox stress
General Reaction of a Cys-thiolate Containing
Protein with H2O2
Comparison of the Rates of Activation of OxyR with the
Rates of Deactivation of Three Mammalian Proteins by
H2O2
Cellular Proteins of Interest in Aging and
Disease that are Under Redox Control
• Nox1 (NADPH oxidase homologue)
- overexpression can result in transformation of NIH3T3
cells
- overexpression increases basal H2O2 levels 10-fold
- sustained increase in H2O2 shown to be essential to
maintain transformed state
• Apoptosis-inducing factor (AIF)
- present in mitochondrion and normally assists in radical
and H2O2 scavenging
- translocation outside mitochondria can trigger cell death
- AIF deficiency associated with neurodegeneration
Cellular Proteins of Interest in Aging and
Disease that are Under Redox Control
• Phosphatases (PTPs, PTEN, Cdc25c)
- contain reactive active site cysteines that are easily
oxidized to sulfenic acids
- cysteine oxidation can alter kinase/phosphatase balance
• p66Shc
- mice lacking p66Shc have increased life span
- cells from p66Shc-/- mice have reduced ROS levels
• C-Myc
- forced expression of c-Myc raises ROS levels
- SOD2 levels are suppressed; peroxiredoxin 3 is increased
Reversible Redox-Dependent Signal Transduction
A Model for Oxidative Stress in Tumor Formation